Abstract:
The invention relates to recombinant polypeptides and peptides and particularly to the polypeptide containing in its polypeptidic chain the following amino acid sequence: the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in FIG.  4   a  and FIG.  4   b . The polypeptides and peptides of the invention can be used for the diagnostic of tuberculosis, and can also be part of the active principle in the preparation of vaccine against tuberculosis.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional application of and claims priority to U.S. application Ser. No. 09/342,673, filed Jun. 29, 1999 now U.S. Pat. No. 6,531,138, which is a continuation application of U.S. application Ser. No. 08/447,430, filed May 22, 1995 now U.S. Pat. No. 5,916,558, which is a file-wrapper-continuation of U.S. application Ser. No. 07/690,949, filed Jul. 8, 1991 now abandoned, based on PCT/EP90/01593, filed Sep. 19, 1990, and British application No. 89402571.7, filed Sep. 19, 1989. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to recombinant polypeptides and peptides, which can be used for the diagnosis of tuberculosis. The invention also relates to a process for preparing the above-said polypeptides and peptides, which are in a state of biological purity such that they can be used as part of the active principle in the preparation of vaccines against tuberculosis. 
     It also relates to nucleic acids coding for said polypeptides and peptides. 
     Furthermore, the invention relates to the in vitro diagnostic methods and kits using the above-said polypeptides and peptides and to the vaccines containing the above-said polypeptides and peptides as active principle against tuberculosis. 
     By “recombinant polypeptides or peptides” it is to be understood that it relates to any molecule having a polypeptidic chain liable to be produced by genetic engineering, through transcription and translation, of a corresponding DNA sequence under the control of appropriate regulation elements within an efficient cellular host. Consequently, the expression “recombinant polypeptides” such as is used herein does not exclude the possibility for the polypeptides to comprise other groups, such as glycosylated groups. 
     The term “recombinant” indeed involves the fact that the polypeptide has been produced by genetic engineering, particularly because it results from the expression in a cellular host of the corresponding nucleic acid sequences which have previously been introduced into the expression vector used in said host. 
     Nevertheless, it must be understood that this expression does not exclude the possibility for the polypeptide to be produced by a different process, for instance by classical chemical synthesis according to methods used in the protein synthesis or by proteolytic cleavage of larger molecules. 
     The expression “biologically pure” or “biological purity” means on the one hand a grade of purity such that the recombinant polypeptide can be used for the production of vaccinating compositions and on the other hand the absence of contaminants, more particularly of natural contaminants. 
     2. Description of the Prior Art 
     Tuberculosis remains a major disease in developing countries. The situation is dramatic in some countries, particularly where high incidence of tuberculosis among AIDS patients represents a new source of dissemination of the disease. 
     Tuberculosis is a chronic infectious disease in which cell-mediated immune mechanisms play an essential role both for protection against and control of the disease. 
     Despite BCG vaccination, and some effective drugs, tuberculosis remains a major global problem. Skin testing with tuberculin PPD (protein-purified derivative) largely used for screening of the disease is poorly specific, due to cross reactivity with other pathogenic or environmental saprophytic mycobacteria. 
     Moreover, tuberculin PPD when used in serological tests (ELISA) does not allow to discriminate between patients who have been vaccinated by BCG, or those who have been primo-infected, from those who are developing evolutive tuberculosis and for whom an early and rapid diagnosis would be necessary. 
     A protein with a molecular weight of 32-kDa has been purified (9) from zinc deficient  Mycobacterium bovis  BCG culture filtrate (8). This 32-kDa protein of  M. bovis  BCG has been purified from Sauton zinc deficient culture filtrate of  M. bovis  BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation has been found to be homogeneous as based on several analyses. This P 32  protein is a constituent of BCG cells grown in normal conditions. It represents about 3% of the soluble fraction of a cellular extract, and appears as the major protein released in normal Sauton culture filtrate. This protein has been found to have a molecular weight of 32 000 by SDS-polyacrylamide gel electrophoresis and by molecular sieving. 
     The NH 2 -terminal amino acid sequence of the 32-kDa protein of  M. bovis  BCG (Phe-Ser-Arg-Pro-Gly-Leu (SEQ ID NO:49)) is identical to that reported for the MPB 59 protein purified from  M. bovis  BCG substrain Tokyo (34). 
     Purified P 32  of  M. bovis  BCG has been tested by various cross immunoelectrophoresis techniques, and has been shown to belong to the antigen 85 complex in the reference system for BCG antigens. It has been more precisely identified as antigen 85A in the Closs reference system for BCG antigens (7). 
     Increased levels of immunoglobulin G antibodies towards the 32-kDa protein of  M. bovis  BCG could be detected in 70% of tuberculous patients (30). 
     Furthermore, the 32-kDa protein of  M. bovis  BCG induces specific lymphoproliferation and interferon-(IFN-γ) production in peripheral blood leucocytes from patients with active tuberculosis (12) and PPD-positive healthy subjects. Recent findings indicate that the amount of 32-kDa protein of  M. bovis  BCG-induced IFN-γ in BCG-sensitized mouse spleen cells is under probable H-2 control (13). Finally, the high affinity of mycobacteria for fibronectin is related to proteins of the BCG 85 antigen complex (1). 
     Matsuo et al. (17) recently cloned the gene encoding the antigen α, a major protein secreted by BCG (substrain Tokyo) and highly homologous to MPB 59 antigen in its NH 2 -terminal amino acid sequence, and even identical for its first 6 amino acids: Phe-Ser-Arg-Pro-Gly-Leu (SEQ ID NO:49). 
     This gene was cloned by using a nucleotide probe homologous to the N-terminal amino acid sequence of antigen α, purified from  M. tuberculosis  as described in Tasaka, H. et al., 1983. “Purification and antigenic specificity of alpha protein (Yoneda and Fukui) from  Mycobacterium tuberculosis  and  Mycobacterium intracellulare . Hiroshima J. Med. Sci. 32, 1–8. 
     The presence of antigens of around 30–32-kDa, named antigen 85 complex, has been revealed from electrophoretic patterns of proteins originating from culture media of mycobacteria, such as  Mycobacterium tuberculosis . By immunoblotting techniques, it has been shown that these antigens cross-react with rabbit sera raised against the 32-kDa protein of BCG (8). 
     A recent study reported on the preferential humoral response to a 30-kDa and 31-kDa antigen in lepromatous leprosy patients, and to a 32-kDa antigen in tuberculoid leprosy patients (24). 
     It has also been found that fibronectin (FN)-binding antigens are prominent components of short-term culture supernatants of  Mycobacterium tuberculosis . In 3-day-old supernatants, a 30-kilodalton (kDa) protein was identified as the major (FN)-binding molecule. In 21-day-old supernatants, FN was bound to a double protein band of around 30 to 32-kDa, as well as to a group of antigens of larger molecular mass (57 to 60 kDa) (1). 
     In other experiments, recombinant plasmids containing DNA from  Mycobacterium tuberculosis  were transformed into  Escherichia coli , and three colonies were selected by their reactivity with polyclonal antisera to  M. tuberculosis . Each recombinant produced 35- and 53-kilodalton proteins (35K and 53K proteins, respectively) (“Expression of Proteins of  Mycobacterium tuberculosis  in  Escherichia coli  and Potential of Recombinant Genes and Proteins for Development of Diagnostic Reagents”, Mitchell L Cohen et al., Journal of Clinical Microbiology, July 1987, p. 1176–1180). 
     Concerning the various results known to date, the physico-chemical characteristics of the antigen P 32  of  Mycobacterium tuberculosis  are not precise and, furthermore, insufficient to enable its unambiguous identifiability, as well as the characterization of its structural and functional elements. 
     Moreover, the pathogenicity and the potentially infectious property of  M. tuberculosis  has hampered research enabling to identify, purify and characterize the constituents as well as the secretion products of this bacteria. 
     SUMMARY OF THE INVENTION 
     An aspect of the invention is to provide recombinant polypeptides which can be used as purified antigens for the detection and control of tuberculosis. 
     Another aspect of the invention is to provide nucleic acids coding for the peptidic chains of biologically pure recombinant polypeptides which enable their preparation on a large scale. 
     Another aspect of the invention is to provide antigens which can be used in serological tests as an in vitro rapid diagnostic of tuberculosis. 
     Another aspect of the invention is to provide a rapid in vitro diagnostic means for tuberculosis, enabling it to discriminate between patients suffering from an evolutive tuberculosis from those who have been vaccinated against BCG or who have been primo-infected. 
     Another aspect of the invention is to provide nucleic probes which can be used as in vitro diagnostic reagent for tuberculosis, as well as in vitro diagnostic reagent for identifying  M. tuberculosis  from other strains of mycobacteria. 
     The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,  
 
and the peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the following properties:
   the polypeptides react with rabbit polyclonal antiserum raised against the protein of 32-kDa of  M. bovis  BCG culture filtrate, and/or   react selectively with human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis at an early stage,   and/or react with the amino acid sequence extending from the extremity constituted by amino acid at position (1), to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b.      

     On  FIGS. 3   a  and  3   b:  
         X represents G or GG,   Y represents C or CC,   Z represents C or G,   W represents C or G and is different from Z,   K represents C or CG,   L represents G or CC,   a 1 -b 1  represents ALA-ARG or GLY-ALA-ALA,   a 2  represents arg or gly,   a 3 -b 3 -c 3 -d 3 -e 3 -f 3 - represents his-trp-val-pro-arg-pro or ala-leu-gly-ala,   a 4  represents pro or pro-asn-thr,   a 5  represents pro or ala-pro.       

     The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,  
 
and the peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the following properties:
   the polypeptides react with rabbit polyclonal antiserum raised against the protein of 32-kDa of  M. bovis  BCG culture filtrate, and/or   react selectively with human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis at an early stage,   and/or react with the amino acid sequence extending from the extremity constituted by amino acid at position (1), to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b.      

     The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−30) to the extremity constituted by amino acid at position (−1) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 ,
 
and the peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the following properties:
   the polypeptides react with rabbit polyclonal antiserum raised against the protein of 32-kDa of  M. bovis  BCG culture filtrate, and/or   react selectively with human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis at an early stage,   and/or react with the amino acid sequence extending from the extremity constituted by amino acid at position (1), to the extremity constituted by amino acid at position (295) represented on  FIG. 5 .   

     Advantageous polypeptides of the invention are characterized by the fact that they react with rabbit polyclonal antiserum raised against the protein of 32-kDa of  M. bovis  BCG culture filtrate, hereafter designated by “P 32  protein of BCG”. 
     Advantageous polypeptides of the invention are characterized by the fact that they selectively react with human sera from tuberculous patients and particularly patients developing an evolutive tuberculosis at an early stage. 
     Hereafter is given, in a non limitative way a process for preparing rabbit polyclonal antiserum raised against the P 32  protein of BCG and a test for giving evidence of the reaction between the polypeptides of the invention and said rabbit polyclonal antiserum raised against the P 32  protein of BCG. 
     1) process for preparing rabbit polyclonal antiserum raised against the P 32  protein of BCG: 
     Purified P 32  protein of BCG from culture filtrate is used. 
     a) Purification of protein P 32  of BCG: 
     P 32  protein can be purified as follows: 
     The bacterial strains used are  M. bovis  BCG substrains 1173P2 (Pasteur Institute, Paris) and GL2 (Pasteur Institute, Brussels). 
     The culture of bacteria is obtained as follows: 
       Mycobacterium bovis  BCG is grown as a pellicle on Sauton medium containing 4 g Aspargine, 57 ml 99% Glycerine (or 60 ml 87% Glycerine), 2 g Citric Acid, 0.5 g K 2 HPO 4 , 0.5 g MgSO 4 , 0.05 g Iron Citrate, 5×10 −6  M Ammonium (17% Fe III) SO 4 Zn-7H 2 O and adjusted to 1 liter distilled water adjusted to pH 7.2 with NH 4 OH, at 37.5° C. for 14 days. As the medium is prepared with distilled water, zinc sulfate is added to the final concentration of 5 μM (normal Sauton medium) (De Bruyn J., Weckx M., Beumer-Jochmans M.-P. Effect of zinc deficiency on  Mycobacterium tuberculosis  var.  bovis  (BCG). J. Gen. Microbiol. 1981; 124:353–7). When zinc deficient medium was needed, zinc sulfate is omitted. 
     The filtrates from zinc deficient cultures are obtained as follows: 
     The culture medium is clarified by decantation. The remaining bacteria are removed by filtration through Millipak 100 filter unit (Millipore Corp., Bedford, Mass.). When used for purification, the filtrate is adjusted to 20 mM in phosphate, 450 mM in NaCl, 1 mM in EDTA, and the pH is brought to 7.3 with 5 M HCl before sterile filtration. 
     The protein analysis is carried out by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done on 13% (w/v) acrylamide-containing gels as described by Laemmli UK. (Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–5). The gels are stained with Coomassie Brilliant Blue R-250 and for quantitative analysis, scanned at 595 nm with a DU8 Beckman spectrophotometer. For control of purity the gel is revealed with silver stain (Biorad Laboratories, Richmond, Calif.). 
     The purification step of P 32  is carried out as follows: 
     Except for hydrophobic chromatography on Phenyl-Sepharose, all buffers contain Tween 80 (0.005% final concentration). The pH is adjusted to 7.3 before sterilization. All purification steps are carried out at +4° C. Elutions are followed by recording the absorbance at 280 nm. The fractions containing proteins are analysed by SDS-PAGE. 
     (i) The treated filtrate from a 4 liters zinc-deficient culture, usually containing 125 to 150 mg protein per liter, is applied to a column (5.0 by 5.0 cm) of Phenyl-Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden), which is previously equilibrated with 20 mM phosphate buffer (PB) containing 0.45 M NaCl and 1 mM EDTA, at a flow rate of 800 ml per hour. The gel is then washed with one column volume of the same buffer to remove unfixed material and successively with 300 ml of 20 mM and 4 mM PB and 10% ethanol (v/v). The P 32  appears in the fraction eluted with 10% ethanol. 
     (ii) After the phosphate concentration of this fraction has been brought to 4 mM, it is applied to a column (2.6 by 10 cm) of DEAE-Sephacel (Pharmacia Fine Chemicals), which is equilibrated with 4 mM PB. After washing with the equilibrating buffer the sample is eluted with 25 mM phosphate at a flow rate of 50 ml per hour. The eluate is concentrated in a 202 Amicon stirred cell equipped with a PM 10 membrane (Amicon Corp., Lexington, Mass.). 
     (iii) The concentrated material is submitted to molecular sieving on a Sephadex G-100 (Pharmacia) column (2.6 by 45 cm) equilibrated with 50 mM PB, at a flow rate of 12 ml per hour. The fractions of the peak giving one band in SDS-PAGE are pooled. The purity of the final preparation obtained is controlled by SDS-PAGE followed by silverstaining and by molecular sieving on a Superose 12 (Pharmacia) column (12.0 by 30 cm) equilibrated with 50 mM PB containing 0.005% Tween 80 at a flow rate of 0.2 ml/min. in the Fast Protein Liquid Chromatography system (Pharmacia). Elution is followed by recording the absorbance at 280 nm and 214 nm. 
     b) Preparation of rabbit polyclonal antiserum raised against the P 32  protein of BCG: 
     400 μg of purified P 32  protein of BCG per ml physiological saline are mixed with one volume of incomplete Freund&#39;s adjuvant. The material is homogenized and injected intradermally in 50 μl doses delivered at 10 sites in the back of the rabbits, at 0, 4, 7 and 8 weeks (adjuvant is replaced by the diluent for the last injection). One week later, the rabbits are bled and the sera tested for antibody level before being distributed in aliquots and stored at −80° C.; 
     2) test for giving evidence of the reaction between the polypeptides of the invention and said rabbit polyclonal antiserum raised against the P 32  protein of BCG: 
     the test used was an ELISA test; the ELISA for antibody determination is based on the method of Engvall and Perlmann (Engvall, E., and P. Perlmann. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871–874) 
     Immulon Microelisa plates (Dynatech, Kloten, Switzerland) are coated by adding to each well 1 μg of one of the polypeptides of the invention in 100 μl Tris hydrochloride buffer 50 mM (pH 8.2). After incubation for 2 h at 27° C. in a moist chamber, the plates are kept overnight at 4° C. They are washed four times with 0.01 M phosphate-buffered saline (pH 7.2) containing 0.05% Tween 20 by using a Titertek microplate washer (Flow Laboratories. Brussels. Belgium). Blocking is done with 0.5% gelatin in 0.06 M carbonate buffer (pH 9.6) for 1 h. Wells are then washed as before, and 100 μl of above mentioned serum diluted in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin is added. According to the results obtained in preliminary experiments, the working dilutions are set at 1:200 for IgG, 1:20 for IgA and 1:80 for IgM determinations. Each dilution is run in duplicate. After 2 h of incubation and after the wells are washed, they are filled with 100 μl of peroxidase-conjugated rabbit immunoglobulins directed against human IgG, IgA or IgM (Dakopatts, Copenhagen, Denmark), diluted 1:400, 1:400 and 1:1,200, respectively in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin and incubated for 90 min. After the wash, the amount of peroxidase bound to the wells is quantified by using a freshly prepared solution of o-phenylenediamine (10 mg/100 ml) and hydrogen peroxide (8 μl of 30% H 2 O 2  per 100 ml) in 0.15 M citrate buffer (pH 5.0) as a substrate. The enzymatic reaction is stopped with 8 N H 2 SO 4  after 15 min. of incubation. The optical density is read at 492 nm with a Titertek Multiskan photometer (Flow Laboratories). 
     Wells without sera are used as controls for the conjugates. Each experiment is done by including on each plate one negative and two positive reference sera with medium and low antibody levels to correct for plate-to-plate and day-to-day variations. The antibody concentrations are expressed as the optical density values obtained after correction of the readings according to the mean variations of the reference sera. 
     Hereafter is also given in a non limitative way, a test for giving evidence of the fact that polypeptides of the invention are recognized selectively by human sera from tuberculous patients. 
     This test is an immunoblotting (Western blotting) analysis, in the case where the polypeptides of the invention are obtained by recombinant techniques. This test can also be used for polypeptides of the invention obtained by a different preparation process. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polypeptides of the invention are blotted onto nitrocellulose membranes (Hybond C. (Amersham)) as described by Towbin et al. (29). The expression of polypeptides of the invention fused to β-galactosidase in  E. coli  Y1089, is visualized by the binding of a polyclonal rabbit anti-32-kDa BCG protein serum (1:1,000) or by using a monoclonal anti-β-galactosidase antibody (Promega). The secondary antibody (alkaline phosphatase anti-rabbit immunoglobulin G and anti-mouse alkaline phosphatase immunoglobulin G conjugates, respectively) is diluted as recommended by the supplier (Promega). 
     In order to identify selective recognition of polypeptides of the invention and of fusion proteins of the invention by human tuberculous sera, nitrocellulose sheets are incubated overnight with these sera (1:50) (after blocking aspecific protein-binding sites). The human tuberculous sera are selected for their reactivity (high or low) against the purified 32-kDa antigen of BCG tested in a dot blot assay as described in document (31) of the bibliography hereafter. Reactive areas on the nitrocellulose sheets are revealed by incubation with peroxidase conjugated goat anti-human immunoglobulin G antibody (Dakopatts, Copenhagen, Denmark) (1:200) for 4 h, and after repeated washings, color reaction is developed by adding peroxidase substrate (α-chloronaphtol) (Bio-Rad Laboratories, Richmond, Calif.) in the presence of peroxidase and hydrogen peroxide. 
     It goes without saying that the free reactive functions which are present in some of the amino acids, which are part of the constitution of the polypeptides of the invention, particularly the free carboxyl groups which are carried by the groups Glu or by the C-terminal amino acid on the one hand and/or the free NH 2  groups carried by the N-terminal amino acid or by amino acid inside the peptidic chain, for instance Lys, on the other hand, can be modified in so far as this modification does not alter the above mentioned properties of the polypeptide. 
     The molecules which are thus modified are naturally part of the invention. The above mentioned carboxyl groups can be acylated or esterified. 
     Other modifications are also part of the invention. Particularly, the amine or ester functions or both of terminal amino acids can be themselves involved in the bond with other amino acids. For instance, the N-terminal amino acid can be linked to a sequence comprising from 1 to several amino acids corresponding to a part of the C-terminal region of another peptide. 
     Furthermore, any peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids of the polypeptides according to the invention are part of the invention in so far as this modification does not alter the above mentioned properties of said polypeptides. 
     The polypeptides according to the invention can be glycosylated or not, particularly in some of their glycosylation sites of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid. 
     Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−49) to to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b.      

     Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−49) to to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b.      

     Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−43) to the extremity constituted by amino acid at position (−1) represented on  FIG. 5 .   

     Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−49) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b.      

     Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−49) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b.      

     Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 ,   the one extending from the extremity constituted by amino acid at position (−30) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 ,   the one extending from the extremity constituted by amino acid at position (−43) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 .   

     Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−49) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b.      

     Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (−294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−49) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b.      

     Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 ,   the one extending from the extremity constituted by amino acid at position (−30) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 ,   the one extending from the extremity constituted by amino acid at position (−43) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 .   

     Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−49) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (−1) represented on  FIG. 3   a  and  FIG. 3   b.      

     Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−59) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−55) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−49) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−47) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−42) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by amino acid at position (−29) to the extremity constituted by amino acid at position (−1) represented on  FIG. 4   a  and  FIG. 4   b.      

     Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
     the one extending from the extremity constituted by amino acid at position (−43) to the extremity constituted by amino acid at position (−1) represented on  FIG. 5 ,   the one extending from the extremity constituted by amino acid at position (−30) to the extremity constituted by amino acid at position (−1) represented on  FIG. 5 .   

     In eukaryotic cells, these polypeptides can be used as signal peptides, the role of which is to initiate the translocation of a protein from its site of synthesis, but which is excised during translocation. 
     Other advantageous peptides of the invention consist in one of the following amino acid sequence:
     the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on  FIG. 3   a  and  FIG. 3   b , or   the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on  FIG. 3   a  and  FIG. 3   b.      

     Other advantageous peptides of the invention consist in one of the following amino acid sequence:
     the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on  FIG. 4   a  and  FIG. 4   b , or   the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on  FIG. 4   a  and  FIG. 4   b.      

     Other advantageous peptides of the invention consist in one of the following amino acid sequence:
     the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on  FIG. 5 , or   the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (295) represented on  FIG. 5 .   

     It is to be noted that the above mentioned polypeptides are derived from the expression products of a DNA derived from the nucleotide sequence coding for a protein of 32-kDa secreted by  Mycobacterium tuberculosis  as explained hereafter in the examples. 
     The invention also relates to the amino acid sequences constituted by the above mentioned polypeptides and a protein or an heterologous sequence with respect to said polypeptide, said protein or heterologous sequence comprising for instance from about 1 to about 1000 amino acids. These amino acid sequences will be called fusion proteins. 
     In an advantageous fusion protein of the invention, the heterologous protein is β-galactosidase. 
     Other advantageous fusion proteins of the invention are the ones containing an heterologous protein resulting from the expression of one of the following plasmids: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pEX1 
                   
               
               
                   
                 pEX2 
               
               
                   
                 pEX3 
               
               
                   
                 pUEX1 
                 pmTNF MPH 
               
               
                   
                 pUEX2 
               
               
                   
                 pUEX3 
               
               
                   
                   
               
             
          
         
       
     
     The invention also relates to any nucleotide sequence coding for a polypeptide of the invention. 
     The invention also relates to nucleic acids comprising nucleotide sequences which hybridize with the nucleotide sequences coding for any of the above mentioned polypeptides under the following hybridization conditions:
     hybridization and wash medium: 3×SSC, 20% formamide (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0),   hybridization temperature (HT) and wash temperature (WT) for the nucleic acids of the invention defined by x-y: i.e. by the sequence extending from the extremity constituted by the nucleotide at position (x) to the extremity constituted by the nucleotide at position (y) represented on  FIG. 3   a  and  FIG. 3   b .   

     
       
         
               
               
             
           
               
                   
               
             
             
               
                  1–182 
                 HT = WT = 69° C. 
               
               
                  1–194 
                 HT = WT = 69° C. 
               
               
                  1–212 
                 HT = WT = 69° C. 
               
               
                  1–218 
                 HT = WT = 69° C. 
               
               
                  1–272 
                 HT = WT = 69° C. 
               
               
                  1–359 
                 HT = WT = 71° C. 
               
               
                  1–1241 
                 HT = WT = 73° C. 
               
               
                  1–1358 
                 HT = WT = 73° C. 
               
               
                 183–359  
                 HT = WT = 70° C. 
               
               
                 183–1241 
                 HT = WT = 73° C. 
               
               
                 183–1358 
                 HT = WT = 73° C. 
               
               
                 195–359  
                 HT = WT = 70° C. 
               
               
                 195–1241 
                 HT = WT = 73° C. 
               
               
                 195–1358 
                 HT = WT = 73° C. 
               
               
                 213–359  
                 HT = WT = 70° C. 
               
               
                 213–1241 
                 HT = WT = 73° C. 
               
               
                 213–1358 
                 HT = WT = 73° C. 
               
               
                 219–359  
                 HT = WT = 71° C. 
               
               
                 219–1241 
                 HT = WT = 73° C. 
               
               
                 219–1358 
                 HT = WT = 73° C. 
               
               
                 234–359  
                 HT = WT = 71° C. 
               
               
                 234–1241 
                 HT = WT = 74° C. 
               
               
                 234–1358 
                 HT = WT = 73° C. 
               
               
                 273–359  
                 HT = WT = 71° C. 
               
               
                 273–1241 
                 HT = WT = 74° C. 
               
               
                 273–1358 
                 HT = WT = 73° C. 
               
               
                 360–1241 
                 HT = WT = 73° C. 
               
               
                 360–1358 
                 HT = WT = 73° C. 
               
               
                 1242–1358  
                 HT = WT = 62° C. 
               
               
                   
               
             
          
         
       
     
     The above mentioned temperatures are to be considered as approximately ±5° C. 
     The invention also relates to nucleic acids comprising nucleotide sequences which are complementary to the nucleotide sequences coding for any of the above mentioned polypeptides. 
     It is to be noted that in the above defined nucleic acids, as well as in the hereafter defined nucleic acids, the nucleotide sequences which are brought into play are such that T can be replaced by U. 
     A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358), wherein N represents one of the five A, T, C, G or I nucleotides, represented in  FIG. 3   a  and  FIG. 3   b,  
 
or above said nucleotide sequences wherein T is replaced by U,
 
or nucleic acids which hybridize with said above mentioned nucleotide sequences or the complementary sequences thereof.
   

     A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358), wherein N represents one of the five A, T, C, G or I nucleotides, represented in  FIG. 4   a  and  FIG. 4   b,  
 
or above said nucleotide sequences wherein T is replaced by U,
 
or nucleic acids which hybridize with said above mentioned nucleotide sequences or the complementary sequences thereof.
   

     A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (219) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1104) to the extremity constituted by nucleotide at position (1299), wherein N represents one of the five A, T, C, G or I nucleotides, represented in  FIG. 5 ,
 
or above said nucleotide sequences wherein T is replaced by U,
 
or nucleic acids which hybridize with said above mentioned nucleotide sequences or the complementary sequences thereof.
   

     Other preferred nucleic acids of the invention comprise one at least of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b.      

     Other preferred nucleic acids of the invention comprise one at least of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b.      

     Another preferred group of nucleic acids of the invention comprises the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented, in  FIG. 3   a  and  FIG. 3   b.      

     Another preferred group of nucleic acids of the invention comprises the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b.      

     According to another advantageous embodiment, nucleic acids of the invention comprises one of the following sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b.      

     According to another advantageous embodiment, nucleic acids of the invention comprises one of the following sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b.      

     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b.      

     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b.      

     These nucleotide sequence can be used as nucleotide signal sequences, coding for the corresponding signal peptide. 
     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b.      

     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b,      the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 3   a  and  FIG. 3   b.      

     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b.      

     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b,      the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358) represented in  FIG. 4   a  and  FIG. 4   b.      

     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (129) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (219) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (219) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 .   

     Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
     the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (129) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (219) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (219) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (219) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1104) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 ,   the one extending from the extremity constituted by nucleotide at position (1104) to the extremity constituted by nucleotide at position (1299) represented in  FIG. 5 .   

     The invention also relates to any recombinant nucleic acids containing at least a nucleic acid of the invention inserted in an heterologous nucleic acid. 
     The invention relates more particularly to recombinant nucleic acid such as defined, in which the nucleotide sequence of the invention is preceded by a promoter (particularly an inducible promoter) under the control of which the transcription of said sequence is liable to be processed and possibly followed by a sequence coding for transcription termination signals. 
     The invention also relates to the recombinant nucleic acids in which the nucleic acid sequences coding for the polypeptide of the invention and possibly the signal peptide, are recombined with control elements which are heterologous with respect to the ones to which they are normally associated within the bacteria gene and, more particularly, the regulation elements adapted to control their expression in the cellular host which has been chosen for their production. 
     The invention also relates to recombinant vectors, particularly for cloning and/or expression, comprising a vector sequence, notably of the type plasmid, cosmid or phage, and a recombinant nucleic acid of the invention, in one of the non essential sites for its replication. 
     Appropriate vectors for expression of the recombinant antigen are the following one: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pEX1 
                 pmTNF 
                 MPH 
               
               
                   
                 pEX2 
                 pIGRI 
               
               
                   
                 pEX3 
               
               
                   
                 pUEX1 
               
               
                   
                 pUEX2 
               
               
                   
                 pUEX3 
               
               
                   
                   
               
             
          
         
       
     
     The pEX1, pEX2 and pEX3 vectors are commercially available and can be obtained from Boehringer Mannheim. 
     The pUEX1, pUEX2 and pUEX3 vectors are also commercially available and can be obtained from Amersham. 
     According to an advantageous embodiment of the invention, the recombinant vector contains, in one of its non essential sites for its replication, necessary elements to promote the expression of polypeptides according to the invention in a cellular host and possibly a promoter recognized by the polymerase of the cellular host, particularly an inducible promoter and possibly a signal sequence and/or an anchor sequence. 
     According to another additional embodiment of the invention, the recombinant vector contains the elements enabling the expression by  E. coli  of a nucleic acid according to the invention inserted in the vector, and particularly the elements enabling the expression of the gene or part thereof of β-galactosidase. 
     The invention also relates to a cellular host which is transformed by a recombinant vector according to the invention, and comprising the regulation elements enabling the expression of the nucleotide sequence coding for the polypeptide according to the invention in this host. 
     The invention also relates to a cellular host chosen from among bacteria such as  E. coli , transformed by a vector as above defined, and defined hereafter in the examples, or chosen from among eukaryotic organism, such as CHO cells, insect cells, Sf9 cells [ Spodoptera frugiperda ] infected by the virus Ac NPV ( Autographa californica  nuclear polyhydrosis virus) containing suitable vectors such as pAc 373 pYM1 or pVC3, BmN [ Bombyx mori ] infected by the virus BmNPV containing suitable vectors such as pBE520 or p89B310. 
     The invention relates to an expression product of a nucleic acid expressed by a transformed cellular host according to the invention. 
     The invention also relates to nucleotidic probes, hybridizing with anyone of the nucleic acids or with their complementary sequences, and particularly the probes chosen among the following nucleotidic sequences gathered in Table 1, and represented in  FIG. 9 . 
                                                                                                                                                                                       TABLE 1                   Probes A(i), A(ii), A(iii), A(iv) and A(v)                A(i)   CAGCTTGTTGACAGGGTTCGTGGC   (SEQ ID NO:1)                   A(ii)   GGTTCGTGGCGCCGTCACG   (SEQ ID NO:2)               A(iii)   CGTCGCGCGCCTAGTGTCGG   (SEQ ID NO:3)               A(iv)   CGGCGCCGTCGGTGGCACGGCGA   (SEQ ID NO:4)               A(v)   CGTCGGCGCGGCCCTAGTGTCGG   (SEQ ID NO:5)                    Probe B                    TCGCCCGCCCTGTACCTG   (SEQ ID NO:6)                            Probe C                    GCGCTGACGCTGGCGATCTATC   (SEQ ID NO:7)                            Probe D                    CCGCTGTTGAACGTCGGCAAG   (SEQ ID NO:8)                            Probe E                    AAGCCGTCGGATCTGGGTGGCAAC   (SEQ ID NO:9)                            Probes F(i), F(ii), F(iii) and F(iv)                F(i)   ACGGCACTGGGTGCCACGCCCAAC   (SEQ ID NO:10)                   F(ii)   ACGCCCAACACCGGGCCCGCCGCA   (SEQ ID NO:11)               F(iii)   ACGGGCACTGGGTGCCACGCCCAAC   (SEQ ID NO:12)               F(iv)   ACGCCCCAACACCGGGCCCGCGCCCCA   (SEQ ID NO:13)                    
or their complementary nucleotidic sequences.
 
     The hybridization conditions can be the following ones:
     hybridization and wash medium: 3×SSC, 20% formamide (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0),   hybridization temperature (HT) and wash temperature (WT):   

     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 (WT) ° C.: 
                 HT and WT (° C.) 
               
               
                   
                   
               
             
             
               
                   
                 A(i) 
                 50 
               
               
                   
                 A(ii) 
                 50 
               
               
                   
                 A(iii) 
                 52 
               
               
                   
                 A(iv) 
                 60 
               
               
                   
                 A(v) 
                 52 
               
               
                   
                 B 
                 48 
               
               
                   
                 C 
                 50 
               
               
                   
                 D 
                 45 
               
               
                   
                 E 
                 52 
               
               
                   
                 F(i) 
                 55 
               
               
                   
                 F(ii) 
                 59 
               
               
                   
                 F(iii) 
                 55 
               
               
                   
                 F(iv) 
                 59 
               
               
                   
                   
               
             
          
         
       
     
     These probes might enable to differentiate  M. tuberculosis  from other bacterial strains and in particular from the following mycobacteria species:
       Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium intracellulare, Mycobacterium xenopi, Mycobacterium gastri, Mycobacterium nonchromogenicum, Mycobacterium terrae  and  Mycobacterium triviale , and more particularly from  M. bovis, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium phlei  and  Mycobacterium fortuitum.      

     The invention also relates to DNA or RNA primers which can be used for the synthesis of nucleotidic sequences according to the invention by PCR (polymerase chain reaction technique), such as described in U.S. Pat. No. 4,683,202 and No. 4,683,195 and European Patent no 200362. 
     The invention also relates to any DNA or RNA primer constituted by about 15 to about 25 nucleotides of a nucleotide sequence coding for a polypeptide according to the invention. 
     The invention also relates to any DNA or RNA primer constituted by about 15 to about 25 nucleotides liable to hybridize with a nucleotide sequence coding for a polypeptide according to the invention. 
     The invention also relates to any DNA or RNA primer constituted by about 15 to about 25 nucleotides complementary to a nucleotide sequence coding for a polypeptide according to the invention. 
     The sequences which can be used as primers are given in Table 2 hereafter (sequences P1 to P6 or their complement) and illustrated in  FIG. 9 : 
     
       
         
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
             
             
               
                 P1 
                 GAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCG 
                 (SEQ ID 
                   
               
               
                   
                   
                 NO:14) 
               
               
                   
               
               
                 P2 
                 ATCAACACCCCGGCGTTCGAGTGGTAC 
                 (SEQ ID 
               
               
                 compl. 
                   
                 NO:15) 
               
               
                   
               
               
                 P2  
                 GTACCACTCGAACGCCGGGCTGTTGAT 
                 (SEQ ID 
               
               
                   
                   
                 NO:16) 
               
               
                   
               
               
                 P3 
                 TGCCAGACTTACAAGTGGGA 
                 (SEQ ID 
               
               
                   
                   
                 NO:17) 
               
               
                   
               
               
                 P3  
                 TCCCACTTGTAAGTCTGGCA 
                 (SEQ ID 
               
               
                 compl. 
                   
                 NO:18) 
               
               
                   
               
               
                 P4 
                 TCCTGACCAGCGAGCTGCCG 
                 (SEQ ID 
               
               
                   
                   
                 NO:19) 
               
               
                   
               
               
                 P4  
                 CGGCAGCTCGCTGGTCAGGA 
                 (SEQ ID 
               
               
                 compl. 
                   
                 NO:20) 
               
               
                   
               
               
                 P5 
                 CCTGATCGGCCTGGCGATGCGTGACGC 
                 (SEQ ID 
               
               
                   
                   
                 NO:21) 
               
               
                   
               
               
                 P5  
                 GCGTCACCCATCGCCAGGCCGATCAGC 
                 (SEQ ID 
               
               
                 compl. 
                   
                 NO:22) 
               
               
                   
               
               
                 P6  
                 GCGCCCCAGTACTCCCAGCTGTGCGT 
                 (SEQ ID 
               
               
                 compl. 
                   
                 NO:23) 
               
               
                   
               
               
                 compl. = complement 
               
             
          
         
       
     
     The sequences can be combined in twelve different primer-sets (given in Table 3) which allow enzymatical amplification by the polymerase chain reaction (PCR) technique of any of the nucleotide sequences of the invention, and more particularly the one extending from the extremity constituted by nucleotide at position 1 to the extremity constituted by nucleotide at position 1358, as well as the nucleotide sequence of antigen a of BCG (17). 
     The detection of the PCR amplified product can be achieved by a hybridization reaction with an oligonucleotide sequence of at least 10 nucleotides which is located between PCR primers which have been used to amplify the DNA. 
     The PCR products of the nucleotide sequences of the invention can be distinguished from the α-antigen gene of BCG or part thereof by hybridization techniques (dot-spot, Southern blotting, etc.) with the probes indicated in Table 3. The sequences of these probes can be found in Table 1 hereabove. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Primer set 
                 Detection with probe 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 P1 and the complement of P2 
                 B 
               
               
                 2. 
                 P1 and the complement of P3 
                 B 
               
               
                 3. 
                 P1 and the complement of P4 
                 B 
               
               
                 4. 
                 P1 and the complement of P5 
                 B or C 
               
               
                 5. 
                 P1 and the complement of P6 
                 B, C, D or E 
               
               
                 6. 
                 P2 and the complement of P5 
                 C 
               
               
                 7. 
                 P2 and the complement of P6 
                 C, D or E 
               
               
                 8. 
                 P3 and the complement of P5 
                 C 
               
               
                 9. 
                 P3 and the complement of P6 
                 C, D or E 
               
               
                 10. 
                 P4 and the complement of P5 
                 C 
               
               
                 11. 
                 P4 and the complement of P6 
                 C, D or E 
               
               
                 12. 
                 P5 and the complement of P6 
                 D or E 
               
               
                   
               
             
          
         
       
     
     It is to be noted that enzymatic amplification can also be achieved with all oligonucleotides with sequences of about 15 consecutive bases of the primers given in Table 2. Primers with elongation at the 5′-end or with a small degree of mismatch may not considerably affect the outcome of the enzymatic amplification if the mismatches do not interfere with the base-pairing at the 3′-end of the primers. 
     Specific enzymatic amplification of the nucleotide sequences of the invention and not of the BCG gene can be achieved when the probes (given in Table 1) or their complements are used as amplification primers. 
     When the above mentioned probes of Table 1 are used as primers, the primer sets are constituted by any of the nucleotide sequences (A, B, C, D, E, F) of Table 1 in association with the complement of any other nucleotide sequence, chosen from A, B, C, D, E or F, it being understood that sequence A means any of the sequences A(i), A(ii), A(iii), A(iv), A(v) and sequence F, any of the sequences F(i), F(ii), F(iii) and F(iv). 
     Advantageous primer sets for enzymatic amplification of the nucleotide sequence of the invention can be one of the following primer sets given in Table 3bis hereafter: 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 3BIS 
               
               
                   
                   
               
             
             
               
                   
                 A(i) 
                   
               
               
                   
                 or A(ii) 
               
               
                   
                 or A(iii) 
                 and the complement of B 
               
               
                   
                 or A(iv) 
               
               
                   
                 or A(v) 
               
               
                   
                 A(i) 
               
               
                   
                 or A(ii) 
               
               
                   
                 or A(iii) 
                 and the complement of C 
               
               
                   
                 or A(iv) 
               
               
                   
                 or A(v) 
               
               
                   
                 B 
                 and the complement of C 
               
               
                   
                 A(i) 
               
               
                   
                 or A(ii) 
               
               
                   
                 or A(iii) 
                 and the complement of F 
               
               
                   
                 or A(iv) 
               
               
                   
                 or A(v) 
               
               
                   
                 A(i) 
               
               
                   
                 or A(ii) 
               
               
                   
                 or A(iii) 
                 and the complement of D 
               
               
                   
                 or A(iv) 
               
               
                   
                 or A(v) 
               
               
                   
                 A(i) 
               
               
                   
                 or A(ii) 
               
               
                   
                 or A(iii) 
                 and the complement of E 
               
               
                   
                 or A(iv) 
               
               
                   
                 or A(v) 
               
               
                   
                 B 
                 and the complement of D 
               
               
                   
                 B 
                 and the complement of E 
               
               
                   
                 B 
                 and the complement of F 
               
               
                   
                 C 
                 and the complement of D 
               
               
                   
                 C 
                 and the complement of E 
               
               
                   
                 C 
                 and the complement of F 
               
               
                   
                 D 
                 and the complement of E 
               
               
                   
                 D 
                 and the complement of F 
               
               
                   
                 E 
                 and the complement of F 
               
               
                   
                   
               
               
                   
                 A(i), A(ii), A(iii), A(iv), A(v), B, C, D, E and F having the nucleotide sequence indicated in Table 1. 
               
             
          
         
       
     
     In the case of amplification of a nucleotide sequence of the invention with any of the above mentioned primer sets defined in Table 3bis hereabove, the detection of the amplified nucleotide sequence can be achieved by a hybridization reaction with an oligonucleotide sequence of at least 10 nucleotides, said sequence being located between the PCR primers which have been used to amplify the nucleotide sequence. An oligonucleotide sequence located between said two primers can be determined from  FIG. 9  where the primers A, B, C, D, E and F are represented by the boxed sequences respectively named probe region A, probe region B, probe region C, probe region D, probe region E and probe region F. 
     The invention also relates to a kit for enzymatic amplification of a nucleotide sequence by PCR technique and detection of the amplified nucleotide sequence containing
     one of the PCR primer sets defined in Table 3 and one of the detection probes of the invention, advantageously the probes defined in Table 1,
 
or one of the PCR primer sets defined in Table 3bis, and a detection sequence consisting for instance in an oligonucleotide sequence of at least 10 nucleotides, said sequence being located ( FIG. 9 ) between the two PCR primers constituting the primer set which has been used for amplifying said nucleotide sequence.
   

     The invention also relates to a process for preparing a polypeptide according to the invention comprising the following steps:
     the culture in an appropriate medium of a cellular host which has previously been transformed by an appropriate vector containing a nucleic acid according to the invention,   the recovery of the polypeptide produced by the above said transformed cellular host from the above said culture medium, and   the purification of the polypeptide produced, eventually by means of immobilized metal ion affinity chromatography (IMAC).   

     The polypeptides of the invention can be prepared according to the classical techniques in the field of peptide synthesis. 
     The synthesis can be carried out in homogeneous solution or in solid phase. 
     For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book titled “Methode der organischen chemie” (Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974. 
     The polypeptides of the invention can also be prepared according to the method described by R. D. MERRIFIELD in the article titled “Solid phase peptide synthesis” (J. Am. Chem. Soc., 45, 2149–2154, 1964). 
     The invention also relates to a process for preparing the nucleic acids according to the invention. 
     A suitable method for chemically preparing the single-stranded nucleic acids (containing at most 100 nucleotides of the invention) comprises the following steps:
         DNA synthesis using the automatic β-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274–325, 1986.       

     In the case of single-stranded DNA, the material which is obtained at the end of the DNA synthesis can be used as such. 
     A suitable method for chemically preparing the double-stranded nucleic acids (containing at most 100 bp of the invention) comprises the following steps:
         DNA synthesis of one sense oligonucleotide using the automatic β-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274–325, 1986, and DNA synthesis of one anti-sense oligonucleotide using said above-mentioned automatic β-cyanoethyl phosphoramidite method,   combining the sense and anti-sense oligonucleotides by hybridization in order to form a DNA duplex,   cloning the DNA duplex obtained into a suitable plasmid vector and recovery of the DNA according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.       

     A method for the chemical preparation of nucleic acids of length greater than 100 nucleotides—or bp, in the case of double-stranded nucleic acids—comprises the following steps:
         assembling of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are compatible with the succession of amino acids in the natural peptide, according to the principle described in Proc. Nat. Acad. Sci. USA 80; 7461–7465, 1983,   cloning the DNA thereby obtained into a suitable plasmid vector and recovery of the desired nucleic acid according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.       

     The invention also relates to antibodies themselves formed against the polypeptides according to the invention. 
     It goes without saying that this production is not limited to polyclonal antibodies. 
     It also relates to any monoclonal antibody produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the purified polypeptide of the invention on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by its ability to produce the monoclonal antibodies recognizing the polypeptide which has been initially used for the immunization of the animals. 
     The invention also relates to any antibody of the invention labeled by an appropriate label of the enzymatic, fluorescent or radioactive type. 
     The peptides which are advantageously used to produce antibodies, particularly monoclonal antibodies, are the following ones gathered in Table 4: 
     
       
         
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4a 
               
             
             
               
                   
               
               
                 (see FIG. 4a and 4b) 
                   
               
             
          
           
               
                 Amino 
                   
                 Amino 
                   
               
               
                 acid 
                   
                 acid 
               
               
                 position 
                   
                 position 
               
               
                 (NH 2 - 
                   
                 (COOH- 
               
               
                 terminal) 
                   
                 terminal) 
               
               
                   
               
             
          
           
               
                  12 
                 QVPSPSMGRDIKVQFQSGGA 
                  31 
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO:24) 
               
               
                  36 
                 LYLLDGLRAQDDFSGWDINT 
                  55 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:25) 
               
               
                  77 
                 SFYSDWYQPACRKAGCQTYK 
                  96 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:26) 
               
               
                 101 
                 LTSELPGWLQANRHVKPTGS 
                 120 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:27) 
               
               
                 175 
                 KASDMWGPKEDPAWQRNDPL 
                 194 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:28) 
               
               
                 211 
                 CGNGKPSDLGGNNLPAKFLE 
                 230 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:29) 
               
               
                 275 
                 KPDLQRHWVPRPTPGPPQGA 
                 294 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:30) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4b 
               
             
             
               
                   
               
               
                 (see FIG. 5) 
                   
               
             
          
           
               
                 Amino 
                   
                 Amino 
                   
               
               
                 acid 
                   
                 acid 
               
               
                 position 
                   
                 position 
               
               
                 (NH 2 - 
                   
                 (COOH- 
               
               
                 terminal) 
                   
                 terminal) 
               
               
                   
               
             
          
           
               
                  77 
                 SFYSDWYQPACGKAGCQTYX 
                  96 
                 (SEQ ID) 
                   
               
               
                   
                   
                   
                 NO:31) 
               
               
                   
               
               
                 276 
                 PDLQRALGATPNTGPAPQCA 
                 295 
                 (SEQ ID) 
               
               
                   
                   
                   
                 NO:32) 
               
               
                   
               
             
          
         
       
     
     The amino acid sequences are given in the 1-letter code. 
     Variations of the peptides listed in Table 4 are also possible depending on their intended use. For example, if the peptides are to be used to raise antisera, the peptides may be synthesized with an extra cysteine residue added. This extra cysteine residue is preferably added to the amino terminus and facilitates the coupling of the peptide to a carrier protein which is necessary to render the small peptide immunogenic. If the peptide is to be labeled for use in radioimmune assays, it may be advantageous to synthesize the protein with a tyrosine attached to either the amino or carboxyl terminus to facilitate iodination. These peptides possess therefore the primary sequence of the peptides listed in Table 4 but with additional amino acids which do not appear in the primary sequence of the protein and whose sole function is to confer the desired chemical properties to the peptides. 
     The invention also relates to a process for detecting in vitro antibodies related to tuberculosis in a human biological sample liable to contain them, this process comprising
     contacting the biological sample with a polypeptide or a peptide according to the invention under conditions enabling an in vitro immunological reaction between said polypeptide and the antibodies which are possibly present in the biological sample and   the in vitro detection of the antigen/antibody complex which may be formed.   

     Preferably, the biological medium is constituted by a human serum. 
     The detection can be carried out according to any classical process. 
     By way of example a preferred method brings into play an immunoenzymatic process according to ELISA technique or immunofluorescent or radioimmunological (RIA) or the equivalent ones. 
     Thus the invention also relates to any polypeptide according to the invention labeled by an appropriate label of the enzymatic, fluorescent, radioactive . . . type. 
     Such a method for detecting in vitro antibodies related to tuberculosis comprises for instance the following steps:
     deposit of determined amounts of a polypeptidic composition according to the invention in the wells of a titration microplate,   introduction into said wells of increasing dilutions of the serum to be diagnosed,   incubation of the microplate,   repeated rinsing of the microplate,   introduction into the wells of the microplate of labeled antibodies against the blood immunoglobulins,   the labeling of these antibodies being carried out by means of an enzyme which is selected from among the ones which are able to hydrolyze a substrate by modifying the absorption of the radiation of this latter at least at a given wave length,   detection by comparing with a control standard of the amount of hydrolyzed substrate.   

     The invention also relates to a process for detecting and identifying in vitro antigens of  M. tuberculosis  in a human biological sample liable to contain them, this process comprising:
     contacting the biological sample with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of  M. tuberculosis  which are possibly present in the biological sample and the in vitro detection of the antigen/antibody complex which may be formed.   

     Preferably, the biological medium is constituted by sputum, pleural effusion liquid, broncho-alveolar washing liquid, urine, biopsy or autopsy material. 
     Appropriate antibodies are advantageously monoclonal antibodies directed against the peptides which have been mentioned in Table 4. 
     The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by  Mycobacterium tuberculosis  comprising the following steps:
     the possible previous amplification of the amount of the nucleotide sequences according to the invention, liable to be contained in a biological sample taken from said patient by means of a DNA primer set as above defined,   contacting the above mentioned biological sample with a nucleotide probe of the invention, under conditions enabling the production of an hybridization complex formed between said probe and said nucleotide sequence,   detecting the above said hybridization complex which has possibly been formed.   

     To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by  Mycobacterium tuberculosis  as above defined, the following necessary or kit can be used, said necessary or kit comprising:
     a determined amount of a nucleotide probe of the invention,   advantageously the appropriate medium for creating an hybridization reaction between the sequence to be detected and the above mentioned probe,   advantageously, reagents enabling the detection of the hybridization complex which has been formed between the nucleotide sequence and the probe during the hybridization reaction.   

     The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by  Mycobacterium tuberculosis  comprising:
     contacting a biological sample taken from a patient with a polypeptide or a peptide of the invention, under conditions enabling an in vitro immunological reaction between said polypeptide or peptide and the antibodies which are possibly present in the biological sample and   the in vitro detection of the antigen/antibody complex which has possibly been formed.   

     To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by  Mycobacterium tuberculosis , the following necessary or kit can be used, said necessary or kit comprising:
     a polypeptide or a peptide according to the invention,   reagents for making a medium appropriate for the immunological reaction to occur,   reagents enabling to detect the antigen/antibody complex which has been produced by the immunological reaction, said reagents possibly having a label, or being liable to be recognized by a labeled reagent, more particularly in the case where the above mentioned polypeptide or peptide is not labeled.   

     The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by  M. tuberculosis , comprising the following steps:
     contacting the biological sample with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of  M. tuberculosis  which are possibly present in the biological sample and—the in vitro detection of the antigen/antibody complex which may be formed.   

     Appropriate antibodies are advantageously monoclonal antibodies directed against the peptides which have been mentioned in Table 4. 
     To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by  Mycobacterium tuberculosis , the following necessary or kit can be used, said necessary or kit comprising:
     an antibody of the invention,   reagents for making a medium appropriate for the immunological reaction to occur,   reagents enabling to detect the antigen/antibody complexes which have been produced by the immunological reaction, said reagent possibly having a label or being liable to be recognized by a label reagent, more particularly in the case where the above mentioned antibody is not labeled.   

     An advantageous kit for the diagnostic in vitro of tuberculosis comprises:
     at least a suitable solid phase system, e.g. a microtiter-plate for deposition thereon of the biological sample to be diagnosed in vitro,   a preparation containing one of the monoclonal antibodies of the invention,   a specific detection system for said monoclonal antibody,   appropriate buffer solutions for carrying out the immunological reaction between a test sample and said monoclonal antibody on the one hand, and the bonded monoclonal antibodies and the detection system on the other hand.   

     The invention also relates to a kit, as described above, also containing a preparation of one of the polypeptides or peptides of the invention, said antigen of the invention being either a standard (for quantitative determination of the antigen of  M. tuberculosis  which is sought) or a competitor, with respect to the antigen which is sought, for the kit to be used in a competition dosage process. 
     The invention also relates to an immunogenic composition comprising a polypeptide or a peptide according to the invention, in association with a pharmaceutically acceptable vehicle. 
     The invention also relates to a vaccine composition comprising among other immunogenic principles anyone of the polypeptides or peptides of the invention or the expression product of the invention, possibly coupled to a natural protein or to a synthetic polypeptide having a sufficient molecular weight so that the conjugate is able to induce in vivo the production of antibodies neutralizing  Mycobacterium tuberculosis , or induce in vivo a cellular immune response by activating  M. tuberculosis  antigen-responsive T cells. 
     The peptides of the invention which are advantageously used as immunogenic principle have one of the following sequences: 
     
       
         
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4a 
               
             
             
               
                   
               
               
                 (see FIG. 4a and 4b) 
                   
               
             
          
           
               
                 Amino 
                   
                 Amino 
                   
               
               
                 acid 
                   
                 acid 
               
               
                 position 
                   
                 position 
               
               
                 (NH 2 - 
                   
                 (COOH- 
               
               
                 terminal) 
                   
                 terminal) 
               
               
                   
               
             
          
           
               
                  12 
                 QVPSPSMGRDIKVQFQSGGA 
                  31 
                 (SEQ ID 
                   
               
               
                   
                   
                   
                 NO:24) 
               
               
                  36 
                 LYLLDGLRAQDDFSGWDINT 
                  55 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:25) 
               
               
                  77 
                 SFYSDWYQPACRKAGCQTYK 
                  96 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:26) 
               
               
                 101 
                 LTSELPGWLQANRHVKPTGS 
                 120 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:27) 
               
               
                 175 
                 KASDMWGPKEDPAWQRNDPL 
                 194 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:28) 
               
               
                 211 
                 CGNGKPSDLGGNNLPAKFLE 
                 230 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:29) 
               
               
                 275 
                 KPDLQRHWVPRPTPGPPQGA 
                 294 
                 (SEQ ID 
               
               
                   
                   
                   
                 NO:30) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4b 
               
             
             
               
                   
               
               
                 (see FIG. 5) 
                   
               
             
          
           
               
                 Amino 
                   
                 Amino 
                   
               
               
                 acid 
                   
                 acid 
               
               
                 position 
                   
                 position 
               
               
                 (NH 2 - 
                   
                 (COOH- 
               
               
                 terminal) 
                   
                 terminal) 
               
               
                   
               
             
          
           
               
                  77 
                 SFYSDWYQPACGKAGCQTYX 
                  96 
                 (SEQ ID) 
                   
               
               
                   
                   
                   
                 NO:31) 
               
               
                   
               
               
                 276 
                 PDLQRALGATPNTGPAPQCA 
                 295 
                 (SEQ ID) 
               
               
                   
                   
                   
                 NO:32) 
               
               
                   
               
             
          
         
       
     
     The amino acid sequences are given in the 1-letter code. 
     Other characteristics and advantages of the invention will appear in the following examples and the figures illustrating the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1(A) and 1(B)  correspond to the identification of six purified λgt11  M. tuberculosis  recombinant clones.  FIG. 1(A)  corresponds to the EcoRI restriction analysis of clone 15, clone 16, clone 17, clone 19, clone 24 and EcoRI-HindIII digested lambda DNA-molecular weight marker lane (in kilobase pairs) (M) (Boehringer). 
         FIG. 1(B)  corresponds to the immunoblotting analysis of crude lysates of  E. coli  lysogenized with clone 15, clone 16, clone 17, clone 19, clone 23 and clone 24. 
       Arrow (←) indicates fusion protein produced by recombinant λgt11- M - tuberculosis  clones. Expression and immunoblotting were as described above. Molecular weight (indicated in kDa) were estimated by comparison with molecular weight marker (High molecular weight-SDS calibration kit, Pharmacia). 
         FIG. 2  corresponds to the restriction map of the DNA inserts in the λgt11  M. tuberculosis  recombinant clones 17 and 24 identified with polyclonal anti-32-kDa (BCG) antiserum as above defined and of clones By1, By2 and By5 selected by hybridization with a 120 bp EcoRI-Kpn I restriction fragment of clone 17. 
       DNA was isolated from λgt11 phage stocks by using the Lambda Sorb Phage Immunoadsorbent, as described by the manufacturer (Promega). Restriction sites were located as described above. Some restriction sites (*) were deduced from a computer analysis of the nucleotide sequence. 
       The short vertical bars 
                                   
represent linker derived EcoRI sites surrounding the DNA inserts of recombinant clones. The lower part represents a magnification of the DNA region containing the antigen of molecular weight of 32-kDa, that has been sequenced. Arrows indicate strategies and direction of dideoxy-sequencing. (→) fragment subcloned in Bluescribe M13; (⇄) fragment subcloned in mp10 and mp11 M13 vectors; (▪→) sequence determined with the use of a synthetic oligonucleotide.
 
         FIGS. 3   a  and  3   b  correspond to the nucleotide (SEQ ID NO:34) and amino acid sequences (SEQ ID NO:35) of the general formula of the antigens of the invention. 
         FIGS. 4   a  and  4   b  correspond to the nucleotide (SEQ ID NO:36) and amino acid sequences (SEQ ID NO:37) of one of the antigens of the invention. 
       Two groups of sequences resembling the  E. coli  consensus promoter sequences are boxed and the homology to the consensus is indicated by italic bold letters. Roman bold letters represent a putative Shine-Dalgarno motif. 
       The NH 2 -terminal amino acid sequence of the mature protein which is underlined with a double line happens to be very homologous—29/32 amino acids—with the one of MPB 59 antigen (34). Five additional ATG codons, upstream of the ATG at position 273 are shown (dotted underlined). Vertical arrows 
                                  
indicate the presumed NH 2  end of clone 17 and clone 24. The option taken here arbitrarily represents the 59 amino acid signal peptide corresponding to ATG 183 .
 
         FIGS. 5   a – 5   c  correspond to the nucleotide (SEQ ID NO:38) and amino acid sequences (SEQ ID NO:39) of the antigen of 32-kDa of the invention. 
       The NH 2 -terminal amino acid sequence of the mature protein which is underlined with a double line happens to be very homologous—29/32 amino acids—with the one of MPB 59 antigen (34). Vertical arrows (↓) indicate the presumed NH 2  end of clone 17 and clone 24. 
         FIG. 6  is the hydropathy pattern of the antigen of the invention of a molecular weight of 32-kDa and of the antigen α of BCG (17). 
         FIGS. 7   a – 7   b  represent the homology between the amino acid sequences of the antigen of 32-kDa (SEQ ID NO:44) of the invention and of antigen α of BCG (SEQ ID NO:45) (revised version). 
       Identical amino acids; (:) evolutionarily conserved replacement of an amino acid (.), and absence of homology ( ) are indicated. Underlined sequence (=) represents the signal peptide, the option taken here arbitrarily representing the 43-amino acid signal peptide corresponding to ATG 91 . Dashes in the sequences indicate breaks necessary for obtaining the optimal alignment. 
         FIG. 8  illustrates the fact that the protein of 32-kDa of the invention is selectively recognized by human tuberculous sera. 
         FIG. 8  represents the immunoblotting with human tuberculous sera, and anti-β-galactosidase monoclonal antibody. Lanes 1 to 6 : E. coli  lysate expressing fusion protein (140 kDa); lanes 7 to 12: unfused β-galactosidase (114 kDa). The DNA insert of clone 17 (2.7 kb) was subcloned into pUEX 2  and expression of fusion protein was induced as described by Bresson and Stanley (4). Lanes 1 and 7 were probed with the anti-β-galactosidase monoclonal antibody: lanes 4, 5, 6 and 10, 11, 12 with 3 different human tuberculous sera highly responding towards purified protein of the invention of 32-kDa; lanes 2 and 3 and 8 and 9 were probed with 2 different low responding sera. 
         FIGS. 9   a – 9   d  represent the nucleic acid sequence alignment of the 32-kDa protein gene of  M. tuberculosis  of the invention (SEQ ID NO:46) (upper line), corresponding to the sequence in  FIG. 5 , of the gene of  FIGS. 4   a  and  4   b  of the invention (SEQ ID NO:47) (middle line), and of the gene for antigen α of BCG (SEQ ID NO:48) (lower line). 
       Dashes in the sequence indicate breaks necessary for obtaining optimal alignment of the nucleic acid sequence. 
         FIG. 9   a  represents part of the nucleic acid sequence of the 32-kDA protein including probe region A and probe region B as well as primer region P1. 
         FIG. 9   b  represents part of the nucleic acid sequence of the 32-kDA protein including Primer regions P2, P3 and P4 and part of probe region C. 
         FIG. 9   c  represents part of the nucleic acid sequence of the 32-kDA protein including part of probe region C, probe regions D and E and primer region P5. 
         FIG. 9   d  represents part of the nucleic acid sequence of the 32 kDA protein including probe region F and primer region P6. 
       The primer regions for enzymatical amplification are boxed (P1 to P6). 
       The specific probe regions are boxed and respectively defined by probe region A, probe region B, probe region C, probe region D, probe region E and probe region F. 
       It is to be noted that the numbering of nucleotides is different from the numbering of  FIG. 3   a  and  FIG. 3   b , and of  FIG. 5 , because nucleotide at position 1 (on  FIG. 9 ) corresponds to nucleotide 234 on  FIG. 3   a , and corresponds to nucleotide 91 on  FIG. 5 . 
         FIG. 10   a  corresponds to the restriction and genetic map of the pIGRI plasmid used in Example IV for the expression of the P 32  antigen of the invention in  E. coli  and contains SEQ ID NO:50. 
       On this figure, underlined restriction sites are unique. 
         FIGS. 10   b – 10   m  correspond to the pIGRI nucleic acid sequence (SEQ ID NO:40). 
       On this figure, the origin of nucleotide stretches used to construct plasmid pIGRI are specified hereafter. 
       
         
           
                 
                 
               
             
                 
                     
                 
                 
                   Position 
                 
                 
                     
                 
               
               
                 
                   3422–206  
                   lambda PL containing EcoRI blunt-MboII 
                 
                 
                     
                   blunt fragment of pPL(λ) (Pharmacia) 
                 
                 
                   207–384 
                   synthetic DNA sequence 
                 
                 
                   228–230 
                   initiation codon ATG of first 
                 
                 
                     
                   cistron 
                 
                 
                   234–305 
                   DNA encoding amino acids 2 to 25 of 
                 
                 
                     
                   mature mouse TNF 
                 
                 
                   306–308 
                   stop codon (TAA) first cistron 
                 
                 
                   311–312 
                   initiation codon (ATG) second 
                 
                 
                     
                   cistron 
                 
                 
                   385–890 
                   rrnBT 1 T 2  containing HindIII-SspI 
                 
                 
                     
                   fragment from pKK223 (Pharmacia) 
                 
                 
                    891–3421 
                   DraI-EcoRI blunt fragment of pAT 153   
                 
                 
                     
                   (Bioexcellence) containing the 
                 
                 
                     
                 
               
            
           
         
           
           
             
               tetracycline resistance gene and the origin of replication. 
             
           
         
      
       Table 5 hereafter corresponds to the complete restriction site analysis of pIGRI. 
       
         
           
                 
               
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
               
                 
               
                 
                 
               
                 
                 
                 
                 
                 
                 
                 
                 
                 
               
                 
                 
               
                 
                 
                 
                 
                 
                 
                 
                 
               
                 
                 
               
             
                 
                   TABLE 5 
                 
                 
                     
                 
                 
                   RESTRICTION-SITE ANALYSIS 
                 
                 
                     
                 
               
               
                 
                   Name of the plasmid: pIGRI 
                 
                 
                   Total number of bases is: 3423. 
                 
                 
                   Analysis done on the complete sequence. 
                 
                 
                     
                 
                 
                   List of cuts by enzyme. 
                 
               
            
             
                 
                   Acc I 
                   370 
                   2765 
                     
                     
                     
                     
                     
                     
                     
                     
                     
                 
                 
                   Acy I 
                   735 
                   2211 
                   2868 
                   2982 
                   3003 
                 
                 
                   Afl III 
                   1645 
                 
                 
                   Aha III 
                   222 
                 
                 
                   Alu I 
                   386 
                   1088 
                   1345 
                   1481 
                   1707 
                   2329 
                   2732 
                   3388 
                   3403 
                 
                 
                   Alw NI 
                   1236 
                 
                 
                   Apa LI 
                   1331 
                 
                 
                   Asp 718I 
                   208 
                 
                 
                   Asu I 
                   329 
                   494 
                   623 
                   713 
                   1935 
                   1977 
                   2156 
                   2280 
                   2529 
                   2617 
                   289 
                 
                 
                     
                   3244 
                 
                 
                   Ava I 
                   1990 
                 
                 
                   Ava II 
                   329 
                   494 
                   1935 
                   1977 
                   2280 
                   2529 
                   2617 
                 
                 
                   Bal I 
                   1973 
                 
                 
                   Bam HI 
                   3040 
                 
                 
                   Bbe I 
                   2214 
                   2871 
                   2985 
                   3006 
                 
                 
                   Bbv I 
                   389 
                   1316 
                   1735 
                   1753 
                   1866 
                   1869 
                   2813 
                   3202 
                 
                 
                   Bbv I* 
                   1017 
                   1223 
                   1226 
                   1973 
                   1997 
                   2630 
                 
                 
                   Bbv II 
                   1822 
                   2685 
                 
                 
                   Bgl I 
                   2253 
                   2487 
                 
                 
                   Bin I 
                   15 
                   903 
                   1001 
                   1087 
                   3048 
                 
                 
                   Bin I* 
                   902 
                   999 
                   2313 
                   3035 
                 
                 
                   Bsp HI 
                   855 
                   925 
                   2926 
                 
                 
                   Bsp MI 
                   382 
                   2361 
                 
                 
                   Bst NI 
                   213 
                   475 
                   585 
                   753 
                   1486 
                   1499 
                   1620 
                   1975 
                   2358 
                   3287 
                 
                 
                   Cau II 
                   4 
                   683 
                   716 
                   1268 
                   1933 
                   2159 
                   2883 
                   3247 
                 
                 
                   Cfr 10I 
                   2132 
                   2486 
                   2646 
                   3005 
                   3014 
                   3255 
                 
                 
                   Cfr I 
                   1971 
                   2476 
                   2884 
                   3016 
                   3120 
                 
                 
                   Cla I 
                   3393 
                 
                 
                   Cvi JI 
                   190 
                   263 
                   270 
                   380 
                   386 
                   391 
                   421 
                   607 
                   625 
                   714 
                   77 
                 
                 
                     
                   791 
                   1088 
                   1117 
                   1160 
                   1171 
                   1236 
                   1315 
                   1340 
                   1345 
                   1481 
                   157 
                 
                 
                     
                   1605 
                   1623 
                   1634 
                   1707 
                   1726 
                   1926 
                   1931 
                   1973 
                   2010 
                   2092 
                   213 
                 
                 
                     
                   2157 
                   2162 
                   2300 
                   2310 
                   2329 
                   2370 
                   2427 
                   2435 
                   2465 
                   2478 
                   249 
                 
                 
                     
                   2544 
                   2588 
                   2732 
                   2748 
                   2804 
                   2822 
                   2886 
                   2894 
                   2932 
                   2946 
                   301 
                 
                 
                     
                   3087 
                   3122 
                   3245 
                   3269 
                   3388 
                   3403 
                 
                 
                   Cvi QI 
                   209 
                   3253 
                 
                 
                   Dde I 
                   133 
                   336 
                   343 
                   518 
                   608 
                   664 
                   962 
                   1371 
                   1835 
                 
                 
                   Dpn I 
                   9 
                   236 
                   897 
                   909 
                   987 
                   995 
                   1006 
                   1081 
                   1957 
                   2274 
                   228 
                 
                 
                     
                   2320 
                   2592 
                   2951 
                   3042 
                   3069 
                 
                 
                   Dra II 
                   1935 
                   1977 
                   2892 
                 
                 
                   Dra III 
                   293 
                 
                 
                   Dsa I 
                   309 
                   1968 
                   2887 
                 
                 
                   Eco 31I 
                   562 
                 
                 
                   Eco 47III 
                   341 
                   1773 
                   2642 
                   2923 
                   3185 
                 
                 
                   Eco 57I 
                   214 
                 
                 
                   Eco 57I* 
                   1103 
                 
                 
                   Eco 78I 
                   2212 
                   2869 
                   2983 
                   3004 
                 
                 
                   Eco NI 
                   196 
                   2792 
                 
                 
                   Eco RII 
                   211 
                   473 
                   583 
                   751 
                   1484 
                   1497 
                   1618 
                   1973 
                   2356 
                   3285 
                 
                 
                   Eco RV 
                   3232 
                 
                 
                   Fnu 4HI 
                   378 
                   479 
                   1031 
                   1237 
                   1240 
                   1305 
                   1448 
                   1603 
                   1721 
                   1724 
                   174 
                 
                 
                     
                   1855 
                   1858 
                   1987 
                   2001 
                   2008 
                   2011 
                   2130 
                   2209 
                   2254 
                   2311 
                   239 
                 
                 
                     
                   2479 
                   2644 
                   2695 
                   2802 
                   2836 
                   2839 
                   3117 
                   3120 
                   3191 
                 
                 
                   Fnu DII 
                   489 
                   1021 
                   1602 
                   1784 
                   1881 
                   2003 
                   2029 
                   2174 
                   2184 
                   2313 
                   237 
                 
                 
                     
                   2440 
                   2445 
                   2472 
                   2601 
                   2716 
                   3072 
                 
                 
                   Fok I 
                   415 
                   799 
                   3317 
                 
                 
                   Fok I* 
                   763 
                   2370 
                   2415 
                   3269 
                 
                 
                   Gsu I 
                   339 
                   2035 
                 
                 
                   Gsu I* 
                   2589 
                 
                 
                   Hae I 
                   775 
                   791 
                   1171 
                   1623 
                   1634 
                   1973 
                   2370 
                   2427 
                   2499 
                 
                 
                   Hae II 
                   343 
                   541 
                   1405 
                   1775 
                   2214 
                   2644 
                   2871 
                   2925 
                   2985 
                   3006 
                   318 
                 
                 
                   Hae III 
                   625 
                   714 
                   775 
                   791 
                   1171 
                   1605 
                   1623 
                   1634 
                   1973 
                   2157 
                   237 
                 
                 
                     
                   2427 
                   2478 
                   2499 
                   2588 
                   2822 
                   2886 
                   2894 
                   3018 
                   3122 
                   3245 
                 
                 
                   Hga I 
                   158 
                   181 
                   743 
                   2035 
                   2185 
                   2776 
                 
                 
                   Hga I* 
                   955 
                   1533 
                   2429 
                   2461 
                   3015 
                 
                 
                   Hgi AI 
                   139 
                   1335 
                   1954 
                   2245 
                   2832 
                   3143 
                 
                 
                   Hgi CI 
                   208 
                   2126 
                   2210 
                   2649 
                   2867 
                   2981 
                   3002 
                   3296 
                   3339 
                 
                 
                   Hgi JII 
                   2934 
                   2948 
                 
                 
                   Hha I 
                   342 
                   489 
                   540 
                   1021 
                   1130 
                   1304 
                   1404 
                   1471 
                   1741 
                   1774 
                   196 
                 
                 
                     
                   2000 
                   2062 
                   2213 
                   2472 
                   2603 
                   2643 
                   2718 
                   2870 
                   2924 
                   2984 
                   300 
                 
                 
                     
                   3158 
                   3186 
                   3318 
                 
                 
                   Hin P1I 
                   340 
                   487 
                   538 
                   1019 
                   1128 
                   1302 
                   1402 
                   1469 
                   1739 
                   1772 
                   196 
                 
                 
                     
                   1998 
                   2060 
                   2211 
                   2470 
                   2601 
                   2641 
                   2716 
                   2868 
                   2922 
                   2982 
                   300 
                 
                 
                     
                   3156 
                   3184 
                   3316 
                 
                 
                   Hind II 
                   107 
                   371 
                   2766 
                 
                 
                   Hind III 
                   384 
                   3386 
                 
                 
                   Hinf I 
                   367 
                   1275 
                   1671 
                   1746 
                   1891 
                   2112 
                   2410 
                   2564 
                   2784 
                 
                 
                   Hpa II 
                   3 
                   682 
                   716 
                   1077 
                   1267 
                   1293 
                   1440 
                   1932 
                   2133 
                   2159 
                   239 
                 
                 
                     
                   2487 
                   2647 
                   2723 
                   2883 
                   3006 
                   3015 
                   3030 
                   3247 
                   3256 
                 
                 
                   Hph I 
                   94 
                   138 
                   181 
                   663 
                   914 
                   1900 
                   2121 
                   2975 
                   3020 
                   3302 
                 
                 
                   Hph I* 
                   6 
                 
                 
                   Kpn I 
                   212 
                 
                 
                   Mae I 
                   364 
                   899 
                   1152 
                   1928 
                   3187 
                 
                 
                   Mae II 
                   274 
                   698 
                   944 
                   1847 
                   1871 
                   2460 
                   2516 
                 
                 
                   Mae III 
                   169 
                   255 
                   304 
                   313 
                   1109 
                   1225 
                   1288 
                   2267 
                   2534 
                   3202 
                   329 
                 
                 
                   Mbo I 
                   7 
                   234 
                   895 
                   907 
                   985 
                   993 
                   1004 
                   1079 
                   1955 
                   2272 
                   228 
                 
                 
                     
                   2318 
                   2590 
                   2949 
                   3040 
                   3067 
                 
                 
                   Mbo II 
                   207 
                   422 
                   917 
                   1779 
                   1827 
                   2419 
                   2690 
                 
                 
                   Mbo II* 
                   988 
                   2944 
                 
                 
                   Mme I* 
                   1252 
                   1436 
                   3112 
                   3199 
                 
                 
                   Mnl I 
                   1218 
                   1542 
                   1948 
                   2446 
                   2630 
                 
                 
                   Mnl I* 
                   208 
                   289 
                   337 
                   711 
                   1467 
                   1750 
                   2116 
                   2143 
                   2181 
                   2242 
                   254 
                 
                 
                     
                   2811 
                   3030 
                   3234 
                   3294 
                 
                 
                   Mse I 
                   179 
                   186 
                   221 
                   433 
                   764 
                   941 
                   3361 
                   3383 
                   3420 
                 
                 
                   Mst I 
                   1963 
                   2061 
                   3157 
                 
                 
                   Nae I 
                   2134 
                   2488 
                   2648 
                   3016 
                 
                 
                   Nar I 
                   2211 
                   2868 
                   2982 
                   3003 
                 
                 
                   Nco I 
                   309 
                 
                 
                   Nhe I 
                   3186 
                 
                 
                   Nla III 
                   166 
                   230 
                   313 
                   512 
                   567 
                   859 
                   929 
                   1649 
                   1828 
                   1962 
                   216 
                 
                 
                     
                   2226 
                   2241 
                   2369 
                   2486 
                   2672 
                   2711 
                   2857 
                   2930 
                   3068 
                   3415 
                 
                 
                   Nla IV 
                   210 
                   330 
                   496 
                   1578 
                   1617 
                   1936 
                   1979 
                   2093 
                   2128 
                   2163 
                   221 
                 
                 
                     
                   2530 
                   2651 
                   2869 
                   2893 
                   2983 
                   3004 
                   3042 
                   3088 
                   3298 
                   3341 
                 
                 
                   Nru I 
                   2445 
                 
                 
                   Nsp BII 
                   1062 
                   1307 
                   2278 
                 
                 
                   Nsp HI 
                   1649 
                   2857 
                 
                 
                   Pfl MI 
                   293 
                   2052 
                   2101 
                 
                 
                   Ple I 
                   375 
                   1754 
                 
                 
                   Ple I* 
                   1269 
                   2778 
                 
                 
                   Ppu MI 
                   1935 
                   1977 
                 
                 
                   Pss I 
                   1938 
                   1980 
                   2895 
                 
                 
                   Pst I 
                   379 
                 
                 
                   Rsa I 
                   210 
                   3254 
                 
                 
                   Sal I 
                   369 
                   2764 
                 
                 
                   Scr FI 
                   4 
                   213 
                   475 
                   585 
                   683 
                   716 
                   753 
                   1268 
                   1486 
                   1499 
                   162 
                 
                 
                     
                   1933 
                   1975 
                   2159 
                   2358 
                   2883 
                   3247 
                   3287 
                 
                 
                   Sdu I 
                   139 
                   1335 
                   1954 
                   2245 
                   2832 
                   2934 
                   2948 
                   3143 
                 
                 
                   Sec I 
                   3 
                   309 
                   1485 
                   1968 
                   2046 
                   2248 
                   2881 
                   2887 
                   3286 
                   3300 
                 
                 
                   Sfa NI 
                   597 
                   765 
                   2392 
                   2767 
                   3178 
                   3291 
                 
                 
                   Sfa NI* 
                   1548 
                   1985 
                   2380 
                   3001 
                   3013 
                   3202 
                 
                 
                   Sph I 
                   2857 
                 
                 
                   Sso II 
                   2 
                   211 
                   473 
                   583 
                   681 
                   714 
                   751 
                   1266 
                   1484 
                   1497 
                   161 
                 
                 
                     
                   1931 
                   1973 
                   2157 
                   2356 
                   2881 
                   3245 
                   3285 
                 
                 
                   Sty I 
                   309 
                   2046 
                 
                 
                   Taq I 
                   252 
                   370 
                   613 
                   1547 
                   2149 
                   2290 
                   2765 
                   3078 
                   3393 
                 
                 
                   Taq IIB 
                   1749 
                 
                 
                   Taq IIB* 
                   2751 
                 
                 
                   TthlllII 
                   38 
                   1054 
                 
                 
                   TthlllII* 
                   633 
                   1022 
                   1061 
                 
                 
                   Xba I 
                   363 
                 
                 
                   Xho II 
                   7 
                   895 
                   907 
                   993 
                   1004 
                   3040 
                 
                 
                   Xma III 
                   2476 
                 
                 
                   Xmn I 
                   414 
                 
               
            
             
                 
                   Total number of cuts is: 705. 
                 
                 
                     
                 
               
            
             
                 
                   Sorted list of enzymes by n* of cuts. 
                     
                 
               
            
             
                 
                   Cvi JI 
                   61 
                   Sdu I 
                   8 
                   TthlllII* 
                   3 
                   Ava I 
                   1 
                     
                 
                 
                   Fnu 4HI 
                   31 
                   Cau II 
                   8 
                   Nsp BII 
                   3 
                   Taq IIB 
                   1 
                 
                 
                   Hha I 
                   25 
                   Bbv I 
                   8 
                   Fok I 
                   3 
                   Alw NI 
                   1 
                 
                 
                   Hin P1I 
                   25 
                   Mbo II 
                   7 
                   Pfl MI 
                   3 
                   Dra III 
                   1 
                 
                 
                   Hae III 
                   21 
                   Ava II 
                   7 
                   Hind II 
                   3 
                   Afl III 
                   1 
                 
                 
                   Nla IV 
                   21 
                   Mae II 
                   7 
                   Dsa I 
                   3 
                   Cla I 
                   1 
                 
                 
                   Nla III 
                   21 
                   Sfa NI 
                   6 
                   Bsp HI 
                   3 
                   Eco 57I* 
                   1 
                 
                 
                   Hpa II 
                   20 
                   Xho II 
                   6 
                   Pss I 
                   3 
                   Nhe I 
                   1 
                 
                 
                   Scr FI 
                   18 
                   Hgi AI 
                   6 
                   Mst I 
                   3 
                   Gsu I* 
                   1 
                 
                 
                   Sso II 
                   18 
                   Sfa NI* 
                   6 
                   Hgi JII 
                   2 
                   Bal I 
                   1 
                 
                 
                   Fnu DII 
                   17 
                   Bbv I* 
                   6 
                   Ple I 
                   2 
                   Eco RV 
                   1 
                 
                 
                   Mbo I 
                   16 
                   Cfr 10I 
                   6 
                   Mbo II* 
                   2 
                   Sph I 
                   1 
                 
                 
                   Dpn I 
                   16 
                   Hga I 
                   6 
                   Cvi QI 
                   2 
                   Xma III 
                   1 
                 
                 
                   Mnl I* 
                   15 
                   Acy I 
                   5 
                   Acc I 
                   2 
                   Hph I* 
                   1 
                 
                 
                   Asu I 
                   12 
                   Bin I 
                   5 
                   Bgl I 
                   2 
                   Taq IIB* 
                   1 
                 
                 
                   Hae II 
                   11 
                   Cfr I 
                   5 
                   Ple I* 
                   2 
                   Eco 57I 
                   1 
                 
                 
                   Mae III 
                   11 
                   Hga I* 
                   5 
                   Gsu I 
                   2 
                   Kpn I 
                   1 
                 
                 
                   Hph I 
                   10 
                   Mae I 
                   5 
                   Ppu MI 
                   2 
                   Xba I 
                   1 
                 
                 
                   Bst NI 
                   10 
                   Eco 47III 
                   5 
                   TthlllII 
                   2 
                   Aha III 
                   1 
                 
                 
                   Eco RII 
                   10 
                   Mnl I 
                   5 
                   Hind III 
                   2 
                   Nru I 
                   1 
                 
                 
                   Sec I 
                   10 
                   Mme I* 
                   4 
                   Nsp HI 
                   2 
                   Bam HI 
                   1 
                 
                 
                   Dde I 
                   9 
                   Eco 78I 
                   4 
                   Rsa I 
                   2 
                   Apa LI 
                   1 
                 
                 
                   Hinf I 
                   9 
                   Nae I 
                   4 
                   Sal I 
                   2 
                   Asp 718I 
                   1 
                 
                 
                   Hae I 
                   9 
                   Bbe I 
                   4 
                   Bbv II 
                   2 
                   Eco 31I 
                   1 
                 
                 
                   Alu I 
                   9 
                   Bin I* 
                   4 
                   Bsp MI 
                   2 
                   Nco I 
                   1 
                 
                 
                   Hgi CI 
                   9 
                   Nar I 
                   4 
                   Sty I 
                   2 
                   Pst I 
                   1 
                 
                 
                   Mse I 
                   9 
                   Fok I* 
                   4 
                   Eco NI 
                   2 
                 
                 
                   Taq I 
                   9 
                   Dra II 
                   3 
                   Xmn I 
                   2 
                 
                 
                     
                 
               
            
             
                 
                   List of non cutting selected enzymes 
                     
                 
               
            
             
                 
                   Aat II, 
                   Afl II, 
                   Apa I, 
                   Asu II, 
                   Avr II, 
                   Bbv II*, 
                   Bcl I 
                     
                 
                 
                   Bql II, 
                   Bsp MI*, 
                   Bsp MII, 
                   Bss HII, 
                   Bst EII, 
                   Bst XI, 
                   Eco 31I* 
                 
                 
                   Eco RI, 
                   Esp I, 
                   Hpa I, 
                   Mlu I, 
                   Mme I, 
                   Nde I, 
                   Not I 
                 
                 
                   Nsi I, 
                   Pma CI, 
                   Pvu I, 
                   Pvu II, 
                   Rsr II, 
                   Sac I, 
                   Sac II 
                 
                 
                   Sau I, 
                   Sca I, 
                   Sci I, 
                   Sfi I, 
                   Sma I, 
                   Sna BI, 
                   Spe I 
                 
                 
                   Spl I, 
                   Ssp I, 
                   Stu I, 
                   Taq IIA, 
                   Taq IIA*, 
                   Tth 111I, 
                   Vsp I 
                 
                 
                   Xca I, 
                   Xho I, 
                   Xma I, 
                 
               
            
             
                 
                   Total number of selected enzymes which do not cut: 45 
                 
                 
                     
                 
               
            
           
         
       
         FIG. 11   a  corresponds to the restriction and genetic map of the pmTNF MPH plasmid used in Example V for the expression of the P 32  antigen of the invention in  E. coli  and contains SEQ ID NO:51 and SEQ ID NO:52. 
         FIGS. 11   b – 11   m  correspond to the pmTNF-MPH nucleic acid sequence (SEQ ID NO:41). 
       On this figure, the origin of nucleotide stretches used to construct plasmid pmTNF-MPH is specified hereafter. 
       
         
           
                 
                 
               
             
                 
                     
                 
                 
                   Position 
                 
                 
                     
                 
               
               
                 
                    1–208 
                   lambda PL containing EcoRI blunt-MboII 
                 
                 
                     
                   blunt fragment of pPL(λ) (Pharmacia) 
                 
                 
                   209–436 
                   synthetic DNA fragment 
                 
                 
                   230–232 
                   initiation codon (ATG) of mTNF 
                 
                 
                     
                   fusion protein 
                 
                 
                   236–307 
                   sequence encoding AA 2 to 25 of 
                 
                 
                     
                   mature mouse TNF 
                 
                 
                   308–384 
                   multiple cloning site containing 
                 
                 
                     
                   His 6  encoding sequence at position 
                 
                 
                     
                   315–332 
                 
                 
                   385–436 
                   HindIII fragment containing  E. coli   
                 
                 
                     
                   trp terminator 
                 
                 
                   437–943 
                   rrnBT 1 T 2  containing HindIII-SspI 
                 
                 
                     
                   fragment from pKK223 (Pharmacia) 
                 
                 
                    944–3474 
                   DraI-EcoRI blunt fragment of pAT 153   
                 
                 
                     
                   (Bioexcellence) containing the 
                 
                 
                     
                   tetracycline resistance gene and the 
                 
                 
                     
                   origin of replication. 
                 
                 
                     
                 
               
            
           
         
       
       Table 6 hereafter corresponds to the complete restriction site analysis of pmTNF-MPH. 
       
         
           
                 
               
                 
               
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
               
                 
               
                 
                 
                 
                 
                 
                 
                 
                 
               
                 
                 
               
             
                 
                   TABLE 6 
                 
                 
                     
                 
                 
                   RESTRICTION-SITE ANALYSIS 
                 
                 
                     
                 
               
               
                 
                   Done on DNA sequence PMTNFMPH. 
                 
                 
                   Total number of bases is: 3474. 
                 
                 
                   Analysis done on the complete sequence. 
                 
                 
                     
                 
               
            
             
                 
                   List of cuts by enzyme. 
                 
               
            
             
                 
                   Acc I 
                   371 
                   2818 
                     
                     
                     
                     
                     
                     
                     
                     
                     
                 
                 
                   Acy I 
                   788 
                   2264 
                   2921 
                   3035 
                   3056 
                 
                 
                   Afl II 
                   387 
                 
                 
                   Afl III 
                   1698 
                 
                 
                   Aha III 
                   224 
                 
                 
                   Alu I 
                   386 
                   439 
                   1141 
                   1398 
                   1534 
                   1760 
                   2382 
                   2785 
                   3441 
                   3456 
                 
                 
                   Alw NI 
                   1289 
                 
                 
                   Apa I 
                   345 
                 
                 
                   Apa LI 
                   1384 
                 
                 
                   Asp 718I 
                   210 
                 
                 
                   Asu I 
                   341 
                   342 
                   547 
                   676 
                   766 
                   1988 
                   2030 
                   2209 
                   2333 
                   2582 
                   267 
                 
                 
                     
                   2945 
                   3297 
                 
                 
                   Ava I 
                   338 
                   2043 
                 
                 
                   Ava II 
                   547 
                   1988 
                   2030 
                   2333 
                   2582 
                   2670 
                 
                 
                   Bal I 
                   2026 
                 
                 
                   Bam HI 
                   334 
                   3093 
                 
                 
                   Bbe I 
                   2267 
                   2924 
                   3038 
                   3059 
                 
                 
                   Bbv I 
                   1369 
                   1788 
                   1806 
                   1919 
                   1922 
                   2866 
                   3255 
                 
                 
                   Bbv I* 
                   1070 
                   1276 
                   1279 
                   2026 
                   2050 
                   2683 
                 
                 
                   Bbv II 
                   1875 
                   2738 
                 
                 
                   Bgl I 
                   2306 
                   2540 
                 
                 
                   Bin I 
                   17 
                   342 
                   956 
                   1054 
                   1140 
                   3101 
                 
                 
                   Bin I* 
                   329 
                   955 
                   1052 
                   2366 
                   3088 
                 
                 
                   Bsp HI 
                   908 
                   978 
                   2979 
                 
                 
                   Bsp MI 
                   2414 
                 
                 
                   Bsp MII 
                   354 
                 
                 
                   Bst NI 
                   215 
                   528 
                   638 
                   806 
                   1539 
                   1552 
                   1673 
                   2028 
                   2411 
                   3340 
                 
                 
                   Cau II 
                   6 
                   339 
                   340 
                   736 
                   769 
                   1321 
                   1986 
                   2212 
                   2936 
                   3300 
                 
                 
                   Cfr 10I 
                   374 
                   2185 
                   2539 
                   2699 
                   3058 
                   3067 
                   3308 
                 
                 
                   Cfr I 
                   2024 
                   2529 
                   2937 
                   3069 
                   3173 
                 
                 
                   Cla I 
                   3446 
                 
                 
                   Cvi JI 
                   192 
                   265 
                   272 
                   343 
                   350 
                   361 
                   386 
                   400 
                   439 
                   444 
                   47 
                 
                 
                     
                   660 
                   678 
                   767 
                   828 
                   844 
                   1141 
                   1170 
                   1213 
                   1224 
                   1289 
                   136 
                 
                 
                     
                   1393 
                   1398 
                   1534 
                   1632 
                   1658 
                   1676 
                   1687 
                   1760 
                   1779 
                   1979 
                   198 
                 
                 
                     
                   2026 
                   2063 
                   2145 
                   2189 
                   2210 
                   2215 
                   2353 
                   2363 
                   2382 
                   2423 
                   248 
                 
                 
                     
                   2488 
                   2518 
                   2531 
                   2552 
                   2597 
                   2641 
                   2785 
                   2801 
                   2857 
                   2875 
                   293 
                 
                 
                     
                   2947 
                   2985 
                   2999 
                   3071 
                   3140 
                   3175 
                   3298 
                   3322 
                   3441 
                   3456 
                 
                 
                   Cvi QI 
                   211 
                   3306 
                 
                 
                   Dde I 
                   135 
                   571 
                   661 
                   717 
                   1015 
                   1424 
                   1888 
                 
                 
                   Dpn I 
                   11 
                   238 
                   336 
                   950 
                   962 
                   1040 
                   1048 
                   1059 
                   1134 
                   2010 
                   232 
                 
                 
                     
                   2342 
                   2373 
                   2645 
                   3004 
                   3095 
                   3122 
                 
                 
                   Dra II 
                   1988 
                   2030 
                   2945 
                 
                 
                   Dra III 
                   295 
                   331 
                 
                 
                   Dsa I 
                   345 
                   2021 
                   2940 
                 
                 
                   Eco 31I 
                   615 
                 
                 
                   Eco 47III 
                   1826 
                   2695 
                   2976 
                   3238 
                 
                 
                   Eco 57I 
                   216 
                 
                 
                   Eco 57I* 
                   1156 
                 
                 
                   Eco 78I 
                   2265 
                   2922 
                   3036 
                   3057 
                 
                 
                   Eco NI 
                   198 
                   2845 
                 
                 
                   Eco RI 
                   309 
                 
                 
                   Eco RII 
                   213 
                   526 
                   636 
                   804 
                   1537 
                   1550 
                   1671 
                   2026 
                   2409 
                   3338 
                 
                 
                   Eco RV 
                   3285 
                 
                 
                   Fnu 4HI 
                   401 
                   417 
                   532 
                   1084 
                   1290 
                   1293 
                   1358 
                   1501 
                   1656 
                   1774 
                   177 
                 
                 
                     
                   1795 
                   1908 
                   1911 
                   2040 
                   2054 
                   2061 
                   2064 
                   2183 
                   2262 
                   2307 
                   236 
                 
                 
                     
                   2447 
                   2532 
                   2697 
                   2748 
                   2855 
                   2889 
                   2892 
                   3170 
                   3173 
                   3244 
                 
                 
                   Fnu DII 
                   542 
                   1074 
                   1655 
                   1837 
                   1934 
                   2056 
                   2082 
                   2227 
                   2237 
                   2366 
                   243 
                 
                 
                     
                   2493 
                   2498 
                   2525 
                   2654 
                   2769 
                   3125 
                 
                 
                   Fok I 
                   468 
                   852 
                   3370 
                 
                 
                   Fok I* 
                   816 
                   2423 
                   2468 
                   3322 
                 
                 
                   Gsu I 
                   2088 
                 
                 
                   Gsu I* 
                   2642 
                 
                 
                   Hae I 
                   361 
                   828 
                   844 
                   1224 
                   1676 
                   1687 
                   2026 
                   2423 
                   2480 
                   2552 
                 
                 
                   Hae II 
                   594 
                   1458 
                   1828 
                   2267 
                   2697 
                   2924 
                   2978 
                   3038 
                   3059 
                   3240 
                 
                 
                   Hae III 
                   343 
                   361 
                   678 
                   767 
                   828 
                   844 
                   1224 
                   1658 
                   1676 
                   1687 
                   202 
                 
                 
                     
                   2210 
                   2423 
                   2480 
                   2531 
                   2552 
                   2641 
                   2875 
                   2939 
                   2947 
                   3071 
                   317 
                 
                 
                     
                   3298 
                 
                 
                   Hga I 
                   160 
                   183 
                   796 
                   2088 
                   2238 
                   2829 
                 
                 
                   Hga I* 
                   1008 
                   1586 
                   2482 
                   2514 
                   3068 
                 
                 
                   Hgi AI 
                   141 
                   1388 
                   2007 
                   2298 
                   2885 
                   3196 
                 
                 
                   Hgi CI 
                   210 
                   2179 
                   2263 
                   2702 
                   2920 
                   3034 
                   3055 
                   3349 
                   3392 
                 
                 
                   Hgi JII 
                   345 
                   2987 
                   3001 
                 
                 
                   Hha I 
                   542 
                   593 
                   1074 
                   1183 
                   1357 
                   1457 
                   1524 
                   1794 
                   1827 
                   2017 
                   205 
                 
                 
                     
                   2115 
                   2266 
                   2525 
                   2656 
                   2696 
                   2771 
                   2923 
                   2977 
                   3037 
                   3058 
                   321 
                 
                 
                     
                   3239 
                   3371 
                 
                 
                   Hin PlI 
                   540 
                   591 
                   1072 
                   1181 
                   1355 
                   1455 
                   1522 
                   1792 
                   1825 
                   2015 
                   205 
                 
                 
                     
                   2113 
                   2264 
                   2523 
                   2654 
                   2694 
                   2769 
                   2921 
                   2975 
                   3035 
                   3056 
                   320 
                 
                 
                     
                   3237 
                   3369 
                 
                 
                   Hind II 
                   109 
                   372 
                   2819 
                 
                 
                   Hind III 
                   384 
                   437 
                   3439 
                 
                 
                   Hinf I 
                   368 
                   1328 
                   1724 
                   1799 
                   1944 
                   2165 
                   2463 
                   2617 
                   2837 
                 
                 
                   Hpa II 
                   5 
                   339 
                   355 
                   375 
                   735 
                   769 
                   1130 
                   1320 
                   1346 
                   1493 
                   198 
                 
                 
                     
                   2186 
                   2212 
                   2450 
                   2540 
                   2700 
                   2776 
                   2936 
                   3059 
                   3068 
                   3083 
                   330 
                 
                 
                     
                   3309 
                 
                 
                   Hph I 
                   96 
                   140 
                   183 
                   716 
                   967 
                   1953 
                   2174 
                   3028 
                   3073 
                   3355 
                 
                 
                   Hph I* 
                   8 
                   305 
                   311 
                   317 
                 
                 
                   Kpn I 
                   214 
                 
                 
                   Mae I 
                   365 
                   952 
                   1205 
                   1981 
                   3240 
                 
                 
                   Mae II 
                   276 
                   330 
                   751 
                   997 
                   1900 
                   1924 
                   2513 
                   2569 
                 
                 
                   Mae III 
                   171 
                   257 
                   1162 
                   1278 
                   1341 
                   2320 
                   2587 
                   3255 
                   3343 
                 
                 
                   Mbo I 
                   9 
                   236 
                   334 
                   948 
                   960 
                   1038 
                   1046 
                   1057 
                   1132 
                   2008 
                   232 
                 
                 
                     
                   2340 
                   2371 
                   2643 
                   3002 
                   3093 
                   3120 
                 
                 
                   Mbo II 
                   209 
                   475 
                   970 
                   1832 
                   1880 
                   2472 
                   2743 
                 
                 
                   Mbo II* 
                   1041 
                   2997 
                 
                 
                   Mme I* 
                   1305 
                   1489 
                   3165 
                   3252 
                 
                 
                   Mnl I 
                   372 
                   1271 
                   1595 
                   2001 
                   2499 
                   2683 
                 
                 
                   Mnl I* 
                   210 
                   291 
                   350 
                   764 
                   1520 
                   1803 
                   2169 
                   2196 
                   2234 
                   2295 
                   259 
                 
                 
                     
                   2864 
                   3083 
                   3287 
                   3347 
                 
                 
                   Mse I 
                   181 
                   188 
                   223 
                   388 
                   486 
                   817 
                   994 
                   3414 
                   3436 
                 
                 
                   Mst I 
                   2016 
                   2114 
                   3210 
                 
                 
                   Nae I 
                   2187 
                   2541 
                   2701 
                   3069 
                 
                 
                   Nar I 
                   2264 
                   2921 
                   3035 
                   3056 
                 
                 
                   Nco I 
                   345 
                 
                 
                   Nhe I 
                   3239 
                 
                 
                   Nla III 
                   168 
                   232 
                   349 
                   382 
                   565 
                   620 
                   912 
                   982 
                   1702 
                   1881 
                   201 
                 
                 
                     
                   2222 
                   2279 
                   2294 
                   2422 
                   2539 
                   2725 
                   2764 
                   2910 
                   2983 
                   3121 
                   346 
                 
                 
                   Nla IV 
                   212 
                   336 
                   343 
                   549 
                   1631 
                   1670 
                   1989 
                   2032 
                   2146 
                   2181 
                   221 
                 
                 
                     
                   2265 
                   2583 
                   2704 
                   2922 
                   2946 
                   3036 
                   3057 
                   3095 
                   3141 
                   3351 
                   339 
                 
                 
                   Nru I 
                   2498 
                 
                 
                   Nsp BII 
                   412 
                   1115 
                   1360 
                   2331 
                 
                 
                   Nsp HI 
                   382 
                   1702 
                   2910 
                 
                 
                   Pfl MI 
                   295 
                   2105 
                   2154 
                 
                 
                   Ple I 
                   376 
                   1807 
                 
                 
                   Ple I* 
                   1322 
                   2831 
                 
                 
                   Pma CI 
                   331 
                 
                 
                   Ppu MI 
                   1988 
                   2030 
                 
                 
                   Pss I 
                   1991 
                   2033 
                   2948 
                 
                 
                   Rsa I 
                   212 
                   3307 
                 
                 
                   Sal I 
                   370 
                   2817 
                 
                 
                   Scr FI 
                   6 
                   215 
                   339 
                   340 
                   528 
                   638 
                   736 
                   769 
                   806 
                   1321 
                   153 
                 
                 
                     
                   1552 
                   1673 
                   1986 
                   2028 
                   2212 
                   2411 
                   2936 
                   3300 
                   3340 
                 
                 
                   Sdu I 
                   141 
                   345 
                   1388 
                   2007 
                   2298 
                   2885 
                   2987 
                   3001 
                   3196 
                 
                 
                   Sec I 
                   5 
                   338 
                   345 
                   1538 
                   2021 
                   2099 
                   2301 
                   2934 
                   2940 
                   3339 
                   335 
                 
                 
                   Sfa NI 
                   650 
                   818 
                   2445 
                   2820 
                   3231 
                   3344 
                 
                 
                   Sfa NI* 
                   420 
                   1601 
                   2038 
                   2433 
                   3054 
                   3066 
                   3255 
                 
                 
                   Sma I 
                   340 
                 
                 
                   Sph I 
                   382 
                   2910 
                 
                 
                   Sso II 
                   4 
                   213 
                   337 
                   338 
                   526 
                   636 
                   734 
                   767 
                   804 
                   1319 
                   153 
                 
                 
                     
                   1550 
                   1671 
                   1984 
                   2026 
                   2210 
                   2409 
                   2934 
                   3298 
                   3338 
                 
                 
                   Stu I 
                   361 
                 
                 
                   Sty I 
                   345 
                   2099 
                 
                 
                   Taq I 
                   254 
                   371 
                   666 
                   1600 
                   2202 
                   2343 
                   2818 
                   3131 
                   3446 
                 
                 
                   Taq IIB 
                   1802 
                 
                 
                   Taq IIB* 
                   2804 
                 
                 
                   TthlllII 
                   40 
                   1107 
                 
                 
                   TthlllII* 
                   686 
                   1075 
                   1114 
                 
                 
                   Xba I 
                   364 
                 
                 
                   Xho II 
                   9 
                   334 
                   948 
                   960 
                   1046 
                   1057 
                   3093 
                 
                 
                   Xma I 
                   338 
                 
                 
                   Xma III 
                   2529 
                 
                 
                   Xmn I 
                   467 
                 
               
            
             
                 
                   Total number of cuts is: 743. 
                 
                 
                     
                 
               
            
             
                 
                   Aat II, 
                   Asu II, 
                   Avr II, 
                   Bbv II*, 
                   Bcl I, 
                   Bgl II, 
                   Bsp MI* 
                     
                 
                 
                   Bss HII, 
                   Bst EII, 
                   Bst XI, 
                   Eco 3lI*, 
                   Esp I, 
                   Hpa I, 
                   Mlu I 
                 
                 
                   Mme I, 
                   Nde I, 
                   Not I, 
                   Nsi I, 
                   Pst I, 
                   Pvu I, 
                   Pvu I 
                 
                 
                   Rsr II, 
                   Sac I, 
                   Sac II, 
                   Sau I, 
                   Sca I, 
                   Sci I, 
                   Sfi I 
                 
                 
                   Sna BI, 
                   Spe I, 
                   Spl I, 
                   Ssp I, 
                   Taq IIA, 
                   Taq IIA*, 
                   Tth lllI 
                 
                 
                   Vsp I, 
                   Xca I, 
                   Xho I 
                 
               
            
             
                 
                   Total number of selected enzymes which do not cut: 38 
                 
                 
                     
                 
               
            
           
         
       
         FIG. 12   a  corresponds to the restriction and genetic map of the plasmid pIG2 used to make the intermediary construct pIG2 Mt32 as described in Example IV for the subcloning of the P 32  antigen in plasmid pIGRI and contains SEQ ID NO:53. 
         FIGS. 12   b – 12   l  correspond to the pIG2 nucleic acid sequence (SEQ ID NO:42). 
       On this figure, the origin of nucleotide stretches used to construct plasmid pIG2 is specified hereafter. 
       
         
           
                 
                 
               
             
                 
                     
                 
                 
                   Position 
                 
                 
                     
                 
               
               
                 
                   3300–206  
                   lambda PL containing EcoRI-MboII blunt 
                 
                 
                     
                   fragment of pPL(λ) (Pharmacia) 
                 
                 
                   207–266 
                   synthetic sequence containing multiple 
                 
                 
                     
                   cloning site and ribosone binding site 
                 
                 
                     
                   of which the ATG initiation codon is 
                 
                 
                     
                   located at position 232–234 
                 
                 
                   267–772 
                   rrnBT 1 T 2  containing HindIII-SspI 
                 
                 
                     
                   fragment from pKK223 (Pharmacia) 
                 
                 
                    773–3300 
                   tetracycline resistance gene and origin 
                 
                 
                     
                   of replication containing EcoRI-DraI 
                 
                 
                     
                   fragment of pAT 153 (Bioexcellence) 
                 
                 
                     
                 
               
            
           
         
       
       Table 7 corresponds to the complete restriction site analysis of pIG2. 
       
         
           
                 
               
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
                 
               
                 
               
                 
                 
               
                 
                 
                 
                 
                 
                 
                 
                 
               
                 
                 
               
             
                 
                   TABLE 7 
                 
                 
                     
                 
                 
                   RESTRICTION-SITE ANALYSIS 
                 
                 
                     
                 
               
               
                 
                   Done on DNA sequence pIG2 
                 
                 
                   Total number of bases is: 3301. 
                 
                 
                   Analysis done on the complete sequence. 
                 
                 
                     
                 
                 
                   List of cuts by enzyme. 
                 
               
            
             
                 
                   Acc I 
                   252 
                   2647 
                     
                     
                     
                     
                     
                     
                     
                     
                     
                 
                 
                   Acy I 
                   617 
                   2093 
                   2750 
                   2664 
                   2885 
                 
                 
                   Afl III 
                   1527 
                 
                 
                   Aha III 
                   222 
                 
                 
                   Alu I 
                   268 
                   970 
                   1227 
                   1363 
                   1589 
                   2211 
                   2614 
                   3270 
                   3285 
                 
                 
                   Alw NI 
                   1118 
                 
                 
                   Apa LI 
                   1213 
                 
                 
                   Asp 718I 
                   208 
                 
                 
                   Asu I 
                   376 
                   505 
                   595 
                   1817 
                   1859 
                   2038 
                   2162 
                   2411 
                   2499 
                   2774 
                   312 
                 
                 
                   Ava I 
                   1872 
                 
                 
                   Ava II 
                   376 
                   1817 
                   1859 
                   2162 
                   2411 
                   2499 
                 
                 
                   Bal I 
                   1855 
                 
                 
                   Bam HI 
                   239 
                   2922 
                 
                 
                   Bbe I 
                   2096 
                   2753 
                   2867 
                   2888 
                 
                 
                   Bbv I 
                   271 
                   1198 
                   1617 
                   1635 
                   1748 
                   1751 
                   2695 
                   3084 
                 
                 
                   Bbv I* 
                   899 
                   1105 
                   1108 
                   1855 
                   1879 
                   2512 
                 
                 
                   Bbv II 
                   1704 
                   2567 
                 
                 
                   Bgl I 
                   2135 
                   2369 
                 
                 
                   Bin I 
                   15 
                   247 
                   785 
                   883 
                   969 
                   2930 
                 
                 
                   Bin I* 
                   234 
                   784 
                   881 
                   2195 
                   2917 
                 
                 
                   Bsp HI 
                   737 
                   807 
                   2808 
                 
                 
                   Bsp MI 
                   264 
                   2243 
                 
                 
                   Bst NI 
                   213 
                   357 
                   467 
                   635 
                   1366 
                   1381 
                   1502 
                   1857 
                   2240 
                   3169 
                 
                 
                   Cau II 
                   4 
                   565 
                   598 
                   1150 
                   1815 
                   2641 
                   2765 
                   3129 
                 
                 
                   Cfr 10I 
                   2014 
                   2368 
                   2528 
                   2887 
                   2896 
                   3137 
                 
                 
                   Cfr I 
                   1853 
                   2358 
                   2766 
                   2898 
                   3002 
                 
                 
                   Cla I 
                   3275 
                 
                 
                   Cvi JI 
                   190 
                   262 
                   268 
                   273 
                   303 
                   489 
                   507 
                   596 
                   657 
                   673 
                   97 
                 
                 
                     
                   999 
                   1042 
                   1053 
                   1118 
                   1197 
                   1222 
                   1227 
                   1363 
                   1461 
                   1487 
                   150 
                 
                 
                     
                   1516 
                   1589 
                   1608 
                   1808 
                   1813 
                   1855 
                   1892 
                   1974 
                   2018 
                   2039 
                   204 
                 
                 
                     
                   2182 
                   2192 
                   2211 
                   2252 
                   2309 
                   2317 
                   2347 
                   2360 
                   2381 
                   2426 
                   247 
                 
                 
                     
                   2614 
                   2630 
                   2686 
                   2704 
                   2768 
                   2776 
                   2814 
                   2828 
                   2900 
                   2969 
                   300 
                 
                 
                     
                   3127 
                   3151 
                   3270 
                   3285 
                 
                 
                   Cvi QI 
                   209 
                   3135 
                 
                 
                   Dde I 
                   133 
                   400 
                   490 
                   546 
                   844 
                   1253 
                   1717 
                 
                 
                   Dpn I 
                   9 
                   241 
                   779 
                   791 
                   869 
                   877 
                   888 
                   963 
                   1839 
                   2156 
                   217 
                 
                 
                     
                   2202 
                   2474 
                   2833 
                   2924 
                   2951 
                 
                 
                   Dra II 
                   1817 
                   1859 
                   2774 
                 
                 
                   Dsa I 
                   230 
                   1850 
                   2769 
                 
                 
                   Eco 31I 
                   444 
                 
                 
                   Eco 47III 
                   1655 
                   2524 
                   2805 
                   3067 
                 
                 
                   Eco 57I 
                   214 
                 
                 
                   Eco 57I* 
                   985 
                 
                 
                   Eco 78I 
                   2094 
                   2751 
                   2865 
                   2886 
                 
                 
                   Eco NI 
                   196 
                   2674 
                 
                 
                   Eco RII 
                   211 
                   355 
                   465 
                   633 
                   1366 
                   1379 
                   1500 
                   1855 
                   2238 
                   3167 
                 
                 
                   Eco RV 
                   3114 
                 
                 
                   Fnu 4HI 
                   260 
                   361 
                   913 
                   1119 
                   1122 
                   1187 
                   1330 
                   1485 
                   1603 
                   1606 
                   162 
                 
                 
                     
                   1737 
                   1740 
                   1869 
                   1883 
                   1890 
                   1893 
                   2012 
                   2091 
                   2136 
                   2193 
                   227 
                 
                 
                     
                   2361 
                   2526 
                   2577 
                   2684 
                   2718 
                   2721 
                   2999 
                   3002 
                   3073 
                 
                 
                   Fnu DII 
                   371 
                   903 
                   1484 
                   1666 
                   1763 
                   1885 
                   1911 
                   2056 
                   2066 
                   2195 
                   226 
                 
                 
                     
                   2322 
                   2327 
                   2354 
                   2483 
                   2598 
                   2954 
                 
                 
                   Fok I 
                   297 
                   681 
                   3199 
                 
                 
                   Fok I* 
                   645 
                   2252 
                   2297 
                   3151 
                 
                 
                   Gsu I 
                   1917 
                 
                 
                   Gsu I* 
                   2471 
                 
                 
                   Hae I 
                   657 
                   673 
                   1053 
                   1505 
                   1516 
                   1855 
                   2252 
                   2309 
                   2381 
                 
                 
                   Hae II 
                   423 
                   1287 
                   1657 
                   2096 
                   2526 
                   2753 
                   2807 
                   2867 
                   2888 
                   3069 
                 
                 
                   Hae III 
                   507 
                   596 
                   657 
                   673 
                   1053 
                   1487 
                   1505 
                   1516 
                   1855 
                   2039 
                   225 
                 
                 
                     
                   2309 
                   2360 
                   2381 
                   2470 
                   2704 
                   2768 
                   2776 
                   2900 
                   3004 
                   3127 
                 
                 
                   Hga I 
                   158 
                   181 
                   625 
                   1917 
                   2067 
                   2658 
                 
                 
                   Hga I* 
                   837 
                   1415 
                   2311 
                   2343 
                   2897 
                 
                 
                   Hgi AI 
                   139 
                   1217 
                   1836 
                   2127 
                   2714 
                   3025 
                 
                 
                   Hgi CI 
                   208 
                   2008 
                   2092 
                   2531 
                   2749 
                   2863 
                   2884 
                   3178 
                   3221 
                 
                 
                   Hgi JII 
                   2816 
                   2830 
                 
                 
                   Hha I 
                   371 
                   422 
                   903 
                   1012 
                   1186 
                   1286 
                   1353 
                   1623 
                   1656 
                   1846 
                   188 
                 
                 
                     
                   1944 
                   2095 
                   2354 
                   2485 
                   2525 
                   2600 
                   2752 
                   2806 
                   2866 
                   2887 
                   304 
                 
                 
                     
                   3068 
                   3200 
                 
                 
                   Hin PlI 
                   369 
                   420 
                   901 
                   1010 
                   1184 
                   1284 
                   1351 
                   1621 
                   1654 
                   1844 
                   188 
                 
                 
                     
                   1942 
                   2093 
                   2352 
                   2483 
                   2523 
                   2598 
                   2750 
                   2804 
                   2864 
                   2885 
                   303 
                 
                 
                     
                   3066 
                   3198 
                 
                 
                   Hind II 
                   107 
                   253 
                   2648 
                 
                 
                   Hind III 
                   266 
                   3268 
                 
                 
                   Hinf I 
                   249 
                   1157 
                   1553 
                   1628 
                   1773 
                   1994 
                   2292 
                   2446 
                   2666 
                 
                 
                   Hpa II 
                   3 
                   564 
                   598 
                   959 
                   1149 
                   1175 
                   1322 
                   1814 
                   2015 
                   2041 
                   227 
                 
                 
                     
                   2369 
                   2529 
                   2605 
                   2765 
                   2888 
                   2897 
                   2912 
                   3129 
                   3138 
                 
                 
                   Hph I 
                   94 
                   138 
                   181 
                   545 
                   796 
                   1782 
                   2003 
                   2857 
                   2902 
                   3184 
                 
                 
                   Hph I* 
                   6 
                 
                 
                   Kpn I 
                   212 
                 
                 
                   Mae I 
                   246 
                   781 
                   1034 
                   1810 
                   3069 
                 
                 
                   Mae II 
                   580 
                   826 
                   1729 
                   1753 
                   2342 
                   2398 
                 
                 
                   Mae III 
                   169 
                   991 
                   1107 
                   1170 
                   2149 
                   2416 
                   3084 
                   3172 
                 
                 
                   Mbo I 
                   7 
                   239 
                   777 
                   789 
                   867 
                   875 
                   886 
                   961 
                   1837 
                   2154 
                   216 
                 
                 
                     
                   2200 
                   2472 
                   2831 
                   2922 
                   2949 
                 
                 
                   Mbo II 
                   207 
                   304 
                   799 
                   1661 
                   1709 
                   2301 
                   2572 
                 
                 
                   Mbo II* 
                   870 
                   2826 
                 
                 
                   Mme I* 
                   1134 
                   1318 
                   2994 
                   3081 
                 
                 
                   Mnl I 
                   253 
                   1100 
                   1424 
                   1830 
                   2328 
                   2512 
                 
                 
                   Mnl I* 
                   208 
                   593 
                   1349 
                   1632 
                   1998 
                   2025 
                   2063 
                   2124 
                   2426 
                   2693 
                   291 
                 
                 
                     
                   3116 
                   3176 
                 
                 
                   Mse I 
                   179 
                   186 
                   221 
                   315 
                   646 
                   823 
                   3243 
                   3265 
                 
                 
                   Mst I 
                   1845 
                   1943 
                   3039 
                 
                 
                   Nae I 
                   2016 
                   2370 
                   2530 
                   2898 
                 
                 
                   Nar I 
                   2093 
                   2750 
                   2864 
                   2885 
                 
                 
                   Nco I 
                   230 
                 
                 
                   Nhe I 
                   3068 
                 
                 
                   Nla III 
                   166 
                   234 
                   394 
                   449 
                   741 
                   811 
                   1531 
                   1710 
                   1844 
                   2051 
                   210 
                 
                 
                     
                   2123 
                   2251 
                   2368 
                   2554 
                   2593 
                   2739 
                   2812 
                   2950 
                   3297 
                 
                 
                   Nla IV 
                   210 
                   241 
                   378 
                   1460 
                   1499 
                   1818 
                   1861 
                   1975 
                   2010 
                   2045 
                   209 
                 
                 
                     
                   2412 
                   2533 
                   2751 
                   2775 
                   2865 
                   2886 
                   2924 
                   2970 
                   3180 
                   3223 
                 
                 
                   Nru I 
                   2327 
                 
                 
                   Nsp BII 
                   944 
                   1189 
                   2160 
                 
                 
                   Nsp HI 
                   1531 
                   2739 
                 
                 
                   Pfl MI 
                   1934 
                   1983 
                 
                 
                   Ple I 
                   257 
                   1636 
                 
                 
                   Ple I* 
                   1151 
                   2660 
                 
                 
                   Ppu MI 
                   1817 
                   1859 
                 
                 
                   Pss I 
                   1820 
                   1862 
                   2777 
                 
                 
                   Pst I 
                   261 
                 
                 
                   Rsa I 
                   210 
                   3136 
                 
                 
                   Sal I 
                   251 
                   2646 
                 
                 
                   Scr FI 
                   4 
                   213 
                   357 
                   467 
                   565 
                   598 
                   635 
                   1150 
                   1368 
                   1381 
                   150 
                 
                 
                     
                   1815 
                   1857 
                   2041 
                   2240 
                   2765 
                   3129 
                   3169 
                 
                 
                   Sdu I 
                   139 
                   1217 
                   1836 
                   2127 
                   2714 
                   2816 
                   2830 
                   3025 
                 
                 
                   Sec I 
                   3 
                   230 
                   1367 
                   1850 
                   1928 
                   2130 
                   2763 
                   2769 
                   3168 
                   3182 
                 
                 
                   Sfa NI 
                   479 
                   647 
                   2274 
                   2649 
                   3060 
                   3173 
                 
                 
                   Sfa NI* 
                   1430 
                   1867 
                   2262 
                   2883 
                   2895 
                   3084 
                 
                 
                   Sph I 
                   2739 
                 
                 
                   Sso II 
                   2 
                   211 
                   355 
                   465 
                   563 
                   596 
                   633 
                   1148 
                   1366 
                   1379 
                   150 
                 
                 
                     
                   1813 
                   1855 
                   2039 
                   2238 
                   2763 
                   3127 
                   3167 
                 
                 
                   Ssp I 
                   226 
                 
                 
                   Sty I 
                   230 
                   1928 
                 
                 
                   Taq I 
                   252 
                   495 
                   1429 
                   2031 
                   2172 
                   2647 
                   2960 
                   3275 
                 
                 
                   Taq IIB 
                   1631 
                 
                 
                   Taq IIB* 
                   2633 
                 
                 
                   TthlllII 
                   38 
                   936 
                 
                 
                   TthlllII* 
                   515 
                   904 
                   943 
                 
                 
                   Xba I 
                   245 
                 
                 
                   Xho II 
                   7 
                   239 
                   777 
                   789 
                   875 
                   886 
                   2922 
                 
                 
                   Xma III 
                   2358 
                 
                 
                   Xmn I 
                   296 
                 
                 
                   EcoRI 
                   3300 
                 
               
            
             
                 
                   Total number of cuts is: 689. 
                 
                 
                     
                 
               
            
             
                 
                   List of non cutting selected enzymes. 
                     
                 
               
            
             
                 
                   Aat II, 
                   Afl II, 
                   Apa I, 
                   Asu II, 
                   Avr II, 
                   Bbv II*, 
                   Bcl I 
                     
                 
                 
                   Bgl II, 
                   Bsp MI*, 
                   Bsp MII, 
                   Bss HII, 
                   Bst EII, 
                   Bst XI, 
                   Dra III 
                 
                 
                   Eco 31I*, 
                   Esp I, 
                   Hpa I, 
                   Mlu I, 
                   Mme I, 
                   Nde I, 
                   Not I 
                 
                 
                   Nsi I, 
                   Pma CI, 
                   Pvu I, 
                   Pvu II, 
                   Rsr II, 
                   Sac I, 
                   Sac II 
                 
                 
                   Sau I, 
                   Sca I, 
                   Sci I, 
                   Sfi I, 
                   Sma I, 
                   Sna BI, 
                   Spe I 
                 
                 
                   Spl I, 
                   Stu I, 
                   Taq IIA, 
                   Taq IIA*, 
                   Tth lllI, 
                   Vsp I, 
                   Xca I 
                 
                 
                   Xho I, 
                   Xma I 
                 
               
            
             
                 
                   Total number of selected enzymes which do not cut: 44 
                 
                 
                     
                 
               
            
           
         
       
         FIG. 13  corresponds to the amino acid sequence of the total fusion protein mTNF-His 6 -P 32  (SEQ ID NO:43). 
       On this figure:
         the continuous underlined sequence ( —   —   —   — _) represents the mTNF sequence (first 25 amino acids),   the dotted underlined sequence (- - - - -) represents the polylinker sequence,   the double underlined sequence           represents the extra amino acids created at cloning site, and   the amino acid marked with nothing is the antigen sequence starting from the amino acid at position 4 of  FIG. 5 .       

         FIGS. 14   a  and  14   b  correspond to the expression of the mTNF-His 6 -P 32  fusion protein in K12ΔH, given in Example VI, with  FIG. 14   a  representing the Coomassie Brilliant Blue stained SDS-PAGE and  14   b  representing immunoblots of the gel with anti-32-kDa and anti-mTNF-antibody. 
       On  FIG. 14   a , the lanes correspond to the following: 
       
         
           
                 
                 
               
                 
                 
                 
                 
               
             
                 
                     
                 
                 
                   Lanes 
                 
                 
                     
                 
               
               
                 
                     
                 
               
            
             
                 
                   1. 
                   protein molecular weight markers 
                     
                     
                 
                 
                   2. 
                   pmTNF-MPH-Mt32 
                   28° C. 
                   1 h induction 
                 
                 
                   3. 
                   ″ 
                   42° C. 
                   ″ 
                 
                 
                   4. 
                   ″ 
                   42° C. 
                   2 h induction 
                 
                 
                   5. 
                   ″ 
                   42° C. 
                   3 h induction 
                 
                 
                   6. 
                   ″ 
                   28° C. 
                   4 h induction 
                 
                 
                   7. 
                   ″ 
                   42° C. 
                   4 h induction 
                 
                 
                   8. 
                   ″ 
                   28° C. 
                   5 h induction 
                 
                 
                   9. 
                   ″ 
                   42° C. 
                   5 h induction 
                 
                 
                     
                 
               
            
           
         
       
       On  FIG. 14   b , the lanes correspond to the following: 
       
         
           
                 
                 
                 
               
                 
                 
                 
                 
                 
               
             
                 
                     
                     
                 
                 
                     
                   Lanes 
                 
                 
                     
                     
                 
               
               
                 
                     
                 
               
            
             
                 
                     
                   1. 
                   pmTNF-MPH-Mt32 
                   28° C. 
                   1 h induction 
                 
                 
                     
                   2. 
                   ″ 
                   42° C. 
                   1 h induction 
                 
                 
                     
                   3. 
                   ″ 
                   28° C. 
                   4 h induction 
                 
                 
                     
                   4. 
                   ″ 
                   42° C. 
                   4 h induction 
                 
                 
                     
                     
                 
               
            
           
         
       
         FIG. 15  corresponds to the IMAC elution profile of the recombinant antigen with decreasing pH as presented in Example VII. 
         FIG. 16  corresponds to the IMAC elution profile of the recombinant antigen with increasing imidazole concentrations as presented in Example VII. 
         FIG. 17  corresponds to the IMAC elution profile of the recombinant antigen with a step gradient of increasing imidazole concentrations as presented in Example VII. 
    
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     EXAMPLE I 
     Material and Methods 
     Screening of the λqt11  M. tuberculosis  Recombinant DNA Library with Anti-32-kDa Antiserum 
     A λgt11 recombinant library constructed from genomic DNA of  M. tuberculosis  (Erdman strain), was obtained from R. Young (35). Screening was performed as described (14,35) with some modifications hereafter mentioned. λgt11 infected  E. coli  Y1090 (10 5  pfu per 150 mm plate) were seeded on NZYM plates (Gibco) (16) and incubated at 42° C. for 24 hrs. To induce expression of the β-galactosidase-fusion proteins the plates were overlaid with isopropyl β-D-thiogalactoside (IPTG)-saturated filters (Hybond C extra, Amersham), and incubated for 2 hrs at 37° C. Screening was done with a polyclonal rabbit anti-32-kDa antiserum. Said polyclonal antiserum rabbit anti-32-kDa antiserum was obtained by raising antiserum against the P 32    M. bovis  BCG (strain 1173P2—Institut Pasteur Paris) as follows: 400 μg (purified P 32  protein of  M. bovis  BCG) per ml physiological saline were mixed with one volume of incomplete Freund&#39;s adjuvant. The material was homogenized and injected intradermally in 50 μl doses, delivered at 10 sites in the back of the rabbits, at 0, 4, 7 and 8 weeks (adjuvant was replaced by the diluent for the last injection). One week later, the rabbits were bled and the sera tested for antibody level before being distributed in aliquots and stored at −80° C. 
     The polyclonal rabbit anti-32-kDa antiserum was pre-absorbed on  E. coli  lysate (14) and used at a final dilution of 1:300. A secondary alkaline-phosphatase anti-rabbit IgG conjugate (Promega), diluted at 1:5000 was used to detect the β-galactosidase fusion proteins. For color development nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were used. Reactive areas on the filter turned deep purple within 30 min. Usually three consecutive purification steps were performed to obtain pure clones. IPTG, BCIP and NBT were from Promega corp. (Madison Wis.). 
     Plaque Screening by Hybridization for Obtaining the Secondary Clones BY1, By2 and By5 Hereafter Defined 
     The procedure used was as described by Maniatis et al. (14). 
     Preparation of Crude Lysates from λgt11 Recombinant Lysogens 
     Colonies of  E. coli  Y1089 were lysogenized with appropriate λgt11 recombinants as described by Hyunh et al. (14). Single colonies of lysogenized  E. coli  Y1089 were inoculated into LB medium and grown to an optical density of 0.5 at 600 nm at 30° C. After a heat shock at 45° C. for 20 min., the production of β-galactosidase-fusion protein was induced by the addition of IPTG to a final concentration of 10 mM. Incubation was continued for 60 min. at 37° C. and cells were quickly harvested by centrifugation. Cells were concentrated 50 times in buffer (10 mM Tris pH 8.0, 2 mM EDTA) and rapidly frozen into liquid nitrogen. The samples were lysed by thawing and treated with 100 μg/ml DNase I in EcoRI restriction buffer, for 5–10 minutes at 37° C. 
     Immunoblotting (Western Blotting) Analysis: 
     After SDS-PAGE electrophoresis, recombinant lysogen proteins were blotted onto nitrocellulose membranes (Hybond C, Amersham) as described by Towbin et al. (29). The expression of mycobacterial antigens, fused to β-galactosidase in  E. coli  Y1089 was visualized by the binding of a polyclonal rabbit anti-32-kDa antiserum (1:1000) obtained as described in the above paragraph “Screening of the λgt11  M. tuberculosis  recombinant DNA library with anti-32-kDa antiserum” and using a monoclonal anti-β-galactosidase antibody (Promega). A secondary alkaline-phosphatase anti-rabbit IgG conjugate (Promega) diluted at 1:5000, was used to detect the fusion proteins. 
     The use of these various antibodies enables to detect the β-galactosidase fusion protein. This reaction is due to the  M. tuberculosis  protein because of the fact that non fused-β-galactosidase is also present on the same gel and is not recognized by the serum from tuberculous patients. 
     In order to identify selective recognition of recombinant fusion proteins by human tuberculous sera, nitrocellulose sheets were incubated overnight with these sera (1:50) (after blocking aspecific protein binding sites). The human tuberculous sera were selected for their reactivity (high or low) against the purified 32-kDa antigen of  M. bovis  BCG tested in a Dot blot assay as previously described (31). Reactive areas on the nitrocellulose sheets were revealed by incubation with peroxidase conjugated goat anti-human IgG antibody (Dakopatts, Copenhagen, Denmark) (1:200) for 4 hrs and after repeated washings color reaction was developed by adding peroxidase substrate (α-chloronaphtol) (Bio-Rad) in the presence of peroxidase and hydrogen peroxide. 
     Recombinant DNA Analysis 
     Initial identification of  M. tuberculosis  DNA inserts in purified λgt11 clones was performed by EcoRI restriction. After digestion, the excised inserts were run on agarose gels and submitted to Southern hybridization. Probes were labeled with α 32 P-dCTP by random priming (10). Other restriction sites were located by single and double digestions of recombinant λgt11 phage DNA or their subcloned EcoRI fragments by HindIII, PstI, KpnI, AccI and SphI. 
     Sequencing 
     Sequence analysis was done by the primer extension dideoxy termination method of Sanger et al. (25) after subcloning of specific fragments in Bluescribe-M13 (6) or in mp10 and mp11 M13 vectors (Methods in Enzymology, vol. 101, 1983, p. 20–89, Joachim Messing, New M13 vectors for cloning, Academic Press). Sequence analysis was greatly hampered by the high GC content of the  M. tuberculosis  DNA (65%). Sequencing reactions were therefore performed with several DNA polymerases: T7 DNA polymerase (“Sequenase” USB), Klenow fragment of DNA polymerase I (Amersham) and in some cases with AMV reverse transcriptase (Super RT, Anglian Biotechnology Ltd.) and sometimes with dITP instead of dGTP. Several oligodeoxynucleotides were synthesized and used to focus ambiguous regions of the sequence. The sequencing strategy is summarized in  FIG. 2 . In order to trace possible artefactual frameshifts in some ambiguous regions, a special program was used to define the most probable open reading frame in sequences containing a high proportion of GC (3). Several regions particularly prone to sequencing artefacts were confirmed or corrected by chemical sequencing (18). For this purpose, fragments were subcloned in the chemical sequencing vector pGV462 (21) and analysed as described previously. Selected restriction fragments of about 250–350 bp were isolated, made blunt-ended by treatment with either Klenow polymerase or Mung bean nuclease, and subcloned in the SmaI or HincII site of pGV462. Both strands of the inserted DNA were sequenced by single-end labeling at Tth 111I or BstEII (32) and a modified chemical degradation strategy (33). 
     Routine computer aided analysis of the nucleic acid and deduced amino acid sequences were performed with the LGBC program from Bellon (2). Homology searches used the FASTA programs from Pearson and Lipman (23) and the Protein Identification Resource (PIR) from the National Biomedical Research Fundation—Washington (NBRF) (NBRF/PIR data bank), release 16 (March 1988). 
     Results 
     Screening of the λgt11M,  M. tuberculosis  Recombinant DNA Library with Polyclonal Anti-32-kDa Antiserum: 
     Ten filters representing 1.5×10 6  plaques were probed with a polyclonal rabbit anti-32-kDa antiserum (8). Following purification, six independent positive clones were obtained. 
     Analysis of Recombinant Clones 
     EcoRI restriction analysis of these 6 purified λgt11 recombinant clones DNA, ( FIG. 1A ) revealed 4 different types of insert. Clone 15 had an insert with a total length of 3.8 kb with two additional internal EcoRI sites resulting in three DNA fragments of 1.8 kb, 1.5 kb and 0.5 kb. The DNA Insert of clone 16 was 1.7 kb long. Clones 17 and 19 had a DNA insert of almost identical length being 2.7 kb and 2.8 kb respectively. 
     Finally, clone 23 (not shown) and clone 24 both contained an insert of 4 kb with one additional EcoRI restriction site giving two fragments of 2.3 kb and 1.7 kb. Southern analysis (data not shown) showed that the DNA inserts of clones 15, 16, 19 and the small fragment (1.7 kb) of clone 24 only hybridized with themselves whereas clone 17 (2.7 kb) hybridized with itself but also equally well with the 2.3 kb DNA fragment of clone 24. Clones 15, 16 and 19 are thus distinct and unrelated to the 17, 23, 24 group. This interpretation was further confirmed by analysis of crude lysates of  E. coli  Y1089 lysogenized with the appropriate λgt11 recombinants and induced with IPTG. Western blot analysis ( FIG. 1B ), showed no fusion protein, either mature or incomplete, reactive with the polyclonal anti-32-kDa antiserum in cells expressing clones 15, 16 and 19. Clones 15, 16 and 19, were thus considered as false positives and were not further studied. On the contrary, cells lysogenized with clone 23 and 24 produced an immunoreactive fusion protein containing about 10 kDa of the 32-kDa protein. Clone 17 finally expressed a fusion protein of which the foreign polypeptide part is about 25 kDa long. The restriction endonuclease maps of the 2.3 kb insert of clone 24 and of the 2.7 kb fragment of clone 17 ( FIG. 2 ) allowed us to align and orient the two inserts suggesting that the latter corresponds to a ±0.5 kb 5′ extension of the first. 
     As clone 17 was incomplete, the same λgt11 recombinant  M. tuberculosis  DNA library was screened by hybridization with a 120 bp EcoRI-Kpnl restriction fragment corresponding to the very 5′ end of the DNA insert of clone 17 (previously subcloned in a Blue Scribe vector commercialized by Vector cloning Systems (Stratagene Cloning System) ( FIG. 2 ). Three 5′-extended clones By1, By2 and By5 were isolated, analyzed by restriction and aligned. The largest insert, By5 contained the information for the entire coding region (see below) flanked by 3.1 kb upstream and 1.1 kb downstream ( FIG. 2 ). 
     DNA Sequencing 
     The 1358 base pairs nucleotide sequence derived from the various λgt11 overlapping clones is represented in  FIG. 3   a  and  FIG. 3   b . The DNA sequence contains a 1059 base pair open reading frame starting at position 183 and ending with a TAG codon at position 1242. It occurs that the NH 2 -terminal amino-acid sequence, (phe-ser-arg-pro-gly-leu-pro-val-glu-tyr-leu-gln-val-pro-ser-pro-ser-met-gly-arg-asp-ile-lys-val-gln-phe-gln-ser-gly-gly-ala-asn; SEQ ID NO:33) which can be located within this open reading frame from the nucleotide sequence beginning with a TTT codon at position 360 corresponds to the same NH 2 -terminal amino acid sequence of the MPB 59 antigen except for the amino acids at position 20, 21, 31, which are respectively gly, cys and asn in the MPB 59 (34). Therefore, the DNA region upstream of this sequence is expected to encode a signal peptide required for the secretion of a protein of 32-kDa. The mature protein thus presumably consists of 295 amino acid residues from the N-terminal Phe (TTT codon) to the C-terminal Ala (GCC codon) (FIG. 5). 
     Six ATG codons were found to precede the TTT at position 360 in the same reading frame. Usage of any of these ATGs in the same reading frame would lead to the synthesis of signal peptides of 29,42,47,49,55 and 59 residues. 
     Hydropathy Pattern 
     The hydropathy pattern coding sequence of the protein of 32-kDa of the invention and that of the antigen α of BCG (17) were determined by the method of Kyte and Doolittle (15). The nonapeptide profiles are shown in  FIG. 6 . Besides the initial hydrophobic signal peptide region, several hydrophilic domains could be identified. It is interesting to note that the overall hydrophilicity pattern of the protein of 32-kDa of the invention is comparable to that of the BCG antigen α. For both proteins, a domain of highest hydrophilicity could be identified between amino acid residues 200 and 250. 
     Homology 
     Matsuo et al. (17) recently published the sequence of a 1095 nucleotide cloned DNA corresponding to the gene coding for the antigen α of BCG. The 978 bp coding region of  M. bovis  antigen α as revised in Infection and Immunity, vol. 58, p. 550–556, 1990, and 1017 bp coding regions of the protein of 32-kDa of the invention show a 77.5% homology, in an aligned region of 942 bp. At the amino acid level both precursor protein sequences share 75.6% identical residues. In addition, 17.6% of the amino acids correspond to evolutionary conserved replacements as defined in the algorithm used for the comparison (PAM250 matrix, ref 23).  FIG. 7  shows sequence divergences in the N-terminal of the signal peptide. The amino terminal sequence—32 amino acids—of both mature proteins is identical except for position 31. 
     Human Sera Recognize the Recombinant 32-kDa Protein 
       FIG. 8  shows that serum samples from tuberculous patients when immunoblotted with a crude  E. coli  extract expressing clone 17 distinctly react with the 140 kDa fusion protein (lanes 4 to 6) contain the protein of 32-kDa of the invention, but not with unfused β-galactosidase expressed in a parallel extract (lanes 10 to 12). Serum samples from two negative controls selected as responding very little to the purified protein of 32-kDa of the invention does neither recognize the 140 kDa fused protein containing the protein of 32-kDa of the invention, nor the unfused β-galactosidase (lanes 2, 3 and 8 and 9). The 140 k-Da fused protein and the unfused β-galactosidase were easily localized reacting with the anti-β-galactosidase monoclonal antibody (lanes 1 to 7). 
     The invention has enabled to prepare a DNA region coding particularly for a protein of 32-kDa (cf.  FIG. 5 ); said DNA region containing an open reading frame of 338 codons (stop codon non included). At position 220 a TTT encoding the first amino acid of the mature protein is followed by the 295 triplets coding for the mature protein of 32-kDa. The size of this open reading frame, the immunoreactivity of the derived fusion proteins, the presence of a signal peptide and, especially, the identification within this gene of a NH 2 -terminal region highly homologous to that found in the MPB 59 antigen (31/32 amino acids homology) and in the BCG antigen α (31/32 amino acids homology) (see  FIG. 7 ), strongly suggest that the DNA fragment described contains the complete cistron encoding the protein of 32-kDa secreted by  M. tuberculosis , and which had never been so far identified in a non ambiguous way. 
     Six ATG codons were found to precede this TTT at position 220 in the same reading frame. Usage of any of these ATGs in the same reading frame would lead to the synthesis of signal peptides of 43, 48, 50, 56 or 60 residues. Among these various possibilities, initiation is more likely to take place either at ATG 91  or ATG 52  because both are preceded by a plausible  E. coli -like promoter and a Shine-Dalgarno motif. 
     If initiation takes place at ATG 91 , the corresponding signal peptide would code for a rather long peptide signal of 43 residues. This length however is not uncommon among secreted proteins from Gram positive bacteria (5). It would be preceded by a typical  E. coli  Shine-Dalgarno motif (4/6 residues homologous to AGGAGG) at a suitable distance. 
     If initiation takes place at ATG 52 , the corresponding signal peptide would code for a peptide signal of 56 residues but would have a less stringent Shine-Dalgarno ribosome binding site sequence. 
     The region encompassing the translation termination triplet was particularly sensitive to secondary structure effects which lead to so-called compressions on the sequencing gels. In front of the TAG termination codon at position 1105, 22 out of 23 residues are G-C base pairs, of which 9 are G&#39;s. 
     Upstream ATG 130 , a sequence resembling an  E. coli  promoter (11) comprising an hexanucleotide (TTGAGA) (homology 5/6 to TTGACA) and a AAGAAT box (homology 4/6 to TATAAT) separated by 16 nucleotides was observed. Upstream the potential initiating codon ATG 91 , one could detect several sequences homologous to the  E. coli “− 35 hexanucleotide box”, followed by a sequence resembling a TATAAT box. Among these, the most suggestive is illustrated on  FIGS. 3   a  and  3   b . It comprises a TTGGCC at position 59 ( FIGS. 3   a  and  3   b ) (homology 4/6 to TTGACA) separated by 14 nucleotides from a GATAAG (homology 4/6 to TATAAT). Interestingly this putative promoter region shares no extensive sequence homology with the promoter region described for the BCG protein α-gene (17) nor with that described for the 65 kDa protein gene (26, 28). 
     Searching the NBRF data bank (issue 16.0) any significant homology between the protein of 32-kDa of the invention and any other completely known protein sequence could not be detected. In particular no significant homology was observed between the 32-kDa protein and α and β subunits of the human fibronectin receptor (1). The NH 2 -terminal sequence of the 32-kDa protein of the invention is highly homologous—29/32 amino acids—to that previously published for BCG MPB 59 antigen (34) and to that of BCG α-antigen—31/32 amino acids—(Matsuo, 17) and is identical in its first 6 amino acids with the 32-kDa protein of  M. bovis  BCG (9). However, the presumed initiating methionine precedes an additional 29 or 42 amino acid hydrophobic sequence which differs from the one of α-antigen (cf.  FIG. 7 ), but displaying all the characteristics attributed to signal sequences of secreted polypeptides in prokaryotes (22). 
     Interestingly, no significant homology between the nucleic acid (1–1358) of the invention (cf.  FIGS. 3   a  and  3   b ) and the DNA of the antigen α of Matsuo exists within their putative promoter regions. 
     EXAMPLE II 
     Construction of a Bacterial Plasmid Containing the Entire Coding Sequence of the 32-kDa Protein of  M. tuberculosis    
     In the previous example, in  FIG. 2 , the various overlapping λgt11 isolates covering the 32-kDa protein gene region from  M. tuberculosis  were described. Several DNA fragments were subcloned from these λgt11 phages in the Blue Scribe M13+ plasmid (Stratagene). Since none of these plasmids contained the entire coding sequence of the 32-kDa protein gene, a plasmid containing this sequence was reconstructed. 
     Step 1: Preparation of the DNA Fragments: 
     1) The plasmid BS-By5-800 obtained by subcloning HindIII fragments of By5 (cf.  FIG. 2 ) into the Blue Scribe M13 +  plasmid (Stratagene), was digested with HindIII and a fragment of 800 bp was obtained and isolated from a 1% agarose gel by electroelution. 
     2) The plasmid BS-4.1 obtained by subcloning the 2.7 kb EcoRI insert from λgt11-17) into the Blue Scribe M13 +  plasmid (Stratagene) (see  FIG. 2  of patent application) was digested with HindIII and SphI and a fragment of 1500 bp was obtained and isolated from a 1% agarose gel by electroelution. 
     3) Blue Scribe M13 +  was digested with HindIII and SphI, and treated with calf intestine alkaline phosphatase (special quality for molecular biology, Boehringer Mannheim) as indicated by the manufacturer. 
     Step 2: Ligation: 
     The ligation reaction contained: 
     125 ng of the 800 bp HindIII fragment (1) 
     125 ng of the 1500 bp SphI-HindIII insert (2) 
     50 ng of the HindIII-SphI digested BSM13 +  vector (3) 
     2 μl of 10 ligation buffer (Maniatis et al., 1982) 
     1 μl of (=2.5 U) of T4 DNA ligase (Amersham) 
     4 μl PEG 6000, 25% (w/v) 
     8 μl H 2 O 
     The incubation was for 4 hours at 16° C. 
     Step 3: Transformation: 
     100 μl of DH5 α E. coli  (Gibco BRL) were transformed with 10 μl of the ligation reaction (step 2) and plated on IPTG, X-Gal ampicillin plates, as indicated by the manufacturer. About 70 white colonies were obtained. 
     Step 4: 
     As the 800 bp fragment could have been inserted in both orientations, plasmidic DNA from several clones were analyzed by digestion with PstI in order to select one clone (different from clone 11), characterized by the presence of 2 small fragments of 229 and 294 bp. This construction contains the HindIII-HindIII-SphI complex in the correct orientation. The plasmid containing this new construction was called: “BS.BK.P 32 .complet”. 
     EXAMPLE III 
     Expression of a Polypeptide of the Invention in  E. coli    
     The DNA sequence coding for a polypeptide, or part of it, can be linked to a ribosome binding site which is part of the expression vector, or can be fused to the information of another protein or peptide already present on the expression vector. 
     In the former case the information is expressed as such and hence devoid of any foreign sequences (except maybe for the aminoterminal methionine which is not always removed by  E. coli ). 
     In the latter case the expressed protein is a hybrid or a fusion protein. 
     The gene, coding for the polypeptide, and the expression vector are treated with the appropriate restriction enzyme(s) or manipulated otherwise as to create termini allowing ligation. The resulting recombinant vector is used to transform a host. The transformants are analyzed for the presence and proper orientation of the inserted gene. In addition, the cloning vector may be used to transform other strains of a chosen host. Various methods and materials for preparing recombinant vectors, transforming them to host cells and expressing polypeptides and proteins are described by Panayatatos, N., in “Plasmids, a practical approach (ed. K. G. Hardy, IRL Press) pp. 163–176, by Old and Primrose, principals of gene manipulation (2d Ed, 1981) and are well known by those skilled in the art. 
     Various cloning vectors may be utilized for expression. Although a plasmid is preferable, the vector may be a bacteriophage or cosmid. The vector chosen should be compatible with the host cell chosen. 
     Moreover, the plasmid should have a phenotypic property that will enable the transformed host cells to be readily identified and separated from those which are not transformed. Such selection genes can be a gene providing resistance to an antibiotic like for instance, tetracycline carbenicillin, kanamycin, chloramphenicol, streptomycin, etc. 
     In order to express the coding sequence of a gene in  E. coli  the expression vector should also contain the necessary signals for transcription and translation. 
     Hence it should contain a promoter, synthetic or derived from a natural source, which is functional in  E. coli . Preferably, although usually not absolutely necessary, the promoter should be controllable by the manipulator. Examples of widely used controllable promoters for expression in  E. coli  are the lac, the trp, the tac and the lambda PL and PR promoter. 
     Preferably, the expression vector should also contain a terminator of transcription functional in  E. coli . Examples of used terminators of transcription are the trp and the rrnB terminators. 
     Furthermore, the expression vector should contain a ribosome binding site, synthetic or from a natural source, allowing translation and hence expression of a downstream coding. sequence. Moreover, when expression devoid of foreign sequences is desired, a unique restriction site, positioned in such a way that it allows ligation of the sequence directly to the initiation codon of the ribosome binding site, should be present. 
     A suitable plasmid for performing this type of expression is pKK233-2 (Pharmacia). This plasmid contains the trc promoter, the lac Z ribosome binding site and the rrnB transcription terminator. 
     Also suitable is plasmid pIGRI (Innogenetics, Ghent, Belgium). This plasmid contains the tetracycline resistance gene and the origin of replication of pAT 153  (available from Bioexcellence, Biores B.V., Woerden, The Netherlands), the lambda PL promoter up to the MboII site in the 5′ untranslated region of the lambda N gene (originating from pPL(λ); Pharmacia). 
     Downstream from the PL promoter, a synthetic sequence was introduced which encodes a “two cistron” translation casette whereby the stop codon of the first cistron (being the first 25 amino acids of TNF, except for Leu at position 1 which is converted to Val) is situated between the Shine-Dalgarno sequence and the initiation codon of the second ribosome binding site. The restriction and genetic map of pIGRI is represented in  FIG. 10   a.    
       FIG. 10   b  and Table 5 represent respectively the nucleic acid sequence and complete restriction site analysis of pIGRI. 
     However, when expression as a hybrid protein is desired, then the expression vector should also contain the coding sequence of a peptide or polypeptide which is (preferably highly) expressed by this vector in the appropriate host. 
     In this case the expression vector should contain a unique cleavage site for one or more restriction endonucleases downstream of the coding sequence. 
     Plasmids pEX1, 2 and 3 (Boehringer, Mannheim) and pUEX1, 2 and 2 (Amersham) are useful for this purpose. 
     They contain an ampicillin resistance gene and the origin of replication of pBR322 (Bolivar at al. (1977) Gene 2, 95–113), the lac Z gene fused at its 5′ end to the lambda PR promoter together with the coding sequence for the 9 first amino acids of its natural gene cro, and a multiple cloning site at the 3′ end of the lac Z coding sequence allowing production of a beta galactosidase fused polypeptide. 
     The pUEX vectors also contain the CI857 allele of the bacteriophage lambda CI repressor gene. 
     Also useful is plasmid pmTNF MPH (Innogenetics). It contains the tetracycline resistance gene and the origin of replication of pAT 153  (obtainable from Bioexcellence, Biores B.V., Woerden. The Netherlands), the lambda PL promoter up to the MboII site in the N gene 5′ untranslated region (originating from pPL(λ); Pharmacia), followed by a synthetic ribosome binding site (see sequence data), and the information encoding the first 25 AA of mTNF (except for the initial Leu which is converted to Val). This sequence is, in turn, followed by a synthetic polylinker sequence which encodes six consecutive histidines followed by several proteolytic sites (a formic acid, CNBr, kallikrein, and  E. coli  protease VII sensitive site, respectively), each accessible via a different restriction enzyme which is unique for the plasmid (SmaI, NcoI, BspMII and StuI, respectively; see restriction and genetic map,  FIG. 11   a ). Downstream from the polylinker, several transcription terminators are present including the  E. coli  trp terminator (synthetic) and the rrnBT 1 T 2  (originating from pKK223-3; Pharmacia). The total nucleic acid sequence of this plasmid is represented in  FIG. 11   b.    
     Table 6 gives a complete restriction site analysis of pmTNF MPH. 
     The presence of 6 successive histidines allows purification of the fusion protein by Immobilized Metal Ion Affinity Chromatography (IMAC). 
     After purification, the foreign part of the hybrid protein can be removed by a suitable protein cleavage method and the cleaved product can then be separated from the uncleaved molecules using the same IMAC based purification procedure. 
     In all the above-mentioned plasmids where the lambda PL or PR promoter is used, the promoter is temperature-controlled by means of the expression of the lambda cI ts 857 allele which is either present on a defective prophage incorporated in the chromosome of the host (K12ΔH, ATCC no. 33767) or on a second compatible plasmid (pACYC derivative). Only in the pUEX vectors is this cI allele present on the vector itself. 
     It is to be understood that the plasmids presented above are exemplary and other plasmids or types of expression vectors maybe employed without departing from the spirit or scope of the present invention. 
     If a bacteriophage or phagemid is used, instead of plasmid, it should have substantially the same characteristics used to select a plasmid as described above. 
     EXAMPLE IV 
     Subcloning of the P32 Antigen in Plasmid pIGRI 
     Fifteen μg of plasmid “BS-BK-P 32  complet” (see Example II) was digested with EclXI and BstEII (Boehringer, Mannheim) according to the conditions recommended by the supplier except that at least 3 units of enzyme were used per μg of DNA. EclXI cuts at position 226 ( FIG. 5 ) and BstEII at position 1136, thus approaching very closely the start and stop codon of the mature P 32  antigen. This DNA is hereafter called DNA coding for the “P 32  antigen fragment”. 
     The DNA coding for the “P 32  antigen fragment” (as defined above) is subcloned in pIGRI (see  FIG. 10   a ) for expression of a polypeptide devoid of any foreign sequences. To bring the ATG codon of the expression vector in frame with the P 32  reading frame, an intermediary construct is made in pIG2 (for restriction and genetic map, see  FIG. 12   a ; DNA sequences, see  FIG. 12   b ; complete restriction site analysis, see Table 7). 
     Five μg of plasmid pIG2 is digested with NcoI. Its 5′ sticky ends are filled in prior to dephosphorylation. 
     Therefore, the DNA was incubated in 40 μl NB buffer (0.05 M Tris-Cl pH 7.4; 10 mM MgCl 2 ; 0.05% β-mercaptoethanol) containing 0.5 mM of all four dXTP (X=A,T,C,G) and 2 μl of Klenow fragment of  E. coli  DNA polymerase I (5 U/μl, Boehringer, Mannheim) for at least 3 h at 15° C. 
     After blunting, the DNA was once extracted with one volume of phenol equilibrated against 200 mM Tris-Cl pH 8, twice with at least two volumes of diethylether and finally collected using the “gene clean kit™” (Bio101) as recommended by the supplier. The DNA was then dephosphorylated at the 5′ ends in 30 μl of CIP buffer (50 mM TrisCl pH 8, 1 mM ZnCl 2 ) and 20 to 25 units of calf intestine phosphatase (high concentration, Boehringer, Mannheim). The mixture was incubated at 37° C. for 30 min, then EGTA (ethyleneglycol bis (β-aminoethylether)-N,N,N′,N′ tetraacetic acid) pH 8 is added to a final concentration of 10 mM. The mixture was then extracted with phenol followed by diethylether as described above, and the DNA was precipitated by addition of 1/10 volume of 3 M KAc (Ac=CH 3 COO) pH 4.8 and 2 volumes of ethanol followed by storage at −20° C. for at least one hour. 
     After centrifugation at 13000 rpm in a Biofuge A (Hereaus) for 5 min the pelleted DNA was dissolved in H 2 O to a final concentration of 0.2 μg/μl. 
     The EclXI-BstEII fragment, coding for the “P 32  antigen fragment” (see above) was electrophoresed on a 1% agarose gel (BRL) to separate it from the rest of the plasmid and was isolated from the gel by centrifugation over a Millipore HVLP filter (φ2 cm) (2 min, 13000 rpm, Biofuge at room temperature) and extracted with Tris equilibrated phenol followed by diethylether as described above. 
     The DNA was subsequently collected using the “Gene clean kit™” (Bio101) as recommended by the supplier. 
     After that, the 5′ sticky ends were blunted by treatment with the Klenow fragment of  E. coli  DNA polymerase I as described above and the DNA was then again collected using the “Gene clean kit™” in order to dissolve it in 7 μl of H 2 O. 
     One μl of vector DNA is added together with one μl of 10× ligase buffer (0.5 M TrisCl pH 7.4, 100 mM MgCl 2 , 5 mM ATP, 50 mM DTT (dithiothreitol)) and 1 μl of T4 DNA ligase (1 unit/μl, Boehringer, Mannheim). Ligation was performed for 6 h at 13° C. and 5 μl of the mixture is then used to transform strain DH1 (lambda) [strain DH1—ATCC No. 33849—lysogenized with wild type bacteriophage λ] using standard transformation techniques as described for instance by Maniatis et al. in “Molecular cloning, a laboratory manual”, Cold Spring Harbor Laboratory (1982). 
     Individual transformants are grown and lysed for plasmid DNA preparation using standard procedures (Experiments with gene fusions, Cold Spring Harbor Laboratory (1984) (T. J. Silhavy, H. L. Berman and L. W. Enquist, eds) and the DNA preparations are checked for the correct orientation of the gene within the plasmid by restriction enzyme analysis. 
     A check for correct blunting is done by verifying the restoration of the NcoI site at the 5′ and 3′ end of the antigen coding sequence. One of the clones containing the P 32  antigen fragment in the correct orientation is kept for further work and designated pIG 2 -Mt32. In this intermediary construct, the DNA encoding the antigen is not in frame with the ATG codon. However, it can now be moved as a NcoI fragment to another expression vector. 
     15 μg of pIG 2 -Mt32 is digested with NcoI. The NcoI fragment encoding the P 32  antigen is gel purified and blunted as described above. After purification, using “gene clear kit TM” it is dissolved in 7 μl of H 2 O. 
     5 μg of plasmid pIGRI is digested with NcoI, blunted and dephosphorylated as described above. After phenol extraction, followed by diethylether and ethanol precipitation, the pellet is dissolved in H 2 O to a final concentration of 0.2 μg/μl. 
     Ligation of vector and “antigen fragment” DNA is carried out as described above. The ligation mixture is then transformed into strain DH1 (lambda) and individual transformants are analysed for the correct orientation of the gene within the plasmid by restriction enzyme analysis. A check for correct blunting is done by verifying the creation of a new NsiI site at the 5′ and 3′ ends of the antigen coding sequence. One of the clones containing the P 32  antigen fragment in the correct orientation is kept for further work and designated pIGRI.Mt32. 
     EXAMPLE V 
     Subcloning of the P32 Antigen in pmTNF MPH 
     Fifteen μg of the plasmid pIG2 Mt32 (see example IV) was digested with the restriction enzyme NcoI (Boehringer, Mannheim), according to the conditions recommended by the supplier except that at least 3 units of enzyme were used per μg of DNA. 
     After digestion, the reaction mixture is extracted with phenol equilibrated against 200 mM TrisCl pH 8, (one volume), twice with diethylether (2 volumes) and precipitated by addition of 1/10 volume of 3 M KAc (Ac=CH 3 COO) pH 4.8 and 2 volumes of ethanol followed by storage at −20° C. for at least one hour. 
     After centrifugation for 5 minutes at 13000 rpm in a Biofuge A (Hereaus) the DNA is electrophoresed on a 1% agarose gel (BRL). 
     The DNA coding for the “P 32  antigen fragment” as described above, is isolated by centrifugation over a Millipore HVLP filter (φ2 cm) (2 minutes, 13000 rpm, Biofuge at room temperature) and extracted one with trisCl equilibrated phenol and twice with diethylether. The DNA is subsequently collected using “Gene clean kit™” (Bio 101) and dissolved in 7 μl of H 2 O. 
     The 5′ overhanging ends of the DNA fragment generated by digestion with NcoI were filled in by incubating the DNA in 40 μl NB buffer (0.05 M Tris-HCl, pH 7.4; 10 mM MgCl 2 ; 0.05% β-mercaptoethanol) containing 0.5 mM of all four dXTPS (X=A, T, C, G) and 2 μl of Klenow fragment of  E. coli  DNA polymerase I (5 units/μl Boehringer Mannheim) for at least 3 h at 15° C. After blunting, the DNA was extracted with phenol, followed by diethylether, and collected using a “gene clean kit™” as described above. 
     Five μg of plasmid pmTNF MPH is digested with StuI, subsequently extracted with phenol, followed by diethylether, and precipitated as described above. The restriction digest is verified by electrophoresis of a 0.5 μg sample on an analytical 1.2% agarose gel. 
     The plasmid DNA is then desphosphorylated at the 5′ ends to prevent self-ligation in 30 μl of CIP buffer (50 mM TrisCl pH 8, 1 mM ZnCl2) and 20 to 25 units of calf intestine phosphatase (high concentration, Boehringer Mannheim). The mixture is incubated at 37° C. for 30 minutes, then EGTA (ethyleneglycol bis (β-aminoethylether)-N,N,N′,N′ tetraacetic acid) pH8 is added to a final concentration of 10 mM. The mixture is extracted with phenol followed by diethylether and the DNA is precipitated as described above. The precipitate is pelleted by centrifugation in a Biofuge A (Hereaus) at 13000 rpm for 10 min at 4° C. and the pellet is dissolved in H 2 O to a final DNA concentration of 0.2 μg/μl. 
     One μl of this vector DNA is mixed with the 7 μl solution containing the DNA fragment coding for the “P32antigen fragment” (as defined above) and 1 μl 10× ligase buffer (0.5 M TrisCl pH7.4, 100 mM MgCl2, 5 mM ATP, 50 mM DTT (dithiothreitol)) plus 1 μl T 4  DNA ligase (1 unit/μl, Boehringer Mannheim) is added. The mixture is incubated at 13° C. for 6 hours and 5 μl of the mixture is then used for transformation into strain DH1 (lambda) using standard transformation techniques are described by for instance Maniatis et al. in “Molecular cloning, a laboratory manual”, Cold Spring Harbor Laboratory (1982). 
     Individual transformants are grown and then lysed for plasmid DNA preparation using standard procedures (Experiments with gene fusions, Cold Spring Harbor Laboratory 1984 (T. J. Silhavy, M. L. Berman and L. W. Enquist eds.)) and are checked for the correct orientation of the gene within the plasmid by restriction enzyme analysis. 
     One of the clones containing the DNA sequence encoding the antigen fragment in the correct orientation was retained for further work and designated pmTNF-MPH-Mt32. It encodes all information of the P 32  antigen starting from position +4 in the amino acid sequence (see  FIG. 5 ). The amino acid sequence of the total fusion protein is represented in  FIG. 13 . 
     EXAMPLE VI 
     Induction of Antigen Expression from pmTNF MPH Mt32 
     A—Material and Methods 
     DNA of pmTNF-MPH-Mt32 is transformed into  E. coli  strain K12ΔH (ATCC 33767) using standard transformation procedures except that the growth temperature of the cultures is reduced to 28° C. and the heat shock temperature to 34° C. 
     A culture of K12ΔH harboring pmTNF-MPH-Mt32, grown overnight in Luria broth at 28° C. with vigorous shaking in the presence of 10 μg/ml tetracycline, is inoculated into fresh Luria broth containing tetracycline (10 μg/ml) and grown to an optical density at 600 nanometers of 0.2 in the same conditions as for the overnight culture. 
     When the optical density at 600 nanometers has reached 0.2 half of the culture is shifted to 42° C. to induce expression while the other half remains at 28° C. as a control. At several time intervals aliquots are taken which are extracted with one volume of phenol equilibrated against M9 salts (0.1% ammonium chloride, 0.3% potassium dihydrogenium phosphate, 1.5% disodium hydrogenium phosphate, 12 molecules of water) and 1% SDS. At the same time, the optical density (600 nm) of the culture is checked. The proteins are precipitated from the phenol phase by addition of two volumes of acetone and storage overnight at −20° C. The precipitate is pelleted (Biofuge A, 5 min., 13000 rpm, room temperature) dried at the air, dissolved in a volume of Laemmli (Nature (1970) 227:680) sample buffer (+β mercapto ethanol) according to the optical density and boiled for 3 min. 
     Samples are then run on a SDS polyacrylamide gel (15%) according to Laemmli (1970). Temperature induction of mTNF-His 6 -P 32  is monitored by both Coomassie Brilliant Blue (CBB) staining and immunoblotting. CBB staining is performed by immersing the gel in a 1/10 diluted CBB staining solution (0.5 g CBB-R250 (Serva) in 90 ml methanol: H 2 O (1:1 v/v) and 10 ml glacial acetic acid) and left for about one hour on a gently rotating platform. After destaining for a few hours in destaining solution (30% methanol, 7% glacial acetic acid) protein bands are visualised and can be scanned with a densitometer (Ultroscan XL Enhanced Laser Densitometer, LKB). 
     For immunoblotting the proteins are blotted onto Hybond C membranes (Amersham) as described by Townbin et al (1979). After blotting, proteins on the membrane are temporarily visualised with Ponceau S (Serva) and the position of the molecular weight markers is indicated. The stain is then removed by washing in H 2 O. A specific protein binding sites are blocked by incubating the blots in 10% non-fat dried milk for about 1 hour on a gently rotating platform. After washing twice with NT buffer (25 mM Tris-HCl, pH 8.0; 150 mM NaCl) blots are incubated with polyclonal rabbit anti-32-kDa antiserum (1:1000), obtained as described in example I (“screening of the λgt11  M. tuberculosis  recombinant DNA library with anti-32-kDa antiserum”) in the presence of  E. coli  lysate or with monoclonal anti-hTNF-antibody which crossreacts with mTNF (Innogenetics, no. 17F5D10) for at least 2 hours on a rotating platform. After washing twice with NT buffer+0.02% Triton.X.100, blots are incubated for at least 1 hour with the secondary antiserum alkaline phosphatase-conjugated swine anti-rabbit immunoglobulins (1/500; Prosan) in the first case, and alkaline phosphatase conjugated rabbit anti-mouse immunoglobulins (1/500; Sigma) in the second case. 
     Blots are washed again twice with NT buffer+0.02% Triton X100 and visualisation is then performed with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) from Promega using conditions recommended by the supplier. 
     B. Results 
     Upon induction of K12ΔH cells containing pmTNF-MPH-Mt32, a clearly visible band of about 35-kDa appears on CBB stained gels, already one hour after start of induction ( FIG. 14   a ). This band, corresponding to roughly 25% of total protein contents of the cell, reacts strongly with anti-32-kDa and anti-mTNF antisera on immunoblots ( FIG. 14   b ). However, this band represents a cleavage product of the original fusion protein, since a minor band, around 37 kDa, is also visible on immunoblots, reacting specifically with both antisera as well. This suggests that extensive cleavage of the recombinant mTNF-His 6 -P 32  takes place about 2–3 kDa from its carboxyterminal end. 
     EXAMPLE VII 
     Purification of Recombinant Antigen on Immobilized Metal Ion Affinity Chromatography (IMAC) 
     The hybrid protein mTNF-His 6 -P 32  (amino acid sequence, see  FIG. 13 ) expressed by K12ΔH cells containing pmTNF.MPH.Mt32, is especially designed to facilitate purification by IMAC, since the 6 successive histidines in the polylinker sequence bring about a strong affinity for metal ions (HOCHULI et al, 1988). 
     a. Preparation of the Crude Cell Extract: 
     12 l of  E. coli  cells K12ΔH containing plasmid pmTNF-MPH-Mt32 were grown in Luria Broth containing tetracycline (10 μg/ml) at 28° C. to an optical density (600 nm) of 0.2 and then induced by shifting the temperature to 42° C. After 3 hours of induction, cells were harvested by centrifugation (Beckman, JA 10 rotor, 7,500 rpm, 10 min). The cell paste was resuspended in lysis buffer (10 mM KCl, 10 mM Tris-HCl pH 6.8, 5 mM EDTA) to a final concentration of 50% (w/v) cells. 
     ε-NH 2 -capronic acid and dithiotreitol (DTT) were added to a final concentration of resp. 20 mM and 1 mM, to prevent proteolytic degradation. This concentrated cell suspension was stored overnight at −70° C. 
     Cells were lysed by passing them three times through a French press (SLM-Aminco) at a working pressure of 800–1000 psi. During and after lysis, cells were kept systematically on ice. 
     The cell lysate was cleared by centrifugation (Beckman, JA 20, 18,000 rpm, 20 min, 4° C.). The supernatant (SN) was carefully taken off and the pellet, containing membranes and inclusion bodies, was kept for further work since preliminary experiments had shown that the protein was mainly localised in the membrane fraction. 
     7 M guanidinium hydrochloride (GuHCl, marketed by ICN) in 100 mM phosphate buffer pH 7.2 was added to the pellet volume to a final concentration of 6 M GuHCl. The pellet was resuspended and extracted in a bounce tissue homogenizer (10 cycles). 
     After clearing (Beckman, JA 20, 18,000 rpm, 20 min, 4° C.), about 100 ml of supernatant was collected (=extract 1) and the removing pellet was extracted again as described above (=extract 2, 40 ml). 
     The different fractions (SN,EX1,EX2) were analysed on SDS-PAGE (Laemmli, Nature 1970; 227:680) together with control samples of the induced culture. Scanning of the gel revealed that the recombinant protein makes up roughly 25% of the total protein content of the induced cell culture. After fractionation most of the protein was found back in the extracts. No difference was noticed between reducing and non-reducing conditions (plus and minus β-mercaptoethanol). 
     b. Preparation of the Ni ++  IDA (Imino Diacetic Acid) Column: 
     5 ml of the chelating gel, Chelating Sepharose 6B (Pharmacia) is washed extensively with water to remove the ethanol in which it is stored and then packed in a “Econo-column” (1×10 cm, Biorad). The top of the column is connected with the incoming fluid (sample, buffer, etc) while the end goes to the UV 280  detector via a peristaltic jump. Fractions are collected using a fraction collector and, when appropriate, pH of the fractions is measured manually. 
     The column is loaded with Ni ++  (6 ml NiCl 2 .6H 2 O; 5 μg/μl) and equilibrated with starting buffer (6 M guanidinium hydrochloride, 100 mM phosphate buffer, pH 7.2). 
     After having applied the sample, the column is washed extensively with starting buffer to remove unbound material. 
     To elute the bound material, 2 different elution procedures are feasible: 
     1) elution by decreasing pH, 
     2) elution by increasing imidazol concentration. 
     Both will be discussed here. 
     To regenerate the column, which has to be done after every 2–3 runs, 20 ml (about 5 column volumes) of the following solutions are pumped successively through the column:
         0.05 M EDTA-0.5 M NaCl   0.1 M NaOH   H 2 O   6 ml NiCl 2 .6H 2 O (5 mg/ml).       

     After equilibrating with starting buffer the column is ready to use again. 
     c. Chromatography: 
     All buffers contained 6 M guanidinium hydrochloride throughout the chromatography. The column was developed at a flow rate of 0.5 ml/min at ambient temperature. Fractions of 2 ml were collected and, when appropriate, further analysed by SDS-PAGE and immunoblotting. Gels were stained with Coomassie Brilliant Blue R250 and silver stain, as described by ANSORGE (1985). Immunoblotting was carried out as described in example I. The primary antiserum used was either polyclonal anti-32kDa-antiserum (1/1000) obtained as described in example I (“screening of the λgt11  M. tuberculosis  recombinant DNA library with anti-32kDa-antiserum”) or anti- E. coli -immunoglobulins (1/500; PROSAN), or monoclonal anti-hTNF-antibody which cross-reacts with mTNF (Innogenetics, No. 17F5D10). The secondary antiserum was alkaline phosphatase conjugated swine anti-rabbit immunoglobulins (1/500, PROSAN), or alkaline phosphatase conjugated rabbit-anti-mouse immunoglobulins (1/500, Sigma). 
     C1. Elution with Decreasing pH: 
     Solutions used: 
     A: 6 M GuHCl 100 mM phosphate pH 7.2 
     B: 6 M GuHCl 25 mM phosphate pH 7.2 
     C: 6 M GuHCl 50 mM phosphate pH 4.2 
     After applying 3 ml of extract 1 (OD 280 =32.0) and extensively washing with solution A, the column is equilibrated with solution B and then developed with a linear pH gradient from 7.2 to 4.2 (25 ml of solution B and 25 ml of solution C were mixed in a gradient former). The elution profile is shown in  FIG. 15 . 
     From SDS-PAGE analysis (Coomassie and silverstain) it was clear that most of the originally bound recombinant protein was eluted in the fractions between pH 5.3 and 4.7. 
     Screening of these fractions on immunoblot with anti-32-kDa and the 17F5D10 monoclonal antibody showed that, together with the intact recombinant protein, also some degradation products and higher aggregation forms of the protein were present, although in much lower amount. Blotting with anti- E. coli  antibody revealed that these fractions (pH 5.3–4.7) still contained immunodetectable contaminating  E. coli  proteins (75, 65, 43, 35 and 31 kDa bands) and lipopolysaccharides. 
     C2. Elution with Increasing Imidazol Concentration: 
     Solutions used: 
     A: 6 M GuHCl 100 mM phosphate pH 7.2 
     B: 6 M GuHCl 50 mM imidazol pH 7.2 
     C: 6 M GuHCl 100 mM imidazol pH 7.2 
     D: 6 M GuHCl 15 mM imidazol pH 7.2 
     E: 6 M GuHCl 25 mM imidazol pH 7.2 
     F: 6 M GuHCl 35 mM imidazol pH 7.2 
     Sample application and washing was carried out as in C1, except that after washing, no equilibration was necessary with 6 M GuHCl 25 mM phosphate. The column was first developed with a linear gradient of imidazol going from 0 to 50 mM (25 ml of solution A and 25 ml of solution B were mixed in a gradient former) followed by a step elution to 100 mM imidazol (solution C). During the linear gradient, proteins were gradually eluted in a broad smear, while the step to 100 mM gave rise to a clear peak ( FIG. 16 ). 
     SDS-PAGE analysis of the fractions revealed that in the first part of the linear gradient (fr 1–24) most contaminating  E. coli  proteins were washed out, while the latter part of the gradient (fr 25–50) and the 100 mM peak contained more than 90% of the recombinant protein. 
     As in C1, these fractions showed, besides a major band of intact recombinant protein, some minor bands of degradation and aggregation products. However, in this case, the region below 24-kDa seemed nearly devoid of protein bands, which suggests that less degradation products co-elute with the intact protein. Also, the same contaminating  E. coli  proteins were detected by immunoblotting, as in C1, although the 31-kDa band seems less intense and even absent in some fractions. 
     In a second stage, we developed the column with a step gradient of increasing imidazol concentrations. After having applied the sample and washed the column, 2 column volumes (about 8 ml) of the following solutions were brought successively onto the column solution D, E, F and finally 4 column volumes of solution C. The step gradient resulted in a more concentrated elution profile ( FIG. 17 ) which makes it more suitable for scaling up purposes. 
     In conclusion, the mTNF-His 6 -P 32  protein has been purified to at least 90% by IMAC. Further purification can be achieved through a combination of the following purification steps:
         IMAC on chelating superose (Pharmacia)   ion exchange chromatography (anion or cation)   reversed phase chromatography   gel filtration chromatography   immunoaffinity chromatography   elution from polyacrylamide gel.       

     These chromatographic methods are commonly used for protein purification. 
     The plasmids of  FIGS. 10   b ,  11   b  and  12   b  are new. 
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