# Patent

## Title
32-kDa protein derived from Mycobacterium tuberculosis and related peptides

## 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. 4a and FIG. 4b. 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.

## Background
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 P32 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 NH2-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 P32 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 NH2-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 P32 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 

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. 3a and FIG. 3b. 


On FIGS. 3a and 3b: 

    
    
        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,
        a1-b1 represents ALA-ARG or GLY-ALA-ALA,
        a2 represents arg or gly,
        a3-b3-c3-d3-e3-f3- represents his-trp-val-pro-arg-pro or ala-leu-gly-ala,
        a4 represents pro or pro-asn-thr,
        a5 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 

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. 4a and FIG. 4b. 


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 “P32 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 P32 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 P32 protein of BCG.
1) process for preparing rabbit polyclonal antiserum raised against the P32 protein of BCG:
Purified P32 protein of BCG from culture filtrate is used.
a) Purification of protein P32 of BCG:
P32 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 K2HPO4, 0.5 g MgSO4, 0.05 g Iron Citrate, 5×10−6 M Ammonium (17% Fe III) SO4Zn-7H2O and adjusted to 1 liter distilled water adjusted to pH 7.2 with NH4OH, 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 P32 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 P32 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 P32 protein of BCG:
400 μg of purified P32 protein of BCG per ml physiological saline are mixed with one volume of incomplete Freund'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 P32 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% H2O2 per 100 ml) in 0.15 M citrate buffer (pH 5.0) as a substrate. The enzymatic reaction is stopped with 8 N H2SO4 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 NH2 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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b. 


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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b. 


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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b. 


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. 3a and FIG. 3b 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 
    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. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 
    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. 4a and FIG. 4b. 


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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b, 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. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b, 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. 4a and FIG. 4b. 


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. 3a and FIG. 3b.















 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. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    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. 3a and FIG. 3b, 

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. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    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. 4a and FIG. 4b, 

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. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b. 


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. 3a and FIG. 3b. 


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. 4a and FIG. 4b. 


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. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b. 


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. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b. 


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. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b. 


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. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
    the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b. 


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. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b. 


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. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
    the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b. 


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


(NH2-

(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


(NH2-

(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


(NH2-

(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


(NH2-

(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. 3a and 3b 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. 4a and 4b 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 NH2-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 NH2 end of clone 17 and clone 24. The option taken here arbitrarily represents the 59 amino acid signal peptide corresponding to ATG183.

FIGS. 5a–5c 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 NH2-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 NH2 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. 7a–7b 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 ATG91. 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 pUEX2 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. 9a–9d 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. 4a and 4b 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. 9a 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. 9b 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. 9c 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. 9d 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. 3a and FIG. 3b, and of FIG. 5, because nucleotide at position 1 (on FIG. 9) corresponds to nucleotide 234 on FIG. 3a, and corresponds to nucleotide 91 on FIG. 5.
FIG. 10a corresponds to the restriction and genetic map of the pIGRI plasmid used in Example IV for the expression of the P32 antigen of the invention in E. coli and contains SEQ ID NO:50.
On this figure, underlined restriction sites are unique.
FIGS. 10b–10m 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
rrnBT1T2 containing HindIII-SspI



fragment from pKK223 (Pharmacia)


 891–3421
DraI-EcoRI blunt fragment of pAT153



(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. 11a corresponds to the restriction and genetic map of the pmTNF MPH plasmid used in Example V for the expression of the P32 antigen of the invention in E. coli and contains SEQ ID NO:51 and SEQ ID NO:52.
FIGS. 11b–11m 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



His6 encoding sequence at position



315–332


385–436
HindIII fragment containing E. coli



trp terminator


437–943
rrnBT1T2 containing HindIII-SspI



fragment from pKK223 (Pharmacia)


 944–3474
DraI-EcoRI blunt fragment of pAT153



(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. 12a 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 P32 antigen in plasmid pIGRI and contains SEQ ID NO:53.
FIGS. 12b–12l 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
rrnBT1T2 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-His6-P32 (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. 14a and 14b correspond to the expression of the mTNF-His6-P32 fusion protein in K12ΔH, given in Example VI, with FIG. 14a representing the Coomassie Brilliant Blue stained SDS-PAGE and 14b representing immunoblots of the gel with anti-32-kDa and anti-mTNF-antibody.
On FIG. 14a, 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. 14b, 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 (105 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 P32 M. bovis BCG (strain 1173P2—Institut Pasteur Paris) as follows: 400 μg (purified P32 protein of M. bovis BCG) per ml physiological saline were mixed with one volume of incomplete Freund'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 α32P-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 1.5×106 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. 3a and FIG. 3b. 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 NH2-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 NH2-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 NH2-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 ATG91 or ATG52 because both are preceded by a plausible E. coli-like promoter and a Shine-Dalgarno motif.
If initiation takes place at ATG91, 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 ATG52, 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's.
Upstream ATG130, 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 ATG91, 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. 3a and 3b. It comprises a TTGGCC at position 59 (FIGS. 3a and 3b) (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 NH2-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. 3a and 3b) 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 H2O
The incubation was for 4 hours at 16° C.
Step 3: Transformation:
100 μl of DH5α 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.P32.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 pAT153 (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. 10a. 
FIG. 10b 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 pAT153 (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. 11a). Downstream from the polylinker, several transcription terminators are present including the E. coli trp terminator (synthetic) and the rrnBT1T2 (originating from pKK223-3; Pharmacia). The total nucleic acid sequence of this plasmid is represented in FIG. 11b. 
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-P32 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 P32 antigen. This DNA is hereafter called DNA coding for the “P32 antigen fragment”.
The DNA coding for the “P32 antigen fragment” (as defined above) is subcloned in pIGRI (see FIG. 10a) for expression of a polypeptide devoid of any foreign sequences. To bring the ATG codon of the expression vector in frame with the P32 reading frame, an intermediary construct is made in pIG2 (for restriction and genetic map, see FIG. 12a; DNA sequences, see FIG. 12b; 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 MgCl2; 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 kitTM” (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 ZnCl2) 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=CH3COO) 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 H2O to a final concentration of 0.2 μg/μl.
The EclXI-BstEII fragment, coding for the “P32 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 kitTM” (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 kitTM” in order to dissolve it in 7 μl of H2O.
One μl of vector DNA is added together with one μl of 10× ligase buffer (0.5 M TrisCl pH 7.4, 100 mM MgCl2, 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 P32 antigen fragment in the correct orientation is kept for further work and designated pIG2-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 pIG2-Mt32 is digested with NcoI. The NcoI fragment encoding the P32 antigen is gel purified and blunted as described above. After purification, using “gene clear kit TM” it is dissolved in 7 μl of H2O.
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 H2O 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 P32 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=CH3COO) 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 “P32 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 kitTM” (Bio 101) and dissolved in 7 μl of H2O.
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 MgCl2; 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 kitTM” 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 H2O 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 T4 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 P32 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-His6-P32 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: H2O (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 H2O. 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. 14a). 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. 14b). 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-His6-P32 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-His6-P32 (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-NH2-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 UV280 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 NiCl2.6H2O; 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
        H2O
        6 ml NiCl2.6H2O (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 (OD280=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-His6-P32 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. 10b, 11b and 12b are new.
BIBLIOGRAPHY


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## Claims

1. A composition comprising an isolated polypeptide, wherein the polypeptide comprises an amino acid sequence set forth in LYLLDGLRAQDDFSGWDINT (SEQ ID NO:25).

2. The composition of claim 1, further comprising a pharmaceutically acceptable vehicle.

3. The composition of claim 1, wherein the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:25.

4. The composition of claim 3, further comprising a pharmaceutically acceptable vehicle.

5. The composition of claim 1, wherein the polypeptide further comprises a cysteine residue added at an amino or carboxyl terminal end of the polypeptide.

6. The composition of claim 1, wherein the polypeptide further comprises a tyrosine residue added at an amino or carboxyl terminal end of the polypeptide.

7. The composition of claim 1, further comprising a heterologous polypeptide sequence comprising 1 to 1000 amino acids.

8. The composition of claim 7, further comprising a pharmaceutically acceptable vehicle.

9. The composition of claim 7, wherein the heterologous polypeptide is a natural or synthetic carrier polypeptide of sufficient molecular weight for the composition to induce a cellular immune response when administered to a mammal.

10. The composition of claim 9, wherein the composition induces a cellular immune response by activating Mycobacterium tuberculosis antigen-responsive T-cells.

11. The composition of claim 9, wherein the composition induces the production of antibodies against Mycobacterium tuberculosis.

12. An immunogenic conjugate comprising a first polypeptide coupled to a second polypeptide, wherein the first polypeptide comprises an amino acid sequence set forth in SEQ ID NO:25, and the second polypeptide is a natural or synthetic polypeptide of sufficient molecular weight for the conjugate to induce a cellular immune response when administered to a mammal.

13. The conjugate of claim 12, wherein the conjugate induces a cellular immune response by activating Mycobacterium tuberculosis antigen-responsive T-cells.

14. The conjugate of claim 12, wherein the composition induces the production of antibodies against Mycobacterium tuberculosis.

15. The conjugate of claim 12, further comprising a pharmaceutically acceptable vehicle.

16. The conjugate of claim 12, wherein the first polypeptide consists of the amino acid sequence set forth in SEQ ID NO:25.