Abstract:
The present invention refers to recombinant DNA molecules encoding fusion peptides from different allergens from  Blomia tropicalis  and  Dermatophagoides pteronyssinus  having potential usefulness in prevention and treatment of allergies caused by domestic mites. Specifically, the invention discloses fusion proteins comprising different fragments of allergens Der p 1, Der p 2, Der p 7, Der p 8, Blo t 5, Blo t 8, Blo t 18, Blo t 12 and Blo t 13 with reduced serum IgE reactivity in allergic and non-allergic individuals. It also discloses methods for production of these molecules in an expression system based on  E. coli  and purification. The invention refers also to effective and safe vaccines.

Description:
This application is a National Stage Application of PCT/IB2012/054994, filed 20 Sep. 2012 and which application is incorporated herein by reference. To the extent appropriate, a claim of priority is made to the above disclosed application. 
     SEQUENCE LISTING 
     The present application includes a Sequence Listing in electronic format as a txt file entitled “Sequence-Listing”, which was created on 9 May 2013 and is 9.31 KB (9,530 bytes) in size. The contents of txt file “Sequence-Listing” were filed electronically with the application on 9 May 2013 and are incorporated herein by reference. 
     TECHNICAL FIELD 
     The present invention relates to design of synthetic nucleotide sequences and to production of recombinant fusion proteins for treatment and prevention of allergies caused by domestic mites, in particular those caused by species  Blomia tropicalis  and  Dermatophagoides pteronyssinus.    
     BACKGROUND OF THE INVENTION 
     Individuals allergic to domestic mites are in general sensitive to mites of genus  Dermatophagoides , being  D. pteronyssinus  and  D. farinae  the most prevalent species worldwide. Additionally, in tropical and sub-tropical regions the presence of  B. tropicalis  is very abundant, being predominant sometimes in the population of mites present in house dust. In these regions, individuals allergic to mites are sensitive mostly to  D. pteronyssinus  and  B. tropicalis  (1). 
     About twenty allergens have been identified among domestic mites. However, only some of those have shown high reactivity frequency in the allergic population and some show cross-reactivity. Amongst  B. tropicalis  allergens, Blo t5 shows the highest reactivity frequency, Blo t12 and Blo t 13 show lower frequency, but induce an intense IgE in many of the sensitive individuals (2, 3). 
     Others as Der p1 and Der p2 are the most important  D. Pteronyssinus , and between 50% and 100% of allergic individuals have IgE specific against these two allergens. 
     Another  D. Pteronyssinus  important allergen is Der p7, being estimated that 50% of individuals allergic to domestic mites shows IgE against this allergen. Allergens of mite groups 8 and 10, such as glutathione S transferase (GST) and tropomyosin, respectively, are of particular importance due to the significant crossed-reactivity observed with homologous derivate from different mite species and other organisms (4). 
     An allergen must produce cross-linking of IgE antibodies linked to the effector cells surface in order to induce cell activation and inflammatory response, requiring this process at least two IgE epitopes on the allergen surface. The IgE antibodies of an allergic patient may recognize continuous epitopes or discontinuous epitopes. Cross-linking of IgE antibodies on mast cells surface induce immediate release of biologically active mediators such as histamine and leukotrienes. 
     Decrease of allergenic capability can be achieved by intervention of IgE epitopes through mechanisms such as point mutation, amino acid deletion and disturbance of the natural molecular bending. Reordering amino acid segments conforming the primary sequence of the allergen also modifies the allergenic capability. 
     In some studies fusion or hybrid proteins have been generated to provide a better allergen-specific immunotherapy. Fusion proteins have been generated by using several portions of different allergens linked to form a unique molecule, intended to obtain vaccines poorly reactive to IgE antibody, which decreases the anaphylaxis risks, and to deliver to the patient only those allergens involved in development of allergic response (5). 
     A number of patents is known in this field related to pollen allergens (U.S. Pat. No. 7,862,828) and to domestic mites, as shown in documents US20080274059; US20110052640; US20070065468; US20060233839; US20090130130 and WO2007140505. 
     In fact, document U.S. Pat. No. 7,862,828 describes a method to prepare fusion allergens, consisting of two or more recombinant polypeptides from grass pollen, to be used as immunotherapeutic agent comprising: (a) to provide a polynucleotide sequence which codifies the fusion allergen, (b) to introduce said polynucleotide sequence into the host cell, (c) to grow the host cell to express the sequence, (d) to recover the fusion allergen expressed from the grown cell, (e) to assay the fusion allergen as a candidate for immunotherapy, by delivering the allergen to a test animal and to select those inducing the strongest immune response and generation of IgE blocking antibodies in comparison to that obtained with the individual components. 
     On this case, the polypeptide has at least two different allergenic proteins or their fragments. The hybrid polypeptide, the polynucleotide and the cell are useful for drug preparation for treatment of allergenic disorders or for prophylactic vaccination. Among allergenic sources for allergenic proteins, grass pollen, mites, bee venom or animal hair allergens may be the most important. Specific examples of allergenic proteins are group 1, group 2, group 4, group 5, group 6, group 11, group 12 and group 13 of allergens from the most important grass pollen, Der p 1 and Der p 2 (mites), phospholipase from bee venom, and Fel d 1 (cat). 
     On the other hand, document US20080274059 reveals fusion protein comprising a group 1 allergen and a group 2 allergen from genus  Dermatophagoides  fused at group 1 allergen N— o C—, where they may be Der p 1 or proDer p 1, and, from group 2, where they may be Der p 2. This document includes claims for the nucleic acid to develop the fusion protein, the expression cassette, the vector containing this cassette and a cell of any type including cassette or expression vector, and a vaccine composition including the fusion protein on a pharmaceutically acceptable support. The fusion protein is applied to prevent or treat allergic reactions to mites. The expression cell may be  Escherichia coli, Pichia pastoris  or  Saccharomyces cerevisiae.    
     The United States Patent Application 20110052640 reports a hypoallergenic hybrid polypeptide comprising an amino acid sequence of at least 50 amino acids of a sequence selected from group 1 and 2 allergens from dust mites ( Dermatophagoides pteronyssinus ), and where a linkage epitope to IgE antibody of said allergen is deleted. Included are either the polypeptide having 70% sequence identity, the corresponding polynucleotide, the vector, the expression host cell including the polynucleotide, the method to produce the polypeptide and the pharmaceutical composition that comprises the polypeptide. The group 1 allergen is Der p 1, and that of group 2 is Der p 2. The obtained polypeptide keeps immunogenic capabilities, being particularly useful for allergy treatment. Additionally, production methods for these polypeptides in heterologous expression systems and efficient purification methods are described 
     Patent US20070065468 reports a product for specific reduction of immunoglobulin E (IgE) as well as the allergic reaction, specifically a chimeric polypeptide having at least two mite allergens. It includes amino acid sequence for those allergens such as Der p 1, Der p 2 and Blo t 5. Allergens can be expressed in  Escherichia coli  or CHO—K1. 
     Patent US20060233839 describes a recombinant protein with an allergen derived from Der p1 where the protein has a reduced allergenic activity, important in relation to the allergen in natural state and comprising three mutation sites. In addition to Der p 1 allergen, ProDer p 1 and Der p 3 with ProDer p 3 and PreProDer p 3 are included. Claims include the nucleic acid molecule, the expression vector, the cell transformed for production of recombinant protein, and the immunological composition used for patient treatment. 
     Document US20090130130 refers to an isolated polypeptide, derivative or fragment composed by (a) a polypeptide, its derivate isoform, or a fragment thereof, comprising a non-helicoidal mutant of at least an allergen from group 5 mites and, (b) a polypeptide, its derivate isoform, or a fragment thereof comprising at least a substituted, added or deleted amino acid, or at least a chemical modification, where the polypeptide exhibits reduction in reactivity equal to or higher than IgE from the natural polypeptide, in subjects allergic to at least one allergen from group 5 mites. This polypeptide from the group 5 allergens may be Blo t 5, Der p 5, Der f 5, or Der m 5. Claims include nucleic acids sequences, a vector/host cell comprising nucleic acid molecules, a pharmaceutical preparation including the polypeptide or a vaccine. 
     Patent WO2007140505 reports a hypoallergenic protein comprising an allergen fused to or conjugated with at least a second non-allergenic protein or a fragment thereof. Included are claims for nucleic acid molecules encoding a hypoallergenic molecule or fusion protein, the vector, the host cell, an antibody addressed against the hypoallergenic or fusion protein, and the vaccine which may comprise it. The protein is derivate from Phl p 5, Fel d 1, Der p 2, Der p 7, Der p 21, Clone 30, Alt a 1, Par j 1, Ole e 1, Fel d 2, Can f 1, Can f 1, Art v 1, Amb a1, Alt a 2 or Alt a 6 having a terminal blunt in C- or N-terminus, and exhibiting reduced capability to IgE in comparison to native Phl p 5. The second protein is a viral, bacterial, fungal, or protozoan protein. For example, it is a virus capsid protein, preferably from picornaviridae. 
     Additionally, it is known that Linhart and collaborators (6) built, by PCR-based recombination, a fusion molecule composed by the most important allergens from timothy grass, in the order Phl p6, Phl p2, Phl p5 y Phl p1, which was expressed in  E. coli  as a 79 kDa protein. This molecule demonstrated to be useful for immunotherapy since it induced lymphoproliferation in a degree similar to that induced by a equimolar mix of the individual allergens in an model of mice allergic to pollen, induced Thl and IgG profile cytokine production, able to block degranulation of mast cells incubated in the presence of the allergen. 
     The concept of fusion proteins has been also explored with mite allergens; a fusion protein composed by Der p1 and Der p2 portions showed lower IgE reactivity in comparison to native allergens and may induce mice to produce blocking IgG, which suggests their usefulness in immunotherapy (7). 
     Recently, Bussieres L. and collaborators (8) built several hybrid proteins composed by Der p1, Der p2 portions and a Der p1 precursor (pro-Der p1), showing that they are able to induce basophile degranulation in individuals allergic to domestic mites, and by “immunoblotting” with polyclonal and monoclonal antibodies, the conservation of B epitopes of native allergens was demonstrated. 
     Nevertheless, there is a non-satisfied need for vaccines based on recombinant molecules that contain several antigenic regions from mites common in tropical and subtropical areas, which may be suitable alternatives to immunotherapy schemes currently known, which are based on application of natural extracts from complete allergens, and which may be able to decrease the anaphylaxis risk and to improve the treatment efficacy. 
     DEFINITIONS 
     The terms used herein will have the following meanings: 
     “allergen”, a substance able to induce an allergic reaction by inducing production of IgE antibodies. 
     “vaccine”, a substance which can be used to stimulate the immune system to develop protection; the substance may be a polypeptide, such as the fusion protein subject of the present invention, in presence or absence of pharmaceutically acceptable carriers or vehicles. 
     “expression”, refers to a gene or a gene product expression including the codified polypeptide. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Schematic representation of peptides comprising the primary sequence of recombinant PF3a. 
         FIG. 2 . Levels of serum IgE specific for PF14c determined by ELISA, in comparison to reactivity levels of natural extracts from  B. tropicalis  and  D. pteronyssinus.    
         FIG. 3 . Schematic representation of peptides comprising the primary sequence of recombinant PF14c. 
         FIG. 4 . Levels of serum IgE specific for PF14c determined by ELISA in comparison to reactivity levels of natural extracts from  B. tropicalis  and  D. pteronyssinus.    
         FIG. 5 . Purification profile for PF3a performed by gel electrophoresis on 15% polyacrylamide, stained with Comassie Blue. 1 is the molecular weight marker, 2 and 3 correspond to non-dialyzed samples, and 4 corresponds to the dialyzed sample in 0.1% TRITON® X-100 (t-octylphenoxypolyethoxyethanol) buffer. 
         FIG. 6 . Purification profile for PF14c performed by gel electrophoresis on 15% polyacrylamide, stained with Comassie Blue. Lane 1 is the molecular weight marker, 2 and 3 represent the purified fractions. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is directed to fusion proteins with different allergen epitopes and reduced linking capability to IgE. The present invention is also directed to a pharmaceutical presentation allowing application within a cutaneous vaccination scheme (“Skin PrickTest”). The composition is: 1. Fusion protein, 2. Glycerol, and 3. Phenol. 
     The invention also reports synthetic nucleotide sequences design and production of recombinant fusion proteins for treatment and prevention of allergies caused by domestic mites, particularly caused by species  Blomia tropicalis  and  Dermatophagoides pteronyssinus.    
     In other embodiment, the invention comprises nucleotide sequences artificially designed, codifying proteins related to domestic mites allergens. 
     The invention also refers to the design of two vectors based on vector pET45b+, the artificially designed nucleotide sequence being inserted such that a suitable reading frame is preserved, which expresses the recombinant protein with a histidine 6× tag, in the amino terminal region. 
     In this sense, the present invention is also addressed to vector pET-45b(+) which is a plasmid vector specifically ordered to Novagen, having a sequence that encodes a histidine 6x tag in the N-terminal region, followed by a sequence for a cleavage site by means of enzyme enterokinase and a sequence for a S-tag in the C-terminal region. It contains 5260 base pairs. The designed nucleotide sequence insertion was ordered for the fusion protein, PF3ao PF14c, at the position flanked by restriction sites Pml 1 and Kpn I, and such that a reading frame (ORF—Open ReadingFrame) was generated to express the protein of interest with nine additional amino acids in the N-terminal region under control of promoter T7. In this way, the invention reports a vector referred as pET45b/PF3a constituted by 5852 base pairs and a vector referred as pET45b/PF14c constituted by 5719 base pairs, which, when introduced into  E. Coli  bacteria, allow expression of recombinant proteins PF3a having 196 amino acids (SEQ ID NO: 2) and PF14 c having 152 amino acids (SEQ ID NO: 4), exhibiting the unique features disclosed in the present invention. 
     The invention also contemplates the possibility to combine, in a sole composition, the pharmaceutical formulation or vaccine including the two recombinant proteins making possible to improve or enhance their efficacy in immunotherapy against domestic mite allergy. 
     The vaccine for immunotherapy against domestic mite allergy according to the present invention is characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 or a mixture thereof. 
     In still another embodiment, the invention refers to a method to obtain a recombinant fusion protein of different allergen segments from  B. tropicalis  expressed in  Escherichia coli , which shows reactivity against IgE antibodies of an allergic population with lower frequency and intensity in comparison to that obtained from the allergenic extract from  B. tropicalis.    
     Even another embodiment of the invention refers to a method to obtain a recombinant fusion protein having several allergen segments from  B. tropicalis  and  D. Pteronyssinus , expressed in  Escherichia coli , which shows reactivity against IgE antibodies of an allergic population with lower frequency and intensity in comparison to that obtained from the corresponding natural extracts. 
     The invention also refers to use of proteins designed according to the invention to prepare a medicament vaccine to treat an individual suffering allergies caused by domestic mites. 
     The invention is also addressed to a treatment method for an individual suffering allergies caused by domestic mites, which comprises administering to said individual a pharmaceutically effective amount of a protein according to the invention or a pharmaceutical composition containing the same. 
     Protein design according to the present invention starts with the selection of different allergens from domestic dust mites  B. tropicalis  and  D. pteronyssinus , as well as of their antigen regions as reported in scientific publications or predicted by our bioinformatic analysis. The different selected regions were linked to form a unique primary protein structure. The probable 3D-structure for the designed protein was modeled from the amino acid sequence. The structure was optimized targeting to a successful expression as recombinant protein. In this manner, respective models were obtained for each of the two fusion proteins subject of the present invention. 
     Prediction for three-dimensional structure was performed by using I-TASSER (9) and Swiss-Model tools and software. (10, 11). 
     Segments combination and order producing a model satisfactory with regard to energy, stereochemistry and structure requirements were selected to order synthesis of an artificial nucleotide sequence encoding the primary sequence. 
     One vector was chosen for expression in  E. Coli , i.e., pET 45b+ (NovagenR, catalog 71327-3), which was inserted in the artificial sequence to obtain a reading frame for expression of the encoded protein at the insertion site, with a histidine 6x tag in the N-terminal region, with no cleavage site to remove the tag. 
     The artificial gene synthesis with codon optimization for expression in bacteria, and its insertion in vector pET 45b+, such that a vector pET 45+b/PF was obtained for expression of fusion protein linked to the histidine 6× tag, was ordered to a US specialized company (GeneScript). 
     Once the artificial gene and the expression vector with insert were received, transformation was initiated in different  E. Coli  strains, according to the requirements of the protein of interest. The positive clones were selected after confirmation of the expected insert by PCR using universal primers, and expression was performed in LB (Luria-Bertani) medium, consistent of 1% tryptone, 0.5% yeast extract and 1% NaCl, pH 7.0, with antibiotic, IPTG (isopropyl-β-D-thiogalactoside) induction, one lactose analog, which acts as a protein expression regulator under lac operon control. Then, the culture was centrifuged for 10 minutes at 4000-6000 r.p.m., and the cell pellet was recovered. A standard procedure was followed (The Condensed Protocols from Molecular Cloning: A Laboratory Manual, Sambrook and Russel, CSHLPress), with modifications. 
     The cell pellet obtained was re-suspended in lysis buffer (NaH2PO4 100 mM, Tris-HCl 10 mM, Urea 8 M, pH 8.0) and stirred at 20 r.p.m. from one to two hours. The mixture was centrifuged 6000 r.p.m. for 10 minutes at 4° C., and the soluble fraction contained in the supernatant was recovered. 8 ml of this supernatant were mixed with Ni-NTA resin previously prepared by washing twice with water and balanced with lysis buffer, pH 6, and was stirred at 20 r.p.m. for two hours. The mixture was centrifuged at 1200-1600 r.p.m. for one minute, and the supernatant containing the fraction not linked to the resin was separated. The resin was washed several times with NaH 2 PO 4  20 mM, Urea 8 My NaCl 500 mM buffer solution at pH 8.0, 6.0 y 5.3 (2 washings at every pH). After every washing, the fraction not linked to the resin was separated by centrifugation at 1200-1600 r.p.m. for one minute and then discarded. The resin was loaded on a column and the fusion protein was recovered by application of a buffer elution solution (NaH 2 PO 4  20 mM, Urea 8 M, NaCl 500 mM at pH 4.0) to the column. Presence of the band of interest in the collected fractions was analyzed by electrophoresis on 15% poly-acrylamide under reductive conditions. Fractions were combined according to the intensity of the band of interest on the electrophoresis gel, and were diluted with the buffer elution solution. 
     To eliminate urea and induce protein re-naturalization, arginine-assisted oxidative re-naturalization was performed. In summary: fractions obtained and diluted after purification were dialyzed in dialysis bags having a 6-8000 kDa limit, against re-naturalization solution (100 mM Tris-HCl, 10 mM EDTA, 0.5 M L-Arginine, 5 mM Cysteine, 1 mM Cystine) at 1:20 ratio, under moderate and constant stirring overnight at 4° C. Then, dialysis was performed against 10 mM Tris-HCl, 0.1% TRITON® X-100 (t-octylphenoxypolyethoxyethanol) buffer solution for 4 hours with exchange at 2 hours. The re-naturalization products were analyzed by electrophoresis on 15% polyacrylamide under reduction conditions, and sorted at −20° C. 
     The linking capability of IgE and IgG antibodies on human sera was demonstrated by ELISA, electrotransfer, and immunodetection techniques, as per a procedure standardized in our laboratory. 
     The method disclosed in the present invention to obtain a recombinant protein from allergen segments of  B. tropicalis  (PF14c) and allergen segments of  B. tropicalis  and  D. pteronyssinus  (PF3a) expressed in  E. Coli , with reactivity against IgE antibodies in allergic populations, with lower frequency and intensity in comparison to natural allergenic extracts from  B. tropicalis  and  D. Pteronyssinus , comprises the following steps: 
     Transformation of  E. Coli  Cells: 
     Vector pET 45b(+)/PF containing the fusion protein segment of encoding nucleotides was used to transform competent One Shot® BL21 STAR™ (DE3) (Invitrogen, Cat. No. C6010-03) and TOP10 (Invitrogen Cat. No. C4040-10)  E. coli  cells, by means of a chemical treatment combined with thermal shock. In summary, 10 ng of vector were mixed and incubated with competent cells for 30 minutes on ice, followed by 30 seconds at 42° C. and then fast cooling on ice. To the foregoing mix, 250 μL of S.O.C optimized medium (consisting of 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 20 mM glucose) were added. Then, the mix was incubated at 37° C., 250 r.p.m. for 30 minutes. The mix was grown on LBA plates (Luria-Bertani, 1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0, con ampicillin 100 mg/mL) at 37° C. overnight. 
     Selection of Transformed Colonies: 
     The presence of inserts in the transformed colonies was determined by PCR (Polymerase Chain Reaction) directly from the colonies, using the universal primers “T7 Promoter” and “T7 Terminator”. For PCR, a reaction mixture was prepared as follows: 
     
       
         
               
               
               
               
             
           
               
                   
               
             
             
               
                   
                 DNA template (colony) 100 ng 
                 2 
                 μL 
               
               
                   
                 dNTPs 50 mM 
                 1 
                 μL 
               
               
                   
                 Primer “T7 terminator” (10 pmol) 
                 1 
                 μL 
               
               
                   
                 Primer “T7 promoter” (10 pmol) 
                 1 
                 μL 
               
               
                   
                 Buffer solution for PCR 10X 
                 5 
                 μL 
               
               
                   
                 MgCl 2  50 mM 
                 1.5 
                 μL 
               
               
                   
                 Taq Polymerase 5 U/μL 
                 1 
                 uL 
               
               
                   
                 (Invitrogen, Cat. No. 116 15-010) 
                   
                   
               
               
                   
                 Deionized water 
                 c.s.p. 50 
                 μL 
               
               
                   
               
             
          
         
       
     
     Amplification schedule consisted of 30 cycles in three steps as follows: 1) 94° C. for one minute, 2) 50° C. for one minute, and 3) 72° C. for 2 minutes, with a 7-minute final extension at 72° C. The reaction was performed in a S-1000™ (BioRad) thermocycler. The amplification product was analyzed by electrophoresis on 2% agarose on a MINI-SUB® DNA Cell (BioRad) instrument at 50 volts. Positive clones were stored at −70° C. in 8% glycerol. 
     Expression of Fusion Protein: 
     The protein linked to the six-residue histidine tag (6xHis-tag) was obtained by induction with IPTG (isopropyl-β-D-thiogalactoside, a galactose analog, protein expression regulator under control of operon lac). Only one colony isolated from a culture of positive clone incubated on a LBA plate was grown on liquid LBA medium and incubated at 37° C., 250 r.p.m. for 12-14 hours. Then, the culture was diluted in liquid LBA (1:20 dilution) and incubated for 3-4 hours under same conditions until a OD600 (optical density at 600 nm) of 0.5-0.8 was accomplished, when IPTG was added and kept in induction for 3-6-hours at 37° C. Then, the culture was centrifuged for 10 minutes at 4000-6000 r.p.m. and the cell pellet was recovered. A standard procedure was followed (The Condensed Protocols from Molecular Cloning: A Laboratory Manual, Sambrook and Russel, CSHL Press), with modifications. 
     Purification and Re-Naturalization of Fusion Proteins: 
     The cell pellet obtained above was re-suspended in lysis buffer (100 mM NaH2PO4, mM Tris-HCl 10.8 M Urea, pH 8.0), stirred at 20 r.p.m. for 1-2 hours. The mix was centrifuged at 6000 r.p.m. for 10 minutes at 4° C., and the soluble fraction contained in the supernatant was recovered. 8 ml of this supernatant were mixed with the affinity resin based on Ni—NTA previously prepared by washing twice with water and balanced with lysis buffer, pH 6, and was stirred at 20 r.p.m. for two hours. The mixture was centrifuged at 1200-1600 r.p.m. for one minute, and the supernatant containing the fraction not linked to the resin was separated. The resin was washed several times with NaH2PO4 20 mM, Urea 8 M y NaCl 500 mM buffer solution at pH 8.0, 6.0 y 5.3 (2 washings at every pH). After every washing, the fraction not linked to the resin was separated by centrifugation at 1200-1600 r.p.m. for one minute and then discarded. The resin was loaded on a column and the fusion protein was recovered by application of a buffer elution solution (NaH2PO4 20 mM, Urea 8 M, NaCl 500 mM at pH 4.0) to the column. Presence of the band of interest in the collected fractions was analyzed by electrophoresis on 15% poly-acrylamide under reduction conditions. Fractions were combined according to the intensity of the band of interest on the electrophoresis gel, and were diluted with the buffer elution solution. 
     To eliminate urea and induce protein re-naturalization, arginine-assisted oxidative re-naturalization was performed. In summary: fractions obtained and diluted after purification were dialyzed in dialysis bags having a 6-8000 kDa limit, against re-naturalization solution (100 mM Tris-HCl, 10 mM EDTA, 0.5 M L-Arginine, 5 mM Cysteine, 1 mM Cystine) at 1:20 ratio, under moderate and constant stirring overnight at 4° C. Then, dialysis was performed against 10 mM Tris-HCl, 0.1% TRITON® X-100 (t-octylphenoxypolyethoxyethanol) buffer solution for 4 hours with exchange at 2 hours. The re-naturalization products were analyzed by electrophoresis on 15% polyacrylamide under reduction conditions, and were stored at −20° C. 
     Features of Recombinant Proteins: 
     PF3a: 
     It is the protein corresponding to SEQ ID NO:2 and comprises 196 amino acids corresponding to different fragments contained in allergens from mites  Blomia tropicalis  and  D. pteronyssinus ; Blo t 5 (Accession Number:O96870.1, GI:14423644 and 2 JRKA GI:193506481), Der p 2 (Accession Number:P49278.1, GI:1352237), Blo t 10 (Accession Number ABU97466.1, GI:156938889), Der p 8 (Accession Number P46419.1, GI:1170095), Blo t 8 (ACV04860.1,GI:256665455) Der p 1 (Accession Number ACG58378.1, GI:195933901), Der p 2 (Accession Number P49278.1, GI:1352237 and Der p 7 (Accession Number 3H4Z-AGI:292659601). This protein with a 196 amino acid sequence, 22.8 KD, has allergenic segments from allergens from mites  Blomia tropicalis  and  Dermatophagoides pteronyssinus.    
     PF14c: 
     This is the protein corresponding to SEQ ID NO: 4, comprising 152 amino acids corresponding to different fragments found in 6 allergens from mite  Blomia tropicalis , Blo t 5 (Accession Number: ABH06352.1, GI:111120436), Blo t 8 (ACV04860.1, GI: 256665455) Blo t 10 (Accession Number ABU97466.1, GI: 156938889), Blo t 12 (Q17282.1, GI: 2498195) and Blo t 13 (Q17284.1, GI:14423698). This 152 amino acid protein, 17.8KD, has allergenic segments from allergens from mite  Blomia tropicalis.