RU2520078C1 - METHOD FOR OBTAINING IMMUNOGENIC COMPOSITION BASED ON Ag85A-DBD HYBRID PROTEIN AND DEXTRANE; pAg85A-DBD RECOMBINANT PLASMIDE; Escherichia coli [pREP4, pAg85A-DBD] STRAIN; Ag85A-DBD CHIMERIC PROTEIN - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention represents Escherichia coli M15 [pREP4, pAg85A-DBD] strain - producer of Ag85A-DBD chimeric protein, as well as a method for immobilisation, concentration and cleaning of protein obtained based on dextrane. Invention relates to a method for obtaining immunogenic composition based on Ag85A-DBD recombinant protein mixed with dextrane and to Ag85A-DBD recombinant protein itself.

EFFECT: invention allows obtaining strain - producer providing high level of production of stable immunogenic proteins that can be obtained, immobilised and cleaned at a single stage, and obtaining effective immunogenic compositions against tuberculosis.

4 cl, 2 dwg, 1 tbl, 5 ex

Description

The technical field to which the invention relates, and the preferred field of use of the invention

The invention relates to genetic engineering, biochemistry, biotechnology and immunology and can be used to obtain a recombinant (chimeric) protein Ag85A-DBD and immunogenic composition containing it, aimed at the induction of immunity against tuberculosis infection. EFFECT: invention makes it possible to obtain a producer strain providing a high level of production of an immunogenic recombinant protein that can be purified and immobilized in one stage, as well as to obtain effective immunogenic compositions against tuberculosis.

Description of the analogues of the invention

Currently, the only vaccine used in the world against tuberculosis is BCG (BCG, Bacillus Calmette-Guerin, 1921), which is a live, attenuated strain of Mycobacterium bovis. It is safe, inexpensive and protects children quite effectively both from infection with tuberculosis and from miliary tuberculosis and tuberculous meningitis. The disadvantage of the BCG vaccine is that it does not protect adults from pulmonary tuberculosis, namely this form of the disease leads to the spread of infection and a serious epidemic in many countries of the world. Hence the high relevance of the development of new vaccines against tuberculosis.

There are several directions for the development of new TB vaccines, alternative to BCG: subunit vaccines, DNA vaccines and live vaccines with improved properties. Subunit vaccines, including preparations consisting of several secreted M. tuberculosis proteins / peptides, are most fully characterized in experimental animal models. Many M. tuberculosis antigens have been identified, potentially important as components of new vaccines. These include, in particular, immunodominant secreted antigens present in the culture filtrate of M. tuberculosis. Among the secreted M. tuberculosis antigens, ESAT-6 (Early Secret Antigen Target-6), CFP-10 (culture-filtrate protein-10) and complex 85 proteins (Ag85A, B, C) are best studied.

The invention is known US 2011/0206713 A1, which describes an immunogenic composition or drugs consisting of a single antigen ESAT-6, Ag85A, Ag85B or TB 10.4 M. tuberculosis; combinations of various chimeric proteins based on these antigens; the use of fusion proteins in various combinations with BCG vaccine.

The invention of US 2011/0027349 A1 relates to an immunogenic composition comprising one of the antigens Ag85A, Ag85B, MPT-64, Pst-S1, Ara, GroES, GroEL, Dnak, CFP-10, Rv083 1c, M. tuberculosis Rv1324, or various combinations thereof; the use of polypeptides as components for prime-boost vaccination in combination with BCG vaccine; the use of various adjuvants.

Also known is the invention of US 2010/0015171 A1 regarding an immunogenic composition comprising antigens of the Ag85 family and 10.4 M. tuberculosis TB; various combinations of these antigens; the use of various adjuvants (DDA, polycationic peptides, oligonucleotides, IC31® adjuvant) for the introduction of antigens.

A common disadvantage of the invention is a complex multistage method for purifying recombinant proteins and formulating an immunogenic composition.

The invention RU (11) 97110087 (13) And relates to a polynucleotide consisting of individual antigens of the Ag85 family M. tuberculosis or their various combinations, which, after introduction into the body, induces a number of immune reactions; a method for introducing an immunogenic composition into vertebrate tissues.

The disadvantage of the invention is the use of only antigens of mycobacteria of the Ag85 family, which casts doubt on the vaccine's effectiveness. DNA vaccine also has low immunogenicity and requires the use of adjuvants, which complicates the formulation of the finished drug.

The invention US 6174700 relates to a method for purification of polypeptides containing a carbohydrate-binding domain. As the carbohydrate binding domain, a cellulose binding domain is described.

The disadvantage of the invention is that the protein purification method given as an example cannot be used for pharmaceutical use due to contamination of the final product with E. coli endotoxins and the difficulty of removing the carbohydrate-binding domain from the protein molecule.

The patent (WO 2009/143413) describes the use of the pneumolysin fusion protein and the mycobacterial proteins ESAT6, mtb or Ag85 conjugated to polysaccharides, including dextran. However, in this patent, carbohydrate-binding protein domains that specifically interact with the polysaccharide are not used to immobilize proteins. The preparations obtained as described in the invention relate to classic conjugate vaccines.

Patents are known which describe the use of a dextran binding domain from the microorganism Leuconostoc mesenteroides as a domain interacting with dextran. Patent RU 2428477 describes a chimeric protein construct of DSD-cn-β-HAL containing a dextran binding domain from the microorganism Leuconostoc mesenteroides, which is genetically engineered via a glycine-serine spacer with thermostable beta-galactosidase from the thermophilic Thermananaerus microorganism. However, the patent does not provide a method for purifying a recombinant protein using dextran as a sorbent, and the example given in the patent for determining the dextran binding properties of a chimeric protein can be used only for purification of thermostable recombinant proteins. It is impossible to use this method to obtain immunogenic components of medical immunobiological preparations due to the thermolability of antigens.

Description of the prototype of the invention

The prototype of the claimed invention is patent RU 2429292, which sets forth a method for producing a recombinant protein Ag85A-CBD, which contains a cellulose-binding domain. The synthetic M. tuberculosis Ag85A gene, optimized for heterologous expression in non-pathogenic laboratory strains, is obtained. Based on it, a recombinant plasmid pAg85A-CBD is obtained, consisting of an artificial bacterial operon of a chimeric protein, including an early promoter of the T5 bacteriophage, a chimeric protein gene and a transcription terminator; the bacterial operon of beta-lactamase and the bacterial replication initiation site of ColE1 type. The invention also includes a strain of E. coli, a producer of the chimeric protein Ag85A-CBD, as well as a method of immobilization, concentration and purification of the obtained protein on cellulose. The invention relates to the most recombinant protein Ag85A-CBD and an immunogenic composition containing it, aimed at the induction of immunity against tuberculosis infection. The invention allows to obtain a producer strain that provides a high level of production of stable immunogenic proteins that can be obtained, immobilized and purified in one stage, as well as to obtain effective immunogenic compositions against tuberculosis.

The disadvantage of this invention is the low biodegradability of cellulose, and, therefore, the inability to use the immunogenic composition "recombinant protein Ag85A-CBD - cellulose" in pharmaceutical practice.

Disclosure of invention

The objective of the invention is to develop vaccines based on individual protein components with a high level of expression and stability of these proteins in the bacterial system. To solve this problem, a dextran domain from the Leuconostoc citreum microorganism is used to obtain an immunogenic composition that is stable for a long time (at least a year) by cloning a gene including the sequence encoding DBD and the sequence of mycobacterial protein (antigen), its expression in E. coli cells and immobilizing the resulting chimeric antigen on a dextran polysaccharide, which is used as an adjuvant.

Recombinant proteins have been created, consisting of two components: M. tuberculosis antigen and the dextran binding domain of the deuctransurase Leuconostoc citreum KM20. Recombinant proteins containing DBD can be immobilized in a single step on a dextran carrier. The result is their simultaneous purification, concentration and stabilization, as well as protection against proteases of producer bacteria. In this case, the initial antigenic properties of the functionally active protein domain are not violated. The use of the dextran binding domain of Leuconostoc citreum dextransurase is determined by the fact that it provides greater stability of recombinant proteins than the dextran binding domain from microorganisms Leuconostoc mesenteroides and Streptococcus sobrinus (S. Suwannarangsee, C. Moulis, G. Potocki . Remaud-Simeon, and W. Chulalaksananukul, "Search for a dextransucrase minimal motif involved in dextran binding", FEBS letters, vol. 581, No. 24, pp. 4675-80, Oct. 2007).

In addition, to ensure a high level of gene expression in a heterologous system, the codons of the Ag85A gene were optimized. As a result, the synthetic Ag85A gene was planned, which differed from the native mycobacterial gene in nucleotide composition with the preserved amino acid sequence of the Ag85A protein encoded by it. This approach made it possible to obtain a highly efficient producer strain synthesizing about 25-30% of the recombinant Ag85A antigen from the total protein of the cell.

Using these approaches, a two-component recombinant protein was obtained. The sequence of the M. tuberculosis gene Ag85A encodes a full-sized protein that forms the first protein domain that determines the functional immunological properties of the complex molecule. The following is the connecting (spacer) amino acid sequence of several alternating glycine and serine residues (Gly-Ser). The second protein domain is encoded by the sequence of the Leuconostoc citreum KM20 dextransurase gene fragment; it determines the ability of a product to interact with a dextran sorbent. The non-covalent interaction of the dextran binding domain with dextran provides a constant, but slow exit of the antigen from the complex with the substrate due to natural dissociation, thereby ensuring a prolonged interaction of the vaccine components with the body's immune system (deposition), sufficient to induce a strong and prolonged immune response.

Protein products are produced in non-pathogenic laboratory strains of E. coli. Antigens immobilized on dextran are a solution of dextran with proteins adsorbed on it. Dextran acts as an adjuvant in immunization, providing for the enlargement and polymerization of the antigen, without which a weaker immune response to protein monoantigens develops.

Thus, the claimed group of inventions allows you to create a strain of E. coli, providing a high level of production of a recombinant protein containing protein M. tuberculosis Ag85A; to obtain a simple and effective scheme for purification of a recombinant protein that specifically and effectively induces an immune response to mycobacterial antigens, including IFN-γ synthesis, which is necessary for the formation of protective immunity against tuberculosis; create immunogenic compositions based on recombinant protein on dextran.

This result is achieved through the synthesis of the recombinant protein Ag85A-DBD in the cells of the recombinant strain of E. coli M15 [pREP4, pAg85A-DBD], carrying the recombinant plasmid pAg85A-DBD. Also, by creating a complex preparation Ag85A-DBD-dextran, in which the recombinant protein Ag85A-DBD (concentration 4 mg / ml) is immobilized on dextran (50 mg / ml) and resuspended in 1 × PBS buffer pH 7.2-7.4 .

SUMMARY OF THE INVENTION

A recombinant plasmid pAg85A-DBD (4362 bp) was created that encodes a bifunctional recombinant protein Ag85A-DBD (sequence No. 1), which has the ability to bind spontaneously to a dextran-containing sorbent. Plasmid pAg85A-DBD (sequence No. 2) contains the following structural elements essential for its functioning:

a) a synthetic gene encoding the sequence of M. tuberculosis Ag85A protein (900 bp);

b) an artificial bacterial operon of a chimeric protein, consisting of:

- the promoter region of the early promoter of the T5 bacteriophage (7-87 bp), which provides efficient transcription of mRNA;

- the recombinant gene encoding the chimeric protein "Ag85A M. tuberculosis - spacer - dextran-binding domain of the dextransurase Leuconostoc citreum KM20" - Ag85A-DBD (117-1460 bp);

- the untranslated region of the termination of transcription of the bacterial operon, which ensures the effective termination of pAg85A-DBD transcription (1500-1596 bp);

c) the bacterial operon bla encoding beta-lactamase protein, a selective marker for transformation of the E. coli strain pAg85A-DBD (3287-4147 bp) of the complementary chain;

d) a bacterial site of ColE1-type replication initiation, which ensures plasmid replication in the E. coli strain: pAg85A-DBD (2529-2539 bp).

The strain producing the recombinant protein Ag85A-DBD (sequence No. 3) is obtained by transformation of E. coli cells of strain M15 [pREP4] with plasmid pAg85A-DBD. The strain provides the production of recombinant protein Ag85A-DBD after the induction procedure of isopropyl-β-D-thio-galactopyranoside (IPTG) up to 12-15% of the total protein of the cell.

The strain E. coli M15 [pREP4], carrying the plasmid pAg85A-DBD, the producer of the bifunctional recombinant protein Ag85A-DBD is characterized by the following features.

Cultural and morphological characters. Cells are straight, rod-shaped, motionless, gram-negative. When sieving on a plate with 2.0% agarized LB medium, growth is in the form of separate colonies, sometimes in the R-form with uneven edges. It grows well on solid and liquid nutrient media (LB-broth, LB-agar, MPA, MPB).

Physiological and biochemical characteristics. Cells grow at temperatures from + 4 ° C to + 42 ° C, with an optimum pH of 6.8-7.5. The strain decomposes glucose, mannitol with the formation of acid, does not decompose sucrose, arabinose, galactose, ferments maltose, xylose, sorbitol, rhamnose. Significant when using this strain is its sensitivity to nalidixic acid (25 mg / ml), streptomycin (20 mg / ml) and rifampicin (25 mg / ml). It is resistant to ampicillin (up to 300 μg / ml) due to the presence of plasmid pAg85A-DBD, and to kanamycin (up to 50 μg / ml) due to the presence of plasmid pREP4.

By creating a complex preparation of recombinant protein and dextran, the technical result is achieved by creating a two-component recombinant protein Ag85A-DBD, consisting of M. tuberculosis Ag85A protein and a gene encoding the dextran binding domain of dextransurase L. citreum KM20. And also by obtaining a complex preparation (immunological composition) in which the recombinant protein (Ag85A-DBD) is immobilized on dextran (composition Ag85A-DBD-dextran).

The recombinant protein Ag85A-DBD incorporates a domain that determines its ability to bind to dextran, which allows concentration, purification and immobilization of the protein product on dextran in one step. Immobilization on dextran is ensured by the presence of the dextran binding domain of dextransurase L. citreum KM20 in the recombinant protein. Since there are no dextran binding proteins in E. coli, the recombinant protein Ag85A-DBD synthesized in E. coli cells transformed with the plasmid pAg85A-DBD is the only protein of the producer strain that binds strongly to dextran. This provides the possibility of a single-stage preparation of a highly purified protein preparation immobilized on a sorbent. Figure 1 shows a diagram of the plasmid pAg85A-DBD.

Comparison of the stability of recombinant mycobacterial proteins fused with dextran binding domains from L. mesenteroides and L. Citreum revealed that proteins fused to the dextran binding domain from L. mesenteroides are unstable and are prone to degradation after storage for more than six months, which does not allow the use of the above purification of these recombinant proteins. The use of DBD L. citreum as a subunit that binds to dextran allows for more stable immunogenic compositions.

The immunogenicity of the complex preparation Ag85A-DBD-dextran in mice was investigated. Mice were immunized under the skin of the back 2 times with a 2-week interval immunogenic and control drug. After 5 weeks, the proliferative response of T cells and the number of T cells producing IFN-gamma were investigated. An increase in IFN-gamma-positive T cells and increased proliferation in the presence of antigen in cell culture were detected compared to control animals.

The technical result achieved by the implementation of the invention is to obtain a producer strain that provides a high level of production of a stable immunogenic protein that can be obtained, immobilized and purified in one stage. An effective immunogenic composition was obtained against tuberculosis.

The invention is illustrated by the following examples below. Genetic engineering and microbiological manipulations, amplification and DNA sequencing were performed according to standard methods (T. Parish, NGStoker. Mycobacterium tuberculosis protocols., Humana Press Inc., 2001, Methods in molecular Medicine, 54; Maniatis T., Fritsch E., Sambrook J. Molecular Cloning, M .: Mir, 1984; DNA Cloning, Methods, Edited by D. Glover, Translated from English, M .: Mir, 1988; Saiki RK, Gelfand DH, Stoffel S. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988, Vol. 239, No. 4839, pp. 487-491; Sanger F., Nicklen S., Coulson AR DNAsequencing with chain-terminating inhibitors (DNA polymerase (nucleotide sequences / bacteriophage 4X174). Proc. Nat. Acad. Sci. USA, 1977, Vol. 74, No. 12, pp. 5463-5467).

Example 1. Obtaining an expression of the genetic construct.

a) Obtaining a fragment of the sp-DBD protein gene followed by its cloned into the pQE6 vector.

The sp-DBD protein gene fragment containing the Gly-Ser spacer (sequence No. 4) and the sequence of the dextran binding domain DBD from L. citreum KM20 (sequence No. 5) was synthetically prepared by Eurogen. The nucleotide sequence encoding the sp-DBD protein was designed using optimal codons for expression in E. coli, taking into account the absence of a pronounced secondary structure of mRNA.

To clone the sp-DBD protein gene, the plasmid vector pQE6 and the plasmid pAT-sp-DBD synthesized by Eurogen were hydrolyzed with restriction endonucleases NcoI and Kpn21 at 37 ° C in a buffer containing 33 mM Tris acetate (pH 7.9 at 37 ° C) , 10 mM magnesium acetate, 66 mM potassium acetate and 0.1 mg / ml BSA for 1 h. Restriction products were separated on a 1.2% agarose gel. A pQE6 vector fragment (3016 bp) isolated from agarose gel was combined with a plasmid fragment pAT-sp-DBD (464 bp). The fragments were then ligated at 25 ° С in a buffer containing 40 mM Tris-HCl, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP (pH 7.8 at 37 ° С) using bacteriophage T4 DNA ligase for 1 h. Reaction products were reprecipitated at

-20 ° C with 96% ethyl alcohol with the addition of ammonium acetate for 1.5 hours. After reprecipitation, the mixture was dissolved in deionized water. Using electroporation, the obtained ligase mixture was used to transform competent E. coli cells of strain M15 [pREP4] (Nal ^S , Str ^S , rif ^S , lac ^- , ara ^- , gal ^- , mtl ^- , F ^- , recA ⁺ , uvr ⁺ ). After transformation, clones were selected on LB agar medium containing antibiotics ampicillin (50 mg / ml) and kanamycin (25 mg / ml). Plasmid DNA from the colonies was isolated by alkaline lysis; were analyzed by restriction mapping with restriction endonucleases BglI, BglII, BspMII and NcoI. Clones of plasmid DNA psp-DBD (3480 bp) containing the sp-DBD protein gene sequence were selected. The primary structure of the resulting constructs was confirmed by sequencing.

b) Obtaining a gene fragment of the protein Ag85A with its subsequent cloned into the plasmid psp-DBD.

A gene fragment of the Ag85A protein from M. tuberculosis was synthetically prepared by Eurogen. The nucleotide sequence encoding the Ag85A protein was designed using optimal codons for expression in E. coli, given the absence of a pronounced secondary structure of mRNA.

To clone the Ag85A-sp-DBD protein gene, psp-DBD plasmid and pAT-Ag85A synthesized at Eurogen were digested with restriction endonucleases NcoI-BamHI and NcoI-BglII, respectively, at 37 ° С in a buffer containing 33 mM Tris-acetate (pH 7.9 at 37 ° C), 10 mM magnesium acetate, 66 mM potassium acetate and 0.1 mg / ml BSA for 1 h. Restriction products were separated on a 1.2% agarose gel. A psp-DBD vector fragment (3469 bp) isolated from agarose gel was combined with a pAT-Ag85A plasmid fragment (893 bp). The fragments were then subsidized at 25 ° C in a buffer containing 40 mM Tris-HCl, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP (pH 7.8 at 37 ° C) using bacteriophage T4 DNA ligase for 1 h. The reaction products were reprecipitated at -20 ° C with 96% ethanol with the addition of ammonium acetate for 1.5 h. After reprecipitation, the mixture was dissolved in deionized water. Using electroporation, the obtained ligase mixture was used to transform competent E. coli cells of strain M15 [pREP4] (Nal ^S , Str ^S , rif ^S , lac ^- , ara ^- , gal ^- , mtl ^- , F ^- , recA ⁺ , uvr ⁺ ). After transformation, clones were selected on LB agar medium containing antibiotics ampicillin (50 mg / ml) and kanamycin (25 mg / ml). Plasmid DNA from the colonies was isolated by alkaline lysis; were analyzed by restriction mapping with restriction endonucleases BglI, BglII, BspMII and NcoI. Clones of plasmid DNA pAg85A-sp-DBD (4362 bp) containing the gene sequence of the protein Ag85A-sp-DBD were selected. The primary structure of the resulting constructs was confirmed by sequencing.

Example 2. Construction of a strain of E. coli, a producer of M. tuberculosis antigen, coupled to a dextran binding domain.

To obtain the E. coli strain, the producer of the recombinant protein Ag85A-DBD, cells of the E. coli strain M15 [pREP4] were transformed with the plasmid pAg85A-DBD. Transformed cells were grown in 3.5 ml of LB medium with ampicillin and kanamycin at 37 ° C to an optical density corresponding to 1 unit. absorption at a wavelength of 550 nm (about 2.5 hours). 3 μl of a 0.1 M IPTG solution was added to the culture and grown for 3 h. To control the production of recombinant proteins in E. coli M15 strain [pREP4, pAg85A-DBD], the Lammley electrophoresis method in the presence of sodium dodecyl sulfate (SDS) was used. Protein separation was carried out on a 12% polyacrylamide gel (PAGE) in a standard buffer system (electrode buffer: 25 mM Tris-HCl, 192 mM glycine, 0.1% sodium dodecyl sulfate, pH 8.3; gel buffer: 375 mM Tris-HCl pH 8.8). At the end of electrophoresis, the gels were stained with 0.15% Coomassie G250 solution in 25% isopropanol and 10% acetic acid and washed in 10% acetic acid.

When comparing the spectrum of proteins in strains of E. coli M15 [pREP4] and E. coli M15 [pREP4, pAg85A-DBD], the appearance of an additional protein band was detected. The molecular weight of the additional band 48.5 kDa was consistent with that calculated for the Ag85A-DBD protein. The level of protein synthesis in E. coli was determined by comparing the staining intensity of the recombinant protein band with the band of the corresponding Unstained Protein Molecular Weight Marker standard protein (Fermentas, Lithuania).

To test the solubility of the synthesized protein, the biomass of the grown strain (E. coli M15 [pREP4, pAg85A-DBD]) was destroyed in 1% TES buffer (25 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 5 mg / ml lysozyme) in a ratio of biomass-buffer 1: 2 and homogenized to a homogeneous mass. After centrifugation, the samples were divided into 2 fractions: soluble cellular proteins (supernatant) and insoluble (precipitate). Both fractions were degraded in lysis buffer (0.05 M Tris-HCl, pH 6.8, 0.5% Triton X-100, 0.5 M NaCl, 0.25 M MgCl ₂ 5 mg / ml lysozyme, 10 mg / ml PMSF, 20 mg / ml DNase) in a 1: 1 ratio; homogenized to a homogeneous mass; warmed at + 95 ° C for 15 minutes The results were monitored by electrophoresis according to Laemmli. It was shown that the recombinant protein Ag85A-DBD is synthesized in E. coli cells both in the soluble fraction and in the form of Taurus inclusions.

Example 3. Obtaining recombinant protein Ag85A-DBD in the free state and in the form of a protein immobilized on dextran.

To obtain a recombinant protein, a culture of E. coli M15 strain [pREP4, pAg85A-DBD] was grown in 1000 ml of LB medium with ampicillin and kanamycin at 37 ° C to an optical density corresponding to 1 unit. absorption at a wavelength of 550 nm. 100 μl of a 0.1 M IPTG solution was added to the medium and grown for 3 hours. Cells were pelleted by centrifugation at 5500 rpm for 15 minutes.

The pellet was resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 0.25 mM NaCl, 5 mM MgCl ₂ , 0.15 mM PMSF, 0.1% Triton-X100, 0.5 mg / ml lysozyme, 20 mg / ml DNase; based on 1 g of biomass per 4 ml of buffer. Additionally, the suspension was treated with ultrasound 3 times for 20 s. After centrifugation at 16,000 rpm for 30 minutes, the fraction of insoluble cellular proteins remained in the sediment, soluble in the supernatant.

The precipitate (insoluble fraction) was suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.002 volume β-mercaptanol) and an equal (by weight) volume of urea was added. After mixing, it was kept in a water bath at + 37 ° C until the urea was completely dissolved. It was centrifuged at 12,000 rpm for 30 minutes and the supernatant was taken. For purification of the recombinant protein, the supernatant was transferred onto a Sephadex G200 column (dextran crosslinked). Elution of proteins from dextran was performed with a gradient urea solution (8-2 M). To remove urea, the samples were dialyzed against PBS buffer at 25 ° C. overnight.

The soluble protein fraction was used for immobilization on dextran. To the supernatant was added dextran with a molecular weight of 500,000 at a concentration of 50 mg / ml in PBS; incubated at 25 ° C for 1 h. The protein concentration in the free and immobilized form was determined by Bradford at a wavelength of 595 nm. Samples were lyophilized. Thus, an immunogenic composition of Ag85A-DBD-dextran was obtained. The recombinant protein purity was at least 95%.

Example 4. Evaluation of the stability of the purified recombinant protein.

Assessment of the stability of recombinant proteins obtained by fusion of the sequence of mycobacterial protein Ag85A and DBD Leuconostoc citreum or DBD Leuconostoc mesenteroides was carried out by electrophoresis in 12% PAG. The preparations of the chimeric proteins Ag85A-DBD Leuconostoc citreum and Ag85A-DBD Leuconostoc mesenteroides were stored at a temperature of 0-4 ° C for 6 and 12 months from the date of lyophilization. Studies have found that the Ag85A-DBD Leuconostoc mesenteroides protein is less stable than the Ag85A-DBD Leuconostoc citreum and after 6 months of storage it begins to denature (see FIG. 2, where 1. Recombinant Ag85A-DBD L. citreum protein after 6 months of storage; 2. Recombinant protein Ag85A-DBD L. citreum after 12 months of storage; M - molecular weight marker; 3. Recombinant protein Ag85A-DBD L. mesenteroides after 6 months of storage; 4. Recombinant protein Ag85A-DBD L. mesenteroides after 12 months of storage ; The arrow indicates the degradation of the recombinant protein).

Example 5. A method for checking the immunogenicity of complex preparations of Ag-DBD-dextran in mice.

The production of IFN-γ by T cells in response to stimulation of mycobacteria by secreted antigens is considered the most important element of the adaptive protective immune response in case of tuberculosis infection, therefore vaccination should first of all stimulate this function.

The immunogenic properties of the sorbent containing the recombinant M. tuberculosis antigen - Ag85A-DBD, was tested on mice of the inbred line C57BI / 6. A positive control was a group of mice vaccinated with BCG. The number of T-lymphocytes producing IFN-γ was determined in the lymph nodes and spleen by ELISPOT.

In immunized mice, the spleen and inguinal lymph nodes were examined; prepared a suspension of cells. We used cloning R4-6A2 monoclonal antibodies to mouse IFN-γ, biotinylated antibodies to mouse IFN-γ clone XMG1.2. Binding monoclonal antibodies were diluted in PBS buffer (working concentration 4 μg / ml), added to the wells and incubated for 18 hours at 4 ° C. After that, the contents of the wells were removed, a suspension of cells (2.5-3.0 × 10 ⁵ cells / well) in the culture medium was introduced and incubated for 2-4 hours at 37 ° C. At this stage and further, before adding each subsequent component, the plate was washed with PBS. The contents of the wells were removed, a sample of the recombinant protein diluted in PBS (10 μg / ml) was added to some of them, PBS was added to the negative control, incubated for 48 h at 37 ° C in 5% CO ₂ . Biotinylated developing antibodies were added to the wells at a concentration of 1 μg / ml, incubated for 2-4 hours at 25 ° C. An alkaline phosphatase conjugate with streptavidin diluted in PBS to a concentration of 1: 1000 (100 μl / well) was added, incubated for 1-2 hours at 25 ° C. Alkaline phosphatase substrate diluted in PBS was added. After 5-60 minutes, the reaction was stopped, the plate was dried, the number of spots in the wells was evaluated. To identify the significance of differences, Student t-test was used (P <0.01). The results are presented in table 1.

Table 1 The number of T cells (× 10 * 6) producing IFN-γ in vitro in response to the Ag85A antigen after double subcutaneous immunization with Ag85A-DBD-dextran Vaccination The number of IFN-gamma producers per 1 million lymph node cells The number of IFN-gamma producers per 1 million spleen cells No antigen in culture 6 ± 0.5 8 ± 1,0 Ag85A-DBD-Dextran 29 ± 1,1 105 ± 2.9 BCG 31 ± 1.4 132 ± 3,5 DBD Dextran 7 ± 0.3 6 ± 0.8

The results show that vaccination with monoantigens bound to dextran via the DBD domain leads to specific activation of IFN-gamma producers. Vaccination with the same antigens, in the same quantities, but without adsorption on dextran, does not cause an immune response.

Claims (4)

1. The method of obtaining purified immobilized recombinant protein Ag85A-DBD on dextran, including:
- growing cells of the strain E. coli M15 [pREP4, pAg85A-DBD];
- immobilization of the Ag85A-DBD protein in the cell extracts of E. coli M15 strain [pREP4, pAg85A-DBD] on a dextran sorbent due to affinity interaction during the incubation procedure, followed by washing from unbound bacterial proteins, while the target product is concentrated and purified;
- selection of the target product. 2. Recombinant E. coli M15 strain [pREP4, pAg85A-DBD], obtained by transformation with plasmid pAg85A-DBD with sequence No. 2, protein producer Ag85A-DBD. 3. Recombinant protein Ag85A-DBD with a molecular weight of 48.5 kDa, including a full-sized protein Ag85A with sequence No. 3, Gly-Ser spacer with sequence No. 4, dextran binding domain of the dextransurase gene L. citreum KM20 with sequence No. 5. 4. Immunogenic composition containing the recombinant protein according to claim 3 of Ag85A-DBD, immobilized on dextran in an effective amount, specifically activating T-lymphocytes that synthesize IFN-γ upon stimulation with mycobacterial antigens.

Images