RU2439155C1 - NUCLEOTIDE SEQUENCE, CODING IMMUNOGENIC POLYPEPTIDE LcrV(G113), CAUSING PROTECTIVE IMMUNE RESPONSE AGAINST Yersinia pestis; RECOMBINANT PLASMID DNA pETV-I-3455, CODING IMMUNOGENIC POLYPEPTIDE LcrV(G113); RECOMBINANT STRAIN Escherichia coli BL21(DE3)/pETV-I-3455 - PRODUCER OF IMMUNOGENIC POLYPEPTIDE LcrV(G113); POLYPEPTIDE LcrV(G113) AND METHOD OF ITS OBTAINING - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention describes nucleotide sequence (dwg.2), coding immunogenic polypeptide LcrV(G113), serving the base for construction of recombinant plasmid DNA pETV-I-3455, with the size 6538 bp, which codes immunogenic polypeptide LcrV(G113). Plasmid consists of plasmid pBR322 replicon, β-lactamase gene, determining resistance to ampicillin, T7-promoter, 1ac-operator, f1-replicon and DNA fragment, flanked by the sites for restrictases Ndel and Hindlll, coding synthesis of protein LcrV(G113), which starts from initiating codon ATG. Described is recombinant strain of bacteria E. coli BL21 (DE3)/pETV-I-3455 - producer of immunogenic polypeptide LcrV(G113) with amino acid sequence, represented on dwg.3, where tryptophan in position 113 (W113) is substituted with glycin. Described is method of obtaining said polypeptide by cultivation of strain E. coli BL21(DE3)/pETV-I-3455. Cells are destroyed in buffer solution by ultrasound and polypeptide is isolated successively by gel-permeation chromatography with application of carrier TSK HW-40, anion-exchanging and hydrophobic chromatography.

EFFECT: invention makes it possible to obtain product with high immunogenic and protective activity.

5 cl, 10 dwg, 2 tbl, 5 ex

Description

The invention relates to biotechnology, in particular to the production of an immunogenic polypeptide that provides the formation of an immune protective response against infection caused by Yersinia pestis.

The specific prophylaxis of especially dangerous infections of bacterial etiology, in particular of the plague, is caused by a tense epidemiological situation, due to the presence of active natural foci of plague on the territory of Russia, the possibility of introducing infection from abroad, and also the real threat of bioterrorism - causes that can cause epidemic manifestations of an emergency nature. Currently, in the Russian Federation, for specific prophylaxis of plague, live vaccines are used, developed in the middle of the last century and characterized by significant reactogenicity. Having the greatest protection, live vaccines are cheap to manufacture, however, due to the presence of a significant number of "ballast" components, they cause a high antigenic load on the body, an increase in allergic reactions, and are not effective enough for revaccinations. These shortcomings are deprived, in particular, of chemical (subunit, molecular) vaccines, which allow the introduction of only immunodominant antigens to significantly reduce the reactogenicity of the drug as a whole; effective for revaccination; can be used against the background of emergency antibiotic prophylaxis; exclude the possibility of an infectious process in people with impaired immune status.

Immunization of animals with live Y. pestis bacteria producing the V antigen [1], as well as a purified native [2] or recombinant [3] V antigen, is known to lead to an antibody response and protect mice from infection with a virulent strain of the plague pathogen.

V antigen (LcrV), one of the “classical” pathogenicity factors of Y. pestis, is encoded by the IcrV gene in the IcrGVH operon [4], located on the calcium-dependent plasmid pCad (pCD1, pVW, pYV or pLcr) present in all yersinia pathogenic to humans [5] was first described by TW Burrows [6]. The V antigen is also produced by two other pathogens of the genus Yersinia: Y. pseudotuberculosis and Y. enterocolitica [5]. The production of V antigen was found to correlate with the virulence of Yersinia spp. [7]. Mutation of the IcrV gene led to a loss of virulence [8]. V antigen regulates the secretion of cytotoxic Yop proteins [9], inhibits neutrophil chemotaxis [10], and has an immunosuppressive effect [11]. The mechanism of the immunosuppressive action of the V antigen is to directly stimulate the synthesis of the anti-inflammatory cytokine, interlekin-10 (IL-10), leading to a general inhibition of anti-inflammatory cytokines [12].

V antigen is a protein including 326 amino acid residues (aa), which corresponds to a molecular weight of 37.2 kDa [13]. The polypeptide forms a mixture of monomers, dimers with a molecular weight of 85-95 kDa and tetramers [14]. Experiments using mass spectrometry determined the molecular weight of the dimers - 75 kDa [2]. The temperature effect (45 ° C, 20 hours) stimulated an increase in the number of dimers [15]. It is found that oligomeric forms have greater LcrV protective ability [16] and stimulate TLR2 (T oll- l ike receptor 2) mediated immune response [14].

Known recombinant plasmid encoding the synthesis of V antigen, causing a protective effect against the action of Y. pestis [17]. However, the IcrV genes of Yersinia spp. encodes various variants of the LcrV polypeptide [18], which differ from each other in amino acid sequence [19]. Amino acid sequences of V antigens of Yersinia spp. are divided into seven classes, one of which is the sequence LcrV Y. pestis and Y. pseudotuberculosis, the rest belong to strains of Y. enterocolitica. A relationship was established between the structure of individual classes of V antigen Y. enterocolitica and the virulence of the strains of this microorganism producing them [20].

For the V antigen of Y. pestis, a high conservativeness of the IcrV gene in strains of the main subspecies was shown [21]. The Y. pestis strains of minor subtypes are characterized by the IcrV gene polymorphism [22], which was detected mainly in four so-called “hot spots” [23], corresponding to amino acid residues 18, 72, 273, and 324-326. According to the results of three-dimensional modeling based on these data, such localization does not significantly affect the structure of the protein and, as a result, does not lead to a change in the chemical and physical properties of the molecule [23].

The native V antigen is sensitive to the action of bacterial proteases, which leads to its low yield upon isolation of yersinia from cells [24]. To solve this problem, fusion proteins resistant to proteolysis were designed: protein A-V antigen or glutathione-S-transferase-V antigen. A significant drawback of this method is the need for proteolytic cleavage of the immunogenic peptide precursor and the introduction of an additional step for removing the cleaved carrier peptide. Glutathione S-transferase is cut off from the fusion protein sequence using factor XA [25]. The use of factor Xa in the purification process is a serious limitation in the use of the immunogenic polypeptide obtained in this way as a component of the vaccine for humans, since factor Xa is obtained from the body of the bulls, and the Russell viper venom is used to activate it.

There is a method of producing an immunogenic polypeptide by affinity chromatography on carriers with immobilized metal chelate groups, which is based on the chemical affinity of a genetically modified immunogenic polypeptide sequence for divalent metal ions, coordination bond acceptors resulting from the introduction of an additional polyhistidine insert into the polypeptide. The strength of the newly formed coordination bond: divalent metal ion (Ni ⁺⁺ ) - polyhistidine (His) _{6 is} sufficient to keep the target protein on the column when washing the latter with various buffer solutions, including in the denatured state, for example, in the presence of concentrated solutions urea or guanidine chloride. The release of bound protein from the column is induced by the redistribution of coordination bonds after the introduction of strong chelating agents into the elution buffer. This allows one-step purification of a protein suitable for immunization of animals [26].

The main disadvantages of this method is, firstly, the forced genetic modification of the V antigen, increasing its primary sequence by at least 6 amino acid residues. Secondly, the need to introduce a site into the primary sequence for subsequent specific proteolytic cleavage of (His) ₆ . Thirdly, the need for additional stages of purification of proteolysis products.

Closest to the proposed method is a method for producing an immunogenic polypetide from E. coli cells, the primary sequence of which is completely identical to the natural one. In this case, E. coli cells are destroyed by pressure, centrifuged and V antigen is isolated from the supernatant fraction using standard chromatographic steps [27]. Initially, part of the ballast proteins is removed from the supernatant by precipitation with 1 M ammonium sulfate. The fraction clarified by centrifugation was passed through a phenyl-sepharose column and the bound proteins were eluted sequentially with a downward linear gradient of ammonium sulfate. The fractions containing the target protein are combined and removed with ammonium sulfate by exhaustive dialysis. The second stage of isolation is carried out by charge on a chromatographic column with DEAE-Sepharose, with which the proteins elute with a buffer solution with an increasing concentration of sodium chloride. The resulting eluate fractions were combined, concentrated and further purified using high performance gel exclusive chromatography on a Hiprep Superdex 75 column (26/60).

The main disadvantage of this method is the mismatch of the steps of chromatographic purification, forcing the authors to introduce intermediate lengthy preparatory steps. In addition, gel-exclusive chromatography on a Hiprep Superdex 75 column is included in the main scheme, which has low productivity, which leads to a 20-fold dilution of the final purified protein preparation.

The present invention is to obtain a new immunogenic polypeptide, promising for the development of a new subunit plague vaccine with increased immunogenicity.

To solve the problem:

- the proposed nucleotide sequence encoding the immunogenic LcrV polypeptide (G113), shown in figure 2;

- designed recombinant plasmid DNA pETV-I-3455, having a size of 6538 bp, encoding an immunogenic LcrV polypeptide (G113) and consisting of the following elements: replicon of plasmid pBR322, β-lactamase gene that determines resistance to ampicillin, T7 promoter, a lac operator, an f1 replicon, and a DNA fragment flanked by restriction enzyme sites NdeI and HindIII, encoding the synthesis of the LcrV protein (G113), starting with the ATG initiation codon;

- proposed a recombinant strain of bacteria E. coli BL21 (DE3) / pETV-I-3455 - producer of the immunogenic LcrV polypeptide (Gl13);

- a method has been developed for the preparation of recombinant LcrV polypetide (G113) by culturing E. coli strain BL21 (DE3) / pETV-I-3455, characterized in that the cultured cells are destroyed in a buffer solution by ultrasound, and the selection begins with gel permeation chromatography using carrier TSK HW-40 and complete sequentially with anion exchange and hydrophobic chromatography;

- proposed immunogenic LcrV polypeptide (G113) produced by a recombinant E. coli strain BL21 (DE3) / pETV-I-3455, having the amino acid sequence shown in figure 3, in which the tryptophan at position 113 (W113) was replaced by glycine.

We found that replacing tryptophan with glycine in the amino acid sequence LcrV (G113) leads to the loss of local hydrophobic and hydrogen bonds, destabilization of the 108-112 loop and a change in its structure and, as a consequence, the structure of the whole protein, which, in turn, leads to a change in the physical properties of the molecule — hydrophobicity, resistance to proteolysis, and the ability to agglomerate. The increased agglomerating ability of LcrV (G113) correlates with an increase in its immunogenicity and protective activity, which is confirmed in the present invention by a significant increase in protection against infection caused by Y. pestis, by introducing a sufficient amount of the immunogenic LcrV (G113) polypeptide on a suitable carrier.

In the process of studying the physicochemical and biological properties of the improved immunogenic LcrV polypeptide (G113), we found that:

- The antigen LcrV (G113) shows a pronounced ability to agglomerate. When conducting denaturing electrophoresis under non-reducing conditions, up to 50% of the protein was located in the band corresponding to the dimeric form.

- Limited proteolysis of V antigens with different amino acid sequences is accompanied by the appearance of various proteolytic fragments, the molecular weights of which depended on the nature of the antigen and the duration of proteolysis.

- Our obtained LcrV polypeptide (G113) induces a more pronounced immune response in animals compared to other structural variants of the LcrV protein studied. In animals vaccinated with LcrV (G113), antibody titers reached 1: 254000, and the immunity index (ratio of LD ₅₀ calculated for vaccinated animals to the same indicator calculated for intact animals) was 5.7 × 10 ⁵ , while how immunization with non-agglomerating structural variants of the LcrV protein (W113) induced low antibody titers (1: 16000) and practically did not protect mice from infection. The absence of pronounced antibody-inducing and protective activities in most of the LcrV types studied by us suggests that the know-how was omitted in the publications of foreign researchers, which makes it possible to increase the degree of agglomeration of the protein included in its composition at the stage of preparation of the vaccine. When animals were infected with a virulent strain of Y. pestis, the LcrV (G113) polypeptide had significantly higher protective properties compared to other structural variants of LcrV - LcrV (W113) and LcrV (E113). It is proposed to consider LcrV (G113) as a promising candidate for the development of a new subunit plague vaccine with increased immunogenicity.

The proposed bacterial strain E. coli BL21 (DE3) / pETV-I-3455 is characterized by the following features.

Morphological signs. According to these characteristics, the producer strain of the recombinant protein LcrV (G113) does not differ from the original E. coli strain BL21 (DE3), which does not contain the plasmid pETV-1-3455.

Cultural signs. According to cultural characteristics, the producer strain of the recombinant protein LcrV (G113) does not differ from the original E. coli strain BL21 (DE3) that does not contain the target plasmid.

Antibiotic resistance. The cells of the producer strain of the recombinant protein LcrV (G113) show resistance to ampicillin, due to the presence of the target plasmid.

A significant difference between the strain E. coli BL21 (DE3) / pETV-I-3455 is that it provides the synthesis of the LcrV polypeptide (G113), which has the properties of an immunogenic antigen with a soluble form of the target protein producing up to 120 mg / L.

The strain E. coli BL21 (DE3) / pETV-I-3455 was deposited in the collection of microorganisms of the Federal State Institution of Science and Science of the Russian Academy of Medical Sciences, catalog number B-6527.

The invention is illustrated by the following graphic materials.

Figure 1. - Organization scheme of the recombinant plasmid pETV-I-3455, where: LcrV is a cloned fragment comprising the complete gene of the LcrV protein (G113); unique sites of restriction endonucleases - HindIII and NdeI, used to create this construct; T7 is the promoter present in the recombinant plasmid; 1acI is a β-lactamase gene that provides transformed bacterial cells with ampicillin resistance.

Figure 2. The nucleotide sequence of the IcrV gene encoding the synthesis of the immunogenic LcrV polypeptide (G113).

Figure 3. Amino acid sequence of the immunogenic LcrV polypeptide (G113).

Figure 4. Comparison of the sequences of IcrV genes of different strains of Y. pestis.

Figure 5. Comparison of the amino acid sequences of the LcrV protein from different strains of Y. pestis.

6. Electrophoretic analysis of V antigens after heat treatment.

7. Electrophoretic analysis of partial trypsinolysis of V antigens.

Fig. 8. Antibody titers in mouse sera after immunization.

For a better understanding of the invention, the following are detailed examples of its specific implementation with reference to the attached tables and figures.

Example 1

SEQUENCE, AMINO ACID COMPOSITION.

To determine the nucleotide sequence of the Y. pestis IcrV gene, a direct sequencing method is used without prior cloning of genomic DNA fragments. The nucleotide sequence of the open reading frame (ORF) of the IcrV gene of strain Y. pestis I-3455 subsp is determined. altaica. The nucleotide sequence is determined using an ABI Applied Biosistems instrument using sequencing oligonucleotide primers complementary to the nucleotide sequence of the IcrG (Forward I primer), IcrV (Forward 2 inside primer), sycD (Reverse primer) genes.

Based on the obtained sequence, the amino acid sequence of the LcrV protein (G113) is determined, which is further used to develop a three-dimensional model of LcrV (G113) and to predict its physicochemical and biological properties (Fig. 3).

The figures (figures 4 and 5) show the results of comparing nucleotide and amino acid sequences from different strains of Y. pestis.

Example 2

CONSTRUCTION OF PLASMIDES.

Plasmid pETV-I-3455 was established as a result of the following manipulations: amplification of the lcrV gene (9Sl n.p.) in PCR on the total DNA matrix of strain Y. pestis I-3455 using the Forward 3 and Reverse 2 cloning primers, PCR-restriction a fragment of NdeI and HindIII endonucleases, ligation of it with restriction of NdeI and HindIII vector DNA endonucleases, under the control of the T7l promoter in the pET32b (+) vector, transformation of cells of the recipient strain; selection on a medium containing 50 μg / ml ampicillin. The selected recombinant E. coli clones show maximum protein production after 3 h of induction with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), the target protein being at least 50% of all cell proteins of the producer strain.

Sequencing primers:

Forward i primer

5 '- cag cct caac atc cct acga

Forward 2 inside primer

5 '- gca aaa tgg cat caa gcg ag

Reverse primer

5 '- tag ttc ccc ctc ctt tta gg-3'

Cloning primers:

Forward 3

5'- gg cat atg att aga gcc tac gaa c-3 '

Reverse 2

5 '- aa aag ctt gtc tgt cgt ctc ttg ttg c-3'

Figure 2 represents the nucleotide sequence of the Y. pestis IcrV gene, size 981 bp, which encodes the synthesis of the immunogenic LcrV polypeptide (Gl 13).

Example 3

OBTAINING A STRAIN PRODUCER OF IMPROVED IMMUNOGENIC POLYPEPTIDE LcrV (G113).

The producer strain of the immunogenic LcrV polypeptide (G113) is constructed on the basis of the protease-deficient strain E. coli BL21 (DE3) [F ^- ompT hsdSB (r _B ^- m _B ^- ) gal dcm (DE3)] carrying the T7 RNA polymerase gene under the control of IPTG- inducible lacUV5 promoter (No-vagen, USA). The strain is obtained as a result of the following manipulations:

amplification of the IcrV Y. pestis I-3455 gene by PCR,

embedding the PCR fragment in the vector pET32b (+),

transformation of the cells of the recipient strain.

As a matrix in PCR, the DNA of Y. pestis I-3455 strain is used.

E. coli BL21 (DE3) cells were transformed with the recombinant plasmid pETV-I-3455 and clones with the Ap ^R phenotype were selected on selective medium with ampicillin (50 μg / ml). The strain was called E. coli BL21 (DE3) / pETV-I-3455.

Example 4

Isolation and purification of the immunogenic polypeptide LcrV (Gl 13).

To isolate the V antigen, E. coli BL21 (DE3) / pETV-I-3455 cells were grown at 37 ° C in Luria-Bertani liquid aerated medium in a New Brunsuik Scientific laboratory fermenter with a working volume of 2 L. The cell suspension is centrifuged at 7000 × g for 10 min and a temperature of 4 ° C, the precipitate is suspended in 10 volumes of 20 mm Tris-Hcl (pH 7.5) buffer solution. Cell lysis is performed on ice using an ultrasonic disintegrator (Virsonic 16-850, USA). The effectiveness of scoring is controlled by microscopy, after which the cell lysate is centrifuged at 15,000 × g for 25 minutes and the supernatant fraction is sterilized by microfiltration (0.22 μm). At the first stage, the filtrate is subjected to gel exclusion chromatography, passing through a column (26/60) packed with TSK HW-40, previously equilibrated with 20 mM Tris-Hcl buffer, pH 8.0, containing 1 mM ED-TA. The same buffer pre-balance the anion-exchange column of the second stage. The target protein is collected in the calculated volume of the eluate, in fractions immediately following the free volume of the column. Thus, at the first stage, low molecular weight (less than 10 kDa) impurities are removed and at the same time the buffer solution is replaced, the properties of which optimally correspond to the conditions of anion exchange chromatography at the second stage of purification. The second purification step begins immediately after selection of the calculated volume of the eluate, which is applied to the column packed with DEAE TSK 650F (26/20). Unbound proteins are separated by washing the column with the same buffer solution, and the V antigen is eluted with a stepwise gradient of sodium chloride. Fractions are analyzed, combined and mixed with an equivalent volume of a 2 M solution of ammonium sulfate. The resulting solution was centrifuged and passed through a phenyl-sepharose column (26/10), previously equilibrated with Tris buffer containing 1 M ammonium sulfate. At the final, third stage of purification, the ballast proteins are removed from the column with the same buffer with ammonium sulfate and the V antigen is eluted with a downward stepwise gradient of this salt. The resulting preparation of highly purified V antigen is dialyzed to remove traces of ammonium sulfate. The yield of V antigen with a purity of at least 95% is 35 mg per liter of culture at a protein production level of 75 mg / L (according to the results of SDS electrophoresis under reducing conditions). The proposed method allows to simplify and speed up the process by eliminating the need for preparation of the drug before each stage of chromatography. So, the initial stage of gel-exclusive chromatography of clarified lysate is at the same time preparation of the preparation for the second stage of anion exchange chromatography. The low efficiency of gel-exclusive chromatography is overcome due to the high flow rate on the TSK HW-40 hard support, which allows proteins with molecular weights from 0.5 kDa to 10 kDa. Thus, the immunogenic polypeptide is rapidly eluted immediately after the free volume of the column and is separated from all impurities whose molecular weights did not exceed 10 kDa. This allows the use of large volumes of lysate application, the limiting value of which reaches 1/3 of the column volume. The release of the drug from low molecular weight impurities that can reduce the capacity of the anion-exchange column allows, at the second stage, anion-exchange chromatography under optimal conditions and to improve the separation of the target protein. The preparation of fractions containing an immunogenic polypeptide for the third stage is essentially reduced to a double dilution with a solution of 2 M ammonium sulfate. Thus, long intermediate stages of dialysis and drug concentration by ultrafiltration are excluded.

Checking the proposed method for compliance with its criterion of "inventive step" showed that neither in the scientific nor in the patent literature revealed a combination of features specified in the characterizing part of the claims.

Example 5. ADDITIONAL CHARACTERISTIC OF THE PRODUCED PRODUCT.

Agglomeration. Solutions of antigens LcrV (W113), LcrV (G113) are normalized by the concentration of total protein to 1 mg / ml and incubated at 45 ° C for 20 hours, after which the samples are analyzed by denaturing electrophoresis in 12% polyacrylamide gel under non-reducing conditions, as well as in a 7% gel, prepared without sodium dodecyl sulfate. Each sample was mixed in a 1: 1 ratio with a buffer solution containing 125 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.02% bromophenol blue, and heated in a boiling water bath for 5 min. in parallel, the samples are subjected to the same treatment with a buffer containing 10% 2-mercaptoethanol; for a 7% gel, the samples are not heated. Proteins in gels stain Coomassie G-250.

Long-term warming contributes to the formation of agglomerated forms of protein in the preparations, as can be seen from the enhancement of high molecular weight bands on electrophoregrams (Fig.6). Processing samples prior to electrophoresis with 2-mercaptoethanol leads to the disappearance of high molecular weight bands in all relevant samples both on native gel (B) and on gel (A) with sodium dodecyl sulfate (Fig.6). Thus, the stability of the agglomerated V antigen is maintained by disulfide bonds, which is confirmed by treatment with 2-mercaptoethanol, leading to the formation of monomers, and the proportion of monomers decreases during the heat treatment of protein solutions.

Proteolysis Proteolytic cleavage is used to analyze the thermal agglomeration of V antigens. The pathway of enzymatic cleavage of protein antigens under non-denaturing conditions is determined by the availability of proteolysis sites on the surface of the protein, which can lead to the formation of various proteolytic fragments. Preparations of V antigens are treated with trypsin at room temperature, the ratio of the mass fractions of the enzyme and the cleaved protein, as well as the duration of proteolysis, are established by preliminary experiments.

It was shown that within 2 hours of treatment with the enzyme, partial proteolysis of V antigens takes place, accompanied by the appearance of various proteolytic fragments (Fig. 7). The molecular weights of the fragments depend on the duration of proteolysis and the nature of the antigen.

Example 6. TESTING IMMUNOGENOUS ACTIVITY

Immunization and infection of mice. The antigens LcrV (W113), LcrV (E113), LcrV (G113) are sorbed on sterile aluminum hydroxide in phosphate-buffered saline with stirring for 2 hours at room temperature. Proteins are pre-sterilized by passing through a membrane with a pore diameter of 0.22 μm. The protein concentration is calculated so that 200 μl of the suspension contains 10 μg of protein. Immunization and infection are carried out according to the following scheme:

Day 0: Immunize mice with 10 μg of LcrV proteins sorbed on aluminum hydroxide.

30th day - re-immunization with the same dose of LcrV protein sorbed on aluminum hydroxide.

62nd day - blood sampling to determine the titers of antibodies of the control group.

64th day - infection of mice with a virulent strain of Y. pestis 231.

85th day (21st day after infection) - the final account of the results of the experiment.

In the experiment, 7–8 weeks old BALB / c female mice were used, immunized with the following proteins: LcrV (W113) with tryptophan at position 113, LcrV (E113) with glutamic acid at position 113, and LcrV (G113) with glycine at position 113. The control group is represented by animals immunized with aluminum hydroxide at a dose equal to the amount of drug administered to animals along with LcrV proteins. Preparations of V antigens are administered to mice subcutaneously in a volume of 200 μl, in the inguinal region.

Determination of antibody titers. Use enzyme-linked immunosorbent assay; V antigens are sorbed into the wells of the plates for 1 h at 37 ° C in 0.1 M carbonate buffer, pH 9.6. At all stages of ELISA, the wells are washed with 10 mM Tris buffer, pH 7.2, containing 0.15 M NaCl and 0.05% Tween-20. The sera of immunized animals are bred with the same buffer, the smallest dilution is 1: 1000, then, starting with this sample, sequentially, in increments of 2.5. The duration of incubation of the working solutions in the wells at all stages is the same and is 1 hour at 37 ° C. Conjugates of anti-mouse antibodies with horseradish peroxidase breed 1: 3000. The development of a color reaction occurs due to a donor-acceptor pair: hydrogen peroxide - o-phenylenediamine in the composition of the substrate solution. The reaction is stopped after 15 minutes by adding 50 μl of a 1 M sulfuric acid solution to the wells and the optical density of the solutions in the samples is measured at 492 nm. The titer is defined as the value of the highest dilution of serum, the optical density of which was two times higher than the optical density of the solution in the corresponding control well.

The results are presented in Fig. 8. The figure is a graph showing the titers of anti-LcrV antibodies in the blood of mice immunized with the immunogenic LcrV polypeptide (G113), compared to antibody titers of mice immunized with other LcrV preparations.

LcrV preparations have varying degrees of immunogenicity and induce the production of antibodies specific for the Lcr protein in mice. Antibody titers determined by the results of enzyme-linked immunosorbent assay are in a wide range, from 1: 8300 to 1: 254000, and show a dependence on the structure of the polypeptides used. The highest degree of immunogenicity was shown by the immunogenic LcrV polypeptide (G113), the titers of antibodies to which reach 1: 254000 (Fig. 8). Antibody titers of the LcrV (W113) and LcrV (E113) proteins were 1: 16000, that is, they have significantly lower immunogenicity.

Definition of protection. The antigenicity of V antigens is determined by infecting pre-immunized mice with a virulent strain of Ypestis 231. Infection is carried out by injecting a ten-fold dilution of the bacterial culture under the skin of the thigh of animals in 0.2 ml of buffered saline. Observation of infected animals is carried out for 21 days. Surviving animals are humanely euthanized by inhalation of CO ₂ .

The calculation of the LD ₅₀ values and the confidence interval (for a probability of 95%) are carried out according to the Karber method in the modification of IP Ashmarin and AA Vorobyov [28].

Tables 1 and 2 show the data on the protectiveness of various structural variants of LcrV.

Table 1 The death of mice previously immunized with various structural variants of the immunogenic LcrV polypeptide after infection with F. pestis 231 (observation period 21 days). Variants of the immunogenic LcrV polypeptide Infecting Dose Y. pestis 231, CFU one 10 10 ² 10 ³ 10 ⁴ 10 ⁵ 10 ⁶ 10 ⁷ LcrV (W113) 2/5 * 0/5 0/5 0/5 0/5 0/5 1/5 LcrV (G113) 5/5 5/5 4/5 3/5 4/5 4/5 5/5 LcrV (E113) 2/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 1 (vaccinated Al (OH) ₃ ) 2/5 2/5 0/5 0/5 - - - - Control 2 (not vaccinated) 5/5 1/5 0/5 0/5 - - - - * survivors after infection / all animals in the group

table 2 The characterization of the protectiveness of various structural variants of the immunogenic LcrV polypeptide. Variants of the immunogenic LcrV polypeptide LD ₅₀ Immunity Index * LcrV (W113) 7 2,3 LcrV (G113) 1.7 × 10 ⁶ 5.7 × 10 ⁵ LcrV (E113) four 1.3 Control 1 (vaccinated Al (OH) ₃ ) one 0.3 Control 2 (not vaccinated) 3 - * Immunity Index - LD ₅₀ for immunized / LD ₅₀ for intact animals

Infection with a virulent strain of Y. pestis 231 leads to almost complete death of animals immunized with LcrV (W113) at all infecting doses (Table 1). LD ₅₀ strain 231 in relation to mice immunized with the LcrV antigen (W113), slightly differs from the control. In groups of mice pre-immunized with the LcrV antigen (G113), a slight death of animals was observed (on average, one individual for each of the infecting doses). For the same group of animals, an increase of LD ₅₀ by six orders of magnitude was noted both in comparison with the control groups and with the groups immunized with other structural variants of LcrV (Table 2).

Thus, a change in the amino acid sequence of LcrV, which consists in replacing tryptophan at position 113 with glycine, ultimately leads to an increase in the ability of this polypeptide to agglomerate, a sharp increase in its immunogenicity and protective activity. A producer of the improved immunogenic LcrV polypeptide (G113) was designed to produce preparative amounts of antigen for use in the subunit plague vaccine.

Claims (5)

1. The nucleotide sequence encoding the immunogenic LcrV polypeptide (G113) is shown in Fig. 2. 2. Recombinant plasmid DNA pETV-I-3455, having a size of 6538 bp, encoding the immunogenic LcrV polypeptide (G113) and consisting of the following elements:
replicon plasmids pBR322,
β-lactamase gene, which determines ampicillin resistance,
T7 promoter
1ac operator
fl-replicon
a DNA fragment flanked by the Ndel and Hindlll restriction enzyme sites encoding the synthesis of the LcrV protein (G113) starting at the ATG initiation codon. 3. The recombinant bacterial strain E. coli BL21 (DE3) / pETV-I-3455 is the producer of the immunogenic LcrV polypeptide (G113), deposited in the FSI SSC PMB (catalog number B-6527). 4. The immunogenic LcrV polypeptide (G113) produced by the recombinant E. coli strain BL21 (DE3) / pETV-I-3455, having the amino acid sequence shown in Fig. 3, in which tryptophan at position 113 (W113) was replaced by glycine. 5. A method of producing a recombinant LcrV polypeptide (G113) by culturing an E. coli strain followed by isolation of the polypeptide, characterized in that the E. coli strain BL21 (DE3) / pETV-I-3455 is cultured, and cultured cells are destroyed in a buffer solution by ultrasound, moreover, the selection of the target product begins with gel permeation chromatography using a TSK HW-40 carrier and is completed sequentially by anion exchange and hydrophobic chromatography.

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