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

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

 

The invention relates to biotechnology, in particular the production of immunogenic polypeptide capable of forming a protective immune response against infections caused by Yersinia pestis.

Specific prophylaxis of especially dangerous infections of bacterial etiology, particularly of the plague, due to intense epidemiological situation, due to the presence on the territory of Russia, an active natural foci of plague, by the importation from abroad, and the real threat of bioterrorism - reasons that can cause epidemic manifestations of an emergency character. Currently in the Russian Federation for the specific prophylaxis of plague used live vaccines developed in the middle of the last century and characterized by a significant reactogenicity. Offering the best produktivnost, live vaccines are cheap to manufacture, but because of the presence of a significant number of "ballast" components cause high antigenic load on the body, increased allergic reactions and ineffective for holding revaccinate. These drawbacks, in particular, chemical (subunit, molecular) vaccines, which allow for the expense of incorporating only the immunodominant antigens significantly reduce reactogenicity of the drug as a whole; the effect is positive for revaccination; can be used against emergency prevention antibiotics; exclude the possibility of the occurrence of infection in persons with impaired immune status.

It is known that immunization of animals live bacteria Y. pestis, producing V antigen [1], as well as purified native [2] or recombinant [3] V antigen led to induction or antibody-based test response and protected mice against infection with a virulent strain of plague.

V antigen (LcrV), one of the "classic" factors of pathogenicity of Y. pestis, is encoded by gene IcrV in the structure of the operon IcrGVH [4]located on the plasmids calcinations pCad (pCD1, pVW, pYV or pLcr)that is present in all pathogenic to humans Yersinia [5], was first described by T.W. Burrows [6]. V antigen produce two other pathogenic for humans species of the genus Yersinia: Y. pseudotuberculosis and Y. enterocolitica [5]. It is established that the products of V antigen correlates with virulence of Yersinia spp. [7]. Mutation in the gene IcrV led to the loss of virulence [8]. V antigen regulates the secretion of cytotoxic Yop proteins [9], inhibits chemotaxis of neutrophils [10], has an immunosuppressive effect [11]. The immunosuppressive mechanism of action of V antigen is direct stimulation of the synthesis of anti-inflammatory cytokine, interlekin-10 (IL-10), leading to a General inhibition of anti-inflammatory cytokines [12].

V is antigen is a protein including 326 amino acid residue (S.A.), which corresponds to a molecular weight in 37.2 kDa [13]. The polypeptide forms a mixture of monomers, dimers with a molecular mass of 85-95 kDa and tetramers [14]. Experiments using mass spectrometry identified the molecular weight of the dimer - 75 kDa [2]. The effects of temperature (45°C, 20 h) stimulated an increase in the number of dimers [15]. Found that oligomeric forms of LcrV are more protective ability [16] and stimulate TLR2 (Toll-like receptor2) mediated immune response [14].

Known recombinant plasmid encoding the synthesis of V antigen that causes a protective effect against the action of Y. pestis [17]. However, IcrV genes of Yersinia spp. encodes different ways of LcrV polypeptide [18], which differ from each other in amino acid sequence [19]. Amino acid sequences of the V antigen of Yersinia spp. divided into seven classes, one of which consists of a sequence LcrV Y. pestis and Y. pseudotuberculosis, the rest belong to the strains of Y. enterocolitica. The interrelation between the structure of individual classes V antigen of Y. enterocolitica and virulence producing their strains of this organism [20].

For V antigen of Y. pestis has been shown highly conservative gene IcrV in strains of the main subspecies [21]. Strains of Y. pestis minor subspecies characterized by polymorphism of the Jena IcrV [22], identified 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 on the basis of these data, this location does not have a significant effect on the protein structure and, consequently, does not change the chemical and physical properties of the molecule [23].

Native V antigen is sensitive to the action of bacterial proteases, which leads to its low yield in the allocation of cells Yersinia [24]. To solve this problem have been constructed resistant to proteolysis "fusion" proteins: protein a-V antigen or glutathione-S-transferase-V antigen. A significant disadvantage of this method is the need for proteolytic cleavage of the precursor of the immunogenic peptide and the introduction of additional removal phase derived peptide carrier. Glutathione-S-transferase cut from a sequence of a fusion protein with factor XA [25]. Use in the process of purification of factor XA is a serious limitation in the application obtained similarly immunogenic polypeptide as a component of the vaccine to the people, as factor XA is obtained from the body of the bulls, and to enable use Viper venom Russell.

A method of obtaining immunogenic polypeptide by the method of affine chromium is ographie media with immobilized metallochelate groups, based on the chemical affinity of genetically modified sequence immunogenic polypeptide ions of bivalent metals, acceptors coordination resulting from the introduction of additional polyhistidine inserted into the polypeptide. The strength of the newly formed coordination: ion of the divalent metal (Ni++- polyhistidine (His)6sufficient to keep the target protein on the column when washing the last of the various buffer solutions, including, in the denatured state, for example, in the presence of concentrated solutions of urea or guanidine chloride. The release of the bound protein from the column induce redistribution of coordination bonds after the introduction of strong chelating substances in lucindy buffer. This allows for one-step protein purification, suitable for immunization of animals [26].

The main disadvantages of this method is, first, forced genetic modification of V antigen, increasing its primary sequence, at least 6 amino acid residues. Secondly, the necessity of introducing into the primary sequence of the website for further specific proteolytic cleavage (His)6. Thirdly the need for additional steps clear the products of proteolysis.

Closest to the proposed method is a method of obtaining immunogenic polypeptide from cells of E. coli, the primary sequence which are identical to natural. In this case, cells of E. coli destroy pressure, centrifuged and allocate V antigen from the supernatant fraction using standard chromatographic steps [27]. Originally from the supernatant fraction removed part of ballast proteins by precipitation of 1 M ammonium sulfate. Clarified by centrifugation fraction is passed through a column of phenyl-separate and sequentially elute associated proteins downward linear gradient of ammonium sulfate. The fractions containing the target protein are pooled and remove the ammonium sulphate exhaustive dialysis. The second stage of selection is carried out by the charge on the chromatographic column with DEAE-separate with which proteins elute buffer solution with increasing concentration of sodium chloride. The obtained fractions of the eluate unite, concentrate, and subjected to additional cleaning using high-performance gel-exclusive chromatography on a column Hiprep Superdex 75" (26/60).

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

The present invention is to obtain a new immunogenic polypeptide that is promising for the development of 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;

- constructed recombinant plasmid DNA pETV-I-3455 sized 6538 P.N. encoding immunogenic LcrV polypeptide(G113) and consisting of the following elements: replicon plasmids pBR322, gene β-lactamase gene determining resistance to ampicillin, T7 - promoter, lac operator, f1 replicon and DNA fragment, flanked by sites for restricted NdeI and HindIII encoding the fusion protein LcrV(G113), beginning with the initiator to don ATG;

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

- developed a method of obtaining a recombinant polypeptide LcrV(G113) under cultivation of E. coli strain BL21(DE3)/pETV-I-3455, wherein the cultured cells destroy in a buffer solution by ultrasound, and the selection start is with phase gel permeation chromatography using a carrier TSK HW-40 and complete in series anion exchange and hydrophobic chromatography;

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

We found that the substitution of tryptophan for glycine in the amino acid sequences of LcrV(G113) leads to the loss of local hydrophobic and hydrogen bonds, destabilizing loops 108-112 and changes in its structure and, as a consequence, the structure of the whole protein, which, in turn, leads to changes in the physical properties of the molecule hydrophobicity, resistance to proteolysis, the ability to agglomeration. Increased agglomerated ability LcrV(G113) correlates with the increase of its immunogenicity and protective activity, which in the present invention is confirmed by the significant strengthening of the protection against infection caused by Y. pestis, by introducing a sufficient number of immunogenic LcrV polypeptide(G113) on a suitable medium.

In the process of studying the physico-chemical and biological properties superior immunogenic LcrV polypeptide(G113), we found that:

Antigen LcrV(G113) shows a pronounced ability to agglomeration. When conducting denaturing electrophoresis in nereguliruemyi conditions up to 50% of the protein was located in the band corresponding to the dimeric form.

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

- We received the LcrV polypeptide(G113) causes a more pronounced immune response in animals compared with the other investigated structural variants LcrV protein. In animals vaccinated with LcrV(G113), the antibody titers reached 1:254000, and the index of immunity (the ratio of the magnitude of LD50designed for vaccinated animals to the same indicator calculated for intact animals) amounted to 5.7×105, while immunization paglalarawan structural variants of the protein LcrV(W113) induced low antibody titers (1:16000) and almost did not protect mice from infection. The absence of expressed antibody-inducing and protective activities most investigated types of LcrV suggests that in the publications of foreign researchers omitted know-how, allowing for preparation of vaccine preparation to increase the degree of agglomeration is included in the composition of the protein. When infected animals virulent strain of Y. pestis LcrV polypeptide(G113) had a significantly higher protective properties compared with other structural variants LcrV - LcrV(W113) and LcrV(E113). Invited to consider LcrV(G113) as a promising candidate for the development of new subunit plague vaccine is increased immunogenicity.

We offer the bacterial strain E. coli BL21(DE3)/pETV-I-3455 characterized by the following features.

Morphological features. These attributes are producing strains of recombinant protein LcrV(G113) is not different from the original strain E. coli BL21(DE3)containing no plasmid V-1-3455.

Cultural characteristics. Cultural characteristics of strain-producer of recombinant protein LcrV(G113) is not different from the original strain E. coli BL21(DE3), not containing target plasmid.

Resistance to antibiotics. Cells of strain-producer of recombinant protein LcrV(G113) are resistant to ampicillin due to the presence of the target plasmid.

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

The strain E. coli BL21(DE3)/pETV-I-3455 deposited in the collection of microorganisms fsri SRC MBP, catalogue number-6527.

The invention is illustrated in the following graphics.

Figure 1. - Organization of the recombinant plasmids pETV-I-3455, where: LcrV-cloned fragment, containing the complete gene protein LcrV(G113); the unique sites of the restriction endonucleases - HindIII and NdeI used to create this design; T7 - promoter present in the recombinant plasmid; 1acI gene β-lacto is the basics, providing the transformed bacterial cells resistant to ampicillin.

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

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

Figure 4. Comparison of gene sequences IcrV different strains of Y. pestis.

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

6. Electrophoretic analysis V antigens after heat treatment.

7. Electrophoretic analysis of partial trypsinolysis V antigens.

Fig. Antibody titers in sera of mice after immunization.

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

Example 1.

SEQUENCE, AMINO ACID COMPOSITION.

To determine the nucleotide sequence of the gene IcrV Y. pestis use the direct method of sequencing without prior cloning of genomic DNA fragments. Determine the nucleotide sequence of an open reading frame (ORF) of the gene IcrV strain Y. pestis And-3455 subsp. altaica. Determination of the nucleotide sequence is performed on the device ABI Applied Biosistems using Sequeira oligonucleotide primers complementary nucleotide sequence is eljnosti genes IcrG (Forward primer I), IcrV (2 inside Forward primer), sycD (Reverse primer).

On the basis of the received sequence determines the amino acid sequence of the protein LcrV(G113), which was then used to develop three-dimensional models LcrV(G113) and predict its physicochemical and biological properties (figure 3).

In figures (4 and 5) presents the results of the comparison of the nucleotide and amino acid sequences from different strains of Y. pestis.

Example 2.

KONSTRUIROVANIE PLASMIDS.

Plasmid pETV-I-3455 construed in the following manipulations: gene amplification lcrV(9Sl n) in PCR on the matrix of total DNA of strain Y. pestis I-3455 using the cloning primers Forward 3 and Reverse 2", PCR restriction fragment endonucleases NdeI and HindIII, ligation with restricciones endonucleases NdeI and HindIII vector DNA under the control of 7l-promoter in the vector 32b(+), transformation of cells of the recipient strain; selection on medium containing 50 μg/ml ampicillin. Selected recombinant clones of E. coli exhibit a maximum production of protein after 3 h induction with 1 mm isopropyl-β-D-thiogalactopyranoside (IPTG), and the target protein is at least 50% of all cellular proteins producer strain.

Sequeira primers:

Forward primer I

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:

p> 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 gene IcrV Y. pestis, the size of 981 BP, which encodes the synthesis of immunogenic LcrV polypeptide(Gl 13).

Example 3

Getting a PRODUCER STRAIN IMPROVED IMMUNOGENIC LcrV POLYPEPTIDE(G113).

Producing strains of immunogenic LcrV polypeptide(G113) design based on processdefinition strain E. coli BL21(DE3) [F-ompT hsdSB (rB-mB-) gal dcm (DE3)], which carries the gene for T7 RNA polymerase under the control of IPTG-inducible lacUV5 promoter (No-vagen, USA). The strain obtained the following manipulations:

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

embed PCR fragment into a vector 32b(+),

transformation of cells of the recipient strain.

As a matrix in PCR using DNA of strain Y. pestis And-3455.

Cells of E. coli BL21(DE3) transformed with recombinant plasmid pETV-I-3455 and select the clones with the phenotype ApRon the selective medium with ampicillin (50 μg/ml). The strain was named E. coli BL21(DE3)/pETV-I-3455.

Example 4.

Isolation AND PURIFICATION of IMMUNOGENIC LcrV POLYPEPTIDE(Gl 13).

To highlight the V antigen of E. coli cells BL21(DE3)/pETV-I-3455 grown at 37°C in aerated liquid medium Luria-Bertani in laboratory fermenter New Brunsuik Scientific displacement of 2 liters of Cell suspension centrifuge holds irout at 7000 × g for 10 min and 4°C, the residue is suspended in 10 volumes of 20 mm Tris-Hcl (pH 7.5) buffer solution. Lysis of the cells is performed on ice using an ultrasonic disintegrator (Virsonic 16-850, USA). The effectiveness of sound control microscopia, then centrifuged cell lysate at 15000 × g for 25 min and the supernatant fraction is sterilized by filtration (0.22 μm). In the first stage, the filtrate is subjected to galaxylife chromatography, passing through the column (26/60), Packed TSK HW-40, pre-equilibrated to 20 mm Tris-Hcl buffer, pH 8.0, containing 1 mm ED TA. The same buffer pre-balancing anion-exchange column of the second stage. The target protein is collected in the estimated volume of the eluate in fractions, following directly behind the free volume of the column. Thus, in the first stage removes low molecular weight (less than 10 kDa) impurities and simultaneously replace the buffer solution, the properties of which in optimum extent consistent with the terms anion-exchange chromatography in the second stage of purification. The second stage of the treatment begin immediately upon selection of the charge volume of the eluate, which is applied on a column Packed with TSK DEAE 650F (26/20). Unbound proteins separated by washing the column with the same buffer solution and elute V antigen of step gradient of sodium chloride. Faction analyze, combine and mix with equivalent is alentum volume of 2 M solution of ammonium sulfate. The resulting solution was centrifuged and passed through a column of phenyl-separate (26/10), pre-equilibrated Tris-buffer containing 1 M ammonium sulphate. The final, third stage of the treatment, ballast proteins are removed from the column with the same buffer with ammonium sulfate and spend the elution of V antigen descending stepwise gradient of this salt. The resulting preparation of high-purity V antigen cialiswhat in order to remove traces of ammonium sulfate. The output of the V antigen purity of at least 95% is 35 mg from one liter of culture at the level of production of the protein of 75 mg/l (based on the results of the LTO-electrophoresis in reducing conditions). The proposed method allows to simplify and speed up the process by eliminating the need for preparation before each phase chromatography. So, the initial stage of a gel-exclusive chromatography clarified lysate is simultaneously the preparation of the preparation for the second phase anion-exchange chromatography. Low efficiency of the gel-exclusive chromatography is overcome due to the high flow speed in hard copy TSK HW-40, allowing proteins with molecular masses ranging from 0.5 kDa to 10 kDa. Thus, the immunogenic polypeptide quickly elute directly behind the free volume of the column and separated from all impurities, molecular weight which does not exceed the Ali 10 kDa. This allows the use of large volumes of application of the lysate, the marginal value of which reaches 1/3 volume of the column. The release of drug from low molecular weight impurities can reduce the capacity of the anion-exchange column, allows the second stage to hold anyone-exchange chromatography under optimal conditions and to improve the separation of the target protein. Preparation of fractions containing immunogenic polypeptide, the third stage is essentially a twofold dilution with a solution of 2 M of ammonium sulfate. Thus eliminating long intermediate stages of dialysis and concentration of the preparation by ultrafiltration.

Validation of the proposed method for compliance with a criterion of "inventive step" showed that neither in science nor in the patent literature have not revealed the combination of features specified in the characterizing part of the claims.

Example 5. ADDITIONAL CHARACTERISTICS OF THE OBTAINED PRODUCT.

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

Long warming contributes to the formation of agglomerated forms of protein drugs, which can be seen to strengthen the high molecular weight bands on electrophoregram (6). Processing of samples before electrophoresis 2-mercaptoethanol leads to the disappearance of high molecular weight bands in all relevant samples as the native gel (B)and the gel (A) with sodium dodecyl sulfate (6). Thus, the stability of the agglomerated V antigen is supported by disulfide bonds, which confirms the processing of 2-mercaptoethanol, leading to the formation of monomers, the percentage of monomers is reduced during the heat treatment of protein solutions.

Proteolysis. Use of proteolytic cleavage analysis of the peculiarities of thermal agglomeration V antigens. The path of enzymatic degradation of protein antigens in adenocarinoma conditions determined by the availability of sites of proteolysis on the surface of the protein, which can lead to the formation of various proteolytic mamantov. Drugs V antigens on srabatyvayut trypsin at room temperature, the mass proportion of the enzyme and cleaved protein, as well as the duration of proteolysis ustanavlivat preliminary experiments.

It is shown that within 2 h of treatment with the enzyme undergoes partial proteolysis V antigens, accompanied by the emergence of various proteolytic fragments (Fig.7). The molecular weight of the fragments depends on the length of proteolysis and the nature of the antigen.

Example 6. TESTING IMMUNOGENIC

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

0-day: immunization of mice 10 µg protein LcrV, adsorbed on aluminium hydroxide.

30-day re-immunization with the same dose of LcrV protein, adsorbed on aluminium hydroxide.

62 day screening of blood for determination of antibody titers in the control group.

64th day - infected mice virulent strain of Y. pestis 231.

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

In the experience of using the RA is divided into groups of female BALB/c mice aged 7-8 weeks, immunized 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 of animals immunized with aluminum hydroxide at a dose equal to the amount of drug administered to the animals with LcrV proteins. Drugs V antigens injected mice subcutaneously in a volume of 200 μl, in the groin area.

Determination of antibody titers. Use solid-phase enzyme-linked immunosorbent assay; wells tablets adsorb V antigens for 1 h at 37°C in 0.1 M carbonate buffer, pH 9,6. At all stages of the ELISA wells are washed with 10 mm Tris buffer, pH of 7.2, containing 0.15 M NaCl and 0.05% tween-20. Serum of immunized animals are bred the same buffer, the lowest dilution is 1:1000, then, from this sample, sequentially, in increments of 2.5. The incubation period of working solutions in the wells at all stages is the same and equals to 1 h at 37°C. the Conjugates artemisinin antibodies to horseradish peroxidase diluted 1:3000. The development of color reaction occurs due to donor-acceptor pairs: hydrogen peroxide - o-phenylenediamine in the composition of the substrate solution. The reaction is stopped after 15 min by adding to the wells, 50 μl of 1 M sulfuric acid solution and measure the optical density of solutions in the samples at 492 nm. Titer is defined as the greates is well the greatest dilution of serum, the optical density of which is two times the optical density of the solution in the appropriate inspection hole.

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

Drugs LcrV have various degrees of immunogenicity and cause the mice to produce antibodies specific to the protein of the Lcr. Antibody titers, determined according to 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 polypeptides used. The greatest degree of immunogenicity showed immunogenic LcrV polypeptide(G113), antibody titers to which reach 1:254000 (Fig). The titers of antibodies to protein LcrV(W113) and LcrV(E113) was the value of 1:16000, that is, they have a far lower immunogenicity.

Definition of protectively. Protectively V antigens determined by infecting pre-immunized mice virulent strain Ypestis 231. Infection is carried out by introduction into the skin of the thigh animals tenfold dilution of bacterial culture in 0.2 ml of buffered saline solution. Observation of infected animals p is avodat within 21 days. Surviving animals humanely evtanaziyu therapy CO2.

Calculation of quantities LD50and a confidence interval for a probability of 95%) is conducted according to the method of Karber modification Ipomaea and GeV [28].

In tables 1 and 2 show data on protectively different structural variants of LcrV.

td align="center"> LcrV(G113)
Table 1
The death of mice pre-immunized with different structural variants immunogenic LcrV polypeptide, after infection F. pestis 231 (observation period of 21 days).
Options immunogenic LcrV polypeptideInfecting dose of Y. pestis 231, CFU
110102103104105106107
LcrV(W113)2/5*0/50/50/50/50/51/5
5/55/54/53/54/54/55/5
LcrV(E113)2/50/50/50/50/50/50/5
Control 1 (vaccinated Al(Oh)3)2/52/50/50/5----
Control 2 (not vaccinated)5/51/50/50/5----
* surviving after infection/total number of animals in the group

Table 2
Characteristics of protectively various structural options immunogenic LcrV polypeptide.
Options immunogenic LcrV polypeptideLD50Index immunity*
LcrV(W113)72,3
LcrV(G113)1,7×106the 5.7×105
LcrV(E113)41,3
Control 1 (vaccinated Al(Oh)3)10,3
Control 2 (not vaccinated)3-
*Index immunity - LD50for immunized / LD50for intact animals

Infection with virulent strain of Y. pestis 231 leads to the almost complete destruction of animals immunized with LcrV(W113), when all infecting doses (table 1). LD50strain 231 in respect of mice immunized with antigen LcrV(W113), not significantly different from the control. In groups of mice pre-immunized with antigen LcrV(G113), a slight case alive is the shaft (on average one individual on each of the infecting dose). For the same group of animals reported increased LD50six orders of magnitude as compared to the control groups and the groups immunized with other structural variants LcrV (table 2).

Thus, the change of the amino acid sequences of LcrV, which consists in the substitution at position 113 of tryptophan for glycine, leads, ultimately, to improve the ability of the polypeptide to agglomeration, a sharp increase its immunogenicity and protective activity. Designed producing superior immunogenic LcrV polypeptide(G113), suitable for preparative production of antigen for use in the subunit composition of plague vaccine.

1. The nucleotide sequence encoding the immunogenic LcrV polypeptide(G113), shown in figure 2.

2. Recombinant plasmid DNA pETV-I-3455 sized 6538 P.N. encoding immunogenic LcrV polypeptide(G113) and consisting of the following elements:
the replicon plasmids pBR322,
gene β-lactamase, which determines resistance to ampicillin,
T7-promoter,
1 ° C-operator,
fl-replicon,
the DNA fragment, flanked by sites for restricted Ndel and Hindlll encoding the fusion protein LcrV(G113), beginning with the initiating ATG codon.

3. Recombinant bacterial strain E. coli BL21(DE3)/pETV-I-3455 - produce the immunogenic LcrV polypeptide(G113), deposited in the fsri SRC MBP (catalog number B-6527).

4. Immunogenic LcrV polypeptide(G113)produced by recombinant strain E. coli BL21(DE3)/pETV-I-3455 having the amino acid sequence shown in Fig.3, in which the replacement of tryptophan at position 113 (W113) glycine.

5. A method of obtaining a recombinant LcrV polypeptide(G113) by cultivation of a strain of E. coli with subsequent isolation of the polypeptide, wherein the cultured strain E. coli BL21(DE3)/pETV-I-3455 and cultivated destroy cells in the buffer solution by ultrasound, and the selection of the target product to start with phase gel permeation chromatography using a carrier TSK HW-40 and complete in series anion exchange and hydrophobic chromatography.



 

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10 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: method involves cultivation of an obligate methanol-assimilating bacterium Methylophilus methylotrophus or Methylobacillus glycogens in a fluid medium with the bacterium secreting an end protein from a bacterial cell where said bacterium has a DNA structure containing a promoter sequence functioning in the methanol-assimilating bacterium, a nucleotide sequence coding a polypeptide containing a signal sequence which functions in the methanol-assimilating bacterium, and a sequence of the end protein functionally connected with the promoter sequence.

EFFECT: method allows producing the protein effectively by means of extracellular secretion, difficult-to-produce by means of secretory production with application of Escherichia coli bacteria.

5 cl, 7 ex

FIELD: medicine.

SUBSTANCE: there are presented version forms of uricase containing sequences referred in the description. Also, there is presented a recovered nucleic acid containing a nucleotide sequence which codes said uricase, and said nucleic acid is operatively bound with a heterological promoter. There is described an expression vector containing said nucleic acid and a host cell containing this vector and being an uricase producer. There are described a method for producing uricase involving stages of cultivation of said host cells under conditions when the nucleic acid sequence is expressed by the host cells, and of uricase recovery.

EFFECT: invention allows relieving hyperuricemia and hyperuricosuria.

13 cl, 8 dwg, 7 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacology, namely a method for producing cytochrome C. The method for producing cytochrome C involving degreasing of pig, cattle and horse hearts, removal of ligaments and vessels, grinding and extraction in a trichloracetic acid solution under certain conditions, separation of the extract from stuffing, sorption of the filtered extract at extract pH in the UNOsphere™ S cation exchanger balanced with a starter buffer solution at pH 3.9-4.2, after completion of the sorption process, the cation exchanger is washed with the starter buffer solution; ballast proteins are removed from the UNOsphere™ S cation exchanger by a tris solution, and it is followed with elution of cytochrome C from the UNOsphere™ S cation exchanger with an ammonium sulphate solution, concentration and dialysis purification to remove ammonium sulphate ions completely.

EFFECT: method allows increasing product yield, providing higher purity and reducing production process.

4 cl, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: method involves: (a) culturing E.coli cells containing polypeptide-coding nucleic acid in a culture medium into which transportable organophosphate is fed, said organophosphate being selected from a group comprising alpha-glycerophosphate, beta-glycerophosphate, glycerol-3-phosphate, and a mixture of glycerol-2-phosphate and glycerol-3-phosphate, such that nucleic acid is expressed, and (b) extracting polypeptide from cells, wherein during the culturing step, inorganic phosphate is added to the culture medium.

EFFECT: improved heterologous protein expression and high output of the end product.

29 cl, 10 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: in invention described is composition for causing immune reaction against Neisseria meningitidis basing on vesicles, separated from bacteria N. meningitidis, in which endogenous gen GNA 1870 is inactivated and which is transformed by gene construction, coding polypeptide GNA 1870. Such bacterium insures super-expression of GNA 1870 polypeptide in vesicles of N. meningitidis. Composition can additionally contain antigen vesicle from second, third bacteria of N. meningitidis, on condition that both bacteria are genetically different from each other and from said first bacteria. GNA 1870 polypeptides, expressed by first, second and third bacteria are also genetically different from each other. Invention describes method of exciting immune reaction against bacteria of species Neisseria by introduction to mammal of composition based on vesicles, containing polypeptide GNA 1870, as well as method of obtaining composition based on vesicles, obtained as a result of culturing in proper way prepared bacteria, by their mixing with pharmaceutically acceptable carrier.

EFFECT: invention provides protective immunity against broad spectrum of N meningitidis strains, including strains N meningitidis of serogroup B.

45 cl, 20 dwg, 5 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: what is offered is a new method for producing human methionine-free interferon-alpha2b. The method starts with recombinant plasmid DNA containing a human interferon-alpha2b gene which is foregone by an enteropeptidase proteolysis site, and used to transform Escherichia coli cells. The cells are cultured, and inclusion bodies of the synthesised predecessor are isolated. It is followed by partial renaturation of the isolated predecessor in the presence of dithioerythrole preventing closure of disulphide bonds. The predecessor is hydrolysed by the enteropeptidase enzyme with producing human methionine-free interferon-alpha2b. After completion of the predecessor hydrolysis reaction, complete renaturation of human interferon-alpha2b is carried out in the presence of a pair of cystine and cysteine compounds promoting closure of disulphide bonds. The produced protein is purified by a chromatography in a KM-sepharose.

EFFECT: higher yield.

5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to recombinant plasmid DNA pER-Hir coding a hybrid protein capable to autocatalytic breakdown to form [Leul, Thr2]-63-desulphatohirudin, to Escherichia coli to an ER2566/pER-Hir strain - a producer of said protein and a method for producing genetically engineered [Leu 1, Thr2]-63-desulphatohirudin. The presented recombinant plasmid DNA consists of the SapI/BamHI fragment of DNA plasmid pTWIN-1 containing a promoter and a terminator of T7-RNA-polymerase transcription, an amplifier of phages T7 gene 10 translation, β-laktamase (Ap) gene, modified mini-intein Ssp DnaB gene, with an integrated sequence of a chitin-binding domain, and the SapI/BamHI-fragment of DNA containing a sequence of a gene of recombinant [Leul, Thr2]-63-desulphatohirudin-1 containing β-laktamase (Ap) gene as a genetic marker, and unique recognition sites of restriction endonucleases located at the following distance to the left from the site BamHI: Nrul - 186 base pairs, Ndel - 594 base pairs, Xbal - 882 base pairs, EcoRV - 2913 base pairs, Hpal - 2966 base pairs.

EFFECT: inventions allow producing said compound which is used as a drug applied to prevent blood hypercoagulation.

3 cl, 1 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: what is offered is a new method for producing human methionine-free interferon-alpha2b. The method starts with recombinant plasmid DNA containing a human interferon-alpha2b gene which is foregone by an enteropeptidase proteolysis site, and used to transform Escherichia coli cells. The cells are cultured, and inclusion bodies of the synthesised predecessor are isolated. It is followed by partial renaturation of the isolated predecessor in the presence of dithioerythrole preventing closure of disulphide bonds. The predecessor is hydrolysed by the enteropeptidase enzyme with producing human methionine-free interferon-alpha2b. After completion of the predecessor hydrolysis reaction, complete renaturation of human interferon-alpha2b is carried out in the presence of a pair of cystine and cysteine compounds promoting closure of disulphide bonds. The produced protein is purified by a chromatography in a KM-sepharose.

EFFECT: higher yield.

5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention can be used for producing recombinant human interleukin-11 (rIL-11). The recombinant plasmid DNA pET32M/mTrx-rhIL-11 is produced by a method which involves synthesis of four fragments of a recombinant human interleukin-11 gene from oligonucleotide primers, cloning of each fragment in a plasmid vector pGEM5z restricted by EcoRV endonuclease, transformation of each of 4 plasmids of E.coli cells of strain DH5a, selection of clones coding the related fragments of the recombinant human interleukin-11 gene, selection of plasmids with the recombinant human interleukin-11 gene without mutations, combination of all fragments of the recombinant human interleukin-11 gene in the plasmid vector pUC19, construction of the vector pET32M of the plasmid pET32b, recloning of the recombinant human interleukin-11 gene in an expression vector pET32M, coding thioredoxin 1 E.coli with substitution of Asn84/Gln. The produced DNA is used to transform the E.coli BL21 (DE3) strain cells to produce the E.coli BL21 (DE3)/pET32M/mTrx-rhIL-11 strain that is a producer of recombinant human interleukin-11 as an ingredient of soluble fused protein mTrx-rhlL-11.

EFFECT: effective production of recombinant interleukin-11 in Escherichia coli cells.

3 cl, 5 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: polypeptide contains extracellular and transmembrane domains of a human tissue factor. A DNA fragment coding this polypeptide, and a plasmid pET28a(+) are used to produce a genetic make-up enabling biosynthesis of the recombinant human tissue factor. Also, the E coli BL21 [DE3]/p6E-tTF strain that is a producer of the recombinant human tissue factor is offered.

EFFECT: high-yield recombinant protein and simplified recovery and purification procedures.

2 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: stable inheritance effect is independent of an insertion position and orientation. The introduction of said sequence essentially reduces the loss frequency of both low-copy, and high-copy vectors. The given invention can find application in plasmid-bearing strain selection. And the cultivation of said strains does not require a selective medium to be used.

EFFECT: higher stability of vector inheritance.

1 dwg, 1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and deals with recombinant plasmid DNA, which contains sequence of gene of mature staphylokinase Staphylococcus aureus with replacement of codons K74, E75 and R77 with triplets, which code Ala, of strain Escherichia coli MZ09 and method of obtaining recombinant protein, which contains sequence of gene of mature staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets. Essence of method includes recombinant plasmid DNA, which codes sequence of gene of mature protein of staphylokinase from Staphylococcus aureus staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets, which has nucleotide sequence, given in dwg.1. Invention also includes strain Esherichia coli MZ09, producent of recombinant staphylokinase, with sequence, given in dwg.1, as well as method of its obtaining.

EFFECT: invention makes it possible to obtain staphylokinase protein which possesses high fibrinogenic activity, with output of recombinant protein to 20% from total amount.

3 cl, 2 dwg

FIELD: medicine.

SUBSTANCE: agent contains mutant luciferase of glowworms Luciola mingrelica (SEQ ID No:2), recovered from recombinant cells E.coli transformed by plasmid pETL7, luciferin, magnesium sulphate, tris-(oxymethyl)-aminomethane, acetic acid, sodium ethylene aminotetraacetate, dithiotreitol, bovine serum albumin, sucrose and water.

EFFECT: higher sensitivity of ATP test, lower enzyme consumption.

2 cl, 3 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: recombinant plasmid pETPHOFus enabling synthesis of an anthrax lethal factor substratum as a part of one polypeptide with a mutated reporter enzyme of alkaline phosphatase Escherichia coli. Said fused polypeptide represents a new high-sensitivity anthrax lethal factor substratum. Besides, an Escherichia coli BL-PHOFus strain being a producer of the fused polypeptide is offered. The strain provides a no-fermentation high-yield synthesized protein not less than 30 mg in 1 litre of a liquid culture. The prepared anthrax lethal factor substratum shows hypersensitiveness and high specificity to LF splitting and is able to detect proteolytic activity of the anthrax lethal factor in concentration up to 1 pM.

EFFECT: invention can find application in clinical recognition of anthrax disease; for detecting hot spots of anthrax, for screening the compound libraries and searching anthrax lethal factor activity inhibitors.

2 cl, 4 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention represents a method of producing a recombinant antibody or its fragment that involves transforming E.coli cell which does not have a pyrC coding gene, or its homologue; a vector including the gene coding said recombinant antibody or its fragment and the wild E.coli pyrC gene, cultivation of specified said E.coli cell in a pyrimidine restriction environment and recovery of said antibody or its fragment.

EFFECT: invention allows high effective production of recombinant antibodies.

10 cl, 5 dwg, 5 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: plasmid vector pE-Trx-Aur is constructed for expression of aurelin in cells of Escherichia coli in composition of hybrid protein Trx-Aur, consisting of two DNA fragments, whose nucleotide sequence is given in description. By means of said vector parent strain of Escherichia coli is transformed, obtaining strain-producent of hybrid protein Trx-Aur. In order to obtain peptide aurilin cultivation of cells of obtained strain-producent is carried out, after that, performed are: cell lysis, affine purification of hybrid protein Trx-Aur on metal-chelate carrier, decomposition of hybrid protein Trx-Aur with bromine cyan by residue of methionine, introduced between sequences of aurelin and thioredoxin, and purification of target peptide by method of reversed-phase HPLC.

EFFECT: invention makes it possible to obtain biologically active aurelin by simplified technology and without application of hard-to-obtain natural raw material.

3 cl, 4 dwg, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: described is an amphiphilic high-polymer yeast RNA which stimulates a leukocytic reaction and a primary humoral immune response. The preparation is obtained from yeast Saccharomyces cerevisiae via extraction with boiling water containing oleic acid titrated with NaOH for 40 minutes at temperature 99-102°C, adding boiling water during evaporation to 0.3 l and NaOH 10, 20 and 30 minutes after the beginning of extraction.

EFFECT: disclosed preparation stimulates leukocytic reaction and a primary humoral immune response.

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