RU2745161C1 - Method of obtainig an attenuated plasmid-free strain of f.tularensis 15 cmsa, synthesizing mycobacterial antigen superoxide dismutase a - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. A method for obtaining a strain is proposed. The strain is obtained from the F. tularensis 15 NIIEG vaccine strain by allelic exchange of one of two copies of the iglC gene for a recombinant operon consisting of the groE promoter region of the F. tularensis operon, the fopL leader sequence of the F. tularensis fopA gene, and the structural part of the modified M. tuberculosis sodA gene.EFFECT: invention makes it possible to obtain an attenuated F. tularensis strain that synthesizes the mycobacterial antigen superoxide dismutase A. to study the prospects for using the strain in the development of a live combined tuberculosis-tularemia vaccine.1 cl, 15 dwg, 1 tbl, 10 ex

Description

The invention relates to the field of medicine and veterinary medicine and can be used in the creation of drugs of microbiological origin for the prevention of tuberculosis and tularemia in health care.

Currently, in the world from tuberculosis, despite large-scale routine immunizations, about 3 million people die every year. This indicates the lack of efficacy of a commercial live vaccine based on the Mycobacterium bovis BCG strain. New approaches for creating highly effective vaccines include the design of vaccine strains based on bacterial strains and viral vectors encoding immunodominant proteins of mycobacteria, the development of DNA vaccines and chemical vaccines.

One of the immunodominant mycobacterial proteins is the enzyme Fe / Mn superoxide dismutase A (Fe / Mn SOD A, EC 1.15.1.1), secreted into the extracellular environment, consisting of four subunits with a molecular weight of 23 kDa and pI 6.0 [Zhang et al., 1991].

This protein is able to stimulate protective cellular immunity and has protective activity in a model of experimental tuberculosis in guinea pigs. Immunization, alone or in combination with other major extracellular proteins of M. tuberculosis, induces significant protective immunity against aerosol infection by virulent M. tuberculosis bacteria in a highly susceptible guinea pig model of pulmonary tuberculosis [Kheraet al., 2005].

It is known that the vaccine strain F. tularensis 15 NIIEG, obtained in the 40s of the last century in Russia and used in the countries of the former USSR, [Olsufiev et al., 1960], is able to activate the immune system of a macroorganism and protect it for a long time not only from tularemia infection, but also to form nonspecific protection against the causative agents of listeriosis and legionellosis [Belyi et al., 1996].

Creation of recombinant strains based on the F. tularensis 15 NIIEG vaccine strain, expressing protective antigens of mycobacteria, makes it possible to study the prospects of creating effective combined anti-tuberculosis and anti-tularemia recombinant vaccines [Kravchenko et al., 2007].

The technical result of the invention consists in the development of a method for obtaining an attenuated plasmid-free strain of F. tularensis 15 CMSA, which synthesizes the mycobacterial antigen superoxide dismutase A. The strain can be used to create promising prototypes of recombinant vaccine strains that form a specific effective protection against intracellular parasites.

The technical result of the invention is achieved by the fact that a suicidal plasmid vector pBCS has been created, which makes it possible to create recombinant operons of genes encoding target antigens located within the region of insertion into the nucleotide sequence of the F. tularensis chromosome.

Suicide plasmid pS51 / ΔiglC with a recombinant operon consisting of the groE promoter region of the F. tularensis operon, the fopL leader sequence of the fop AF gene. tularensis and the structural part of the modified M. tuberculosis sodA gene, flanked by the nucleotide sequences of the F. tularensis genome adjacent to the structural part of the iglC gene, makes it possible to replace one copy of the iglC gene in the F. tularensisna chromosome. -SodA.

The method for creating the F. tularensis 15 CMSA strain consists in using a recombinant DNA fragment created in vitro, consisting of the groE promoter region of the F. tularensis operon, the fopL leader sequence of the fop AF gene. tularensis and the structural part of the modified M. tuberculosis sodA gene, flanked by the nucleotide sequences of the F. tularensis genome adjacent to the structural part of the iglC gene, for allele exchange of the homologous native chromosome fragment of the F. tularensis 15 NIIEG vaccine strain for a recombinant DNA fragment.

The difference between the proposed method and the previously used method of allelic exchange in the F. tularensis genome is the inclusion in the nucleotide sequence of F. tularensis DNA of recombinant heterogeneous operons, which are functionally active in the tularemia microbe, and the replacement of the pPV2 and pGM5 vectors with the pBCS vector, which we constructed on the basis of the plasmid pBlueScriptII SK (+) with introduced sacB gene and cat gene from plasmid Sa.

As a result, an attenuated plasmid-free strain of F. tularensis 15 CMSA was obtained, which synthesizes the enzymatically inactive mycobacterial protein Sod A.

The developed method allows one to create attenuated plasmid-free F. tularensis strains without additional antibiotic resistance genes, synthesizing heterogeneous protective antigens, which opens up wide possibilities for using attenuated F. tularensis strains to create promising effective recombinant vaccines to protect human and animal organisms from intracellular infections.

Main properties of F. tularensis 15 CMSA strain.

Cultural and morphological.

Bacteria of the F. tularensis 15 CMSA strain are small coccoid and rod-shaped cells measuring 0.2-0.7 microns, immobile, gram-negative, polymorphic, do not form spores and capsules. Aerobes, do not grow on simple media without yeast extract, auxotrophs, are cultivated at temperatures from 15 ° C to 42 ° C in high-grade media with glucose and an increased concentration of cysteine. The optimum growing temperature is 37 ° C, pH 7.0-7.5.

The medium for cultivation of the F. tularensis 15 CM8A strain: cultivated on a solid nutrient medium FT-agar (FBUN SSC PMB, Obolensk); dense nutrient medium based on erythritol agar with the addition of dried blood of cattle (FBUN GNTs PMB, Obolensk), and liquid nutrient medium: (medium composition per 1 l) 5 g of enzymatic hydrolyzate of casein, 5 g of yeast extract, 5 g of sodium chloride , 12 g of monosubstituted potassium phosphate, pH7.2, 1% glucose, 10 mg of cysteine, 10 mg of ferric chloride [Lapin et al., 2009]. Colonies appear on FT-agar at 37 ° C after 48 hours.On a dense medium based on erythritol-agar, colonies with a diameter of 2.0-2.5 mm are formed in 72 hours, convex, shiny, smooth, bluish-white, opaque, homogeneous.

Stability. When subcultured on solid nutrient media, the F. tularensis 15 CMSA strain is stable and does not dissociate.

Biochemical properties The cells of the F. tularensis 15 CMSA strain utilize cysteine to form hydrogen sulfide, ferment glucose and maltose on Downs medium, do not ferment glycerin, sucrose and lactose. Resistant to erythromycin, polymyxin, penicillin, ampicillin, sensitive to streptomycin, tetracycline, doxycycline, gentamycin, kanamycin, nalidixic acid, do not have citrulline ureidase and phosphatase activity.

Storage conditions of the strain culture: the culture of the F. tularensis 15 CMSA strain is stored at + 4 ° C on schools of FT-agar medium for up to 1 month.

Method of storage of strain F. tularensis 15 CMSA: in 40% sucrose-gelatin protective medium in a lyophilic state, storage at a temperature of (4-8) ° С, recommended re-laying after 5 years.

Technological features during cultivation. There are no technological features in the cultivation of the F. tularensis 15 CMSA strain in comparison with the F. tularensis 15 NIIEG vaccine strain.

The invention is illustrated by the following figures.

FIG. 1 - Nucleotide sequences of the native sodAM gene. tuberculosis (A), modified sodATA gene (B), and a fragment of the pPMC1 plasmid with the groE gene promoter region (B).

FIG. 2 - Schematic of the structure of the plasmid pFSC24.

FIG. 3 - Scheme of the structure of the plasmid pFSC51.

FIG. 4 - Scheme of the pBCS suicide vector.

FIG. 5 - Scheme of the suicide plasmid pS51 / ΔiglC.

FIG. 6 - Electropherogram of PCR products obtained on a DNA template of F. tularensis15 CMSA mutant variants.

FIG. 7. - Electropherogram (10% PAGE) of the induction of the synthesis of the recombinant protein rSodA in the cells of the E. coli pET23b (+) SodA strain in the presence of the inducer IPTG.

FIG. 8. - Electropherogram of the renatured recombinant protein rSodA.

Fig. 9. - Electrophoretogram (A) and immunoblot (B) of native and recombinant rSodA proteins with rabbit polyclonal antiserum to the recombinant rSodA protein.

FIG. 10. - Zymogram of native and recombinant proteins rSodA.

FIG. 11. - Immunoblotting of lysates of recombinant clones of the F. tularensis FSC24 strain with rabbit monospecific polyclonal serum to the recombinant rSodA protein.

FIG. 12. - Immunoblotting of lysates of recombinant clones of F. tularensis FSC24 and FSC51 with rabbit monospecific polyclonal serum to the recombinant protein rSodA.

FIG. 13. - Immunoblotting of lysates of recombinant clones of F. tularensis 15 CMSA after passage in the body of white mice with rabbit monospecific polyclonal serum to the recombinant protein rSodA.

FIG. 14. - Analysis of the enzymatic activity of the cloned SodAe protein in recombinant clones of F. tularensis 15 CMSA.

FIG. 15. - Immunoblotting of lysates of recombinant clones of F. tularensis 15 CMSA after passage in the body of white mice with rabbit monospecific polyclonal serum to the recombinant protein rSodA.

Example 1. Construction of a modified sodA24 gene obtained by introducing into the original sodAM gene. tuberculosis, additional nucleotides and substitutions of a number of nucleotides, leading to adaptation of the expression of a heterologous gene in tularemia microbe cells.

The cloned sodAM gene. tuberculosis (nucleotide sequence of the soda gene (NCBIGenBank: AL123456.3 _gene_4033) [locus_tag = Rv3846] [location = 4320704..4321327] to create a recognition site for the BglII restriction enzyme, AGATCT nucleotides were added. the native sodA gene by two codons for the amino acids Arg and Ser. In addition, codons rarely found in F. tularensis messenger RNAs were replaced at the end of the native sodA gene, which resulted in the appearance of a PstI site in the recombinant sodA gene at position 504 (Fig. . 1, A).

An amplicon with the left structural part of the sodA gene was synthesized by PCR using primers Sod-Left / F and BglII-Left / R; using a pair of primers BglII-Right / F and Sod-Right-Pst / R, an amplicon with the central part of the sodA gene was synthesized, and using a pair of primers Sod-Right-Pst / F and Sod-Right / R, an amplicon with a right structural part of the sodA gene (Fig. 1, B). In all cases, M. tuberculosis H37Rv DNA was used as a template.

After combining these three amplicons at the BglII and PstI sites, a 627 bp DNA fragment was obtained that encodes the modified sodA24 gene containing recognition sites for the BglII and PstI restriction enzymes, flanked by the recognition sites for the NdeI and BamHI restriction enzymes.

A pro amplicon containing the promoter region of the groEF gene was synthesized by PCR using a pair of primers Xpr / F and Ppr / R1. tularensis (Fig. 1, B). The pPMC1 plasmid DNA was used as a template [Kravchenko et al., 2007].

The obtained DNA fragments were combined at the NdeI site, which led to the appearance of a DNA fragment of 974 bp with a modified sodA2A operon. The created operon was inserted into the vector plasmid pPMC1 between the XhoI and BamHI sites. As a result, the plasmid pFSC24 with a size of 5845 bp was obtained. (Fig. 2), which by transformation was transferred into the cells of the vaccine strain F. tularensis 15 NIIEG. The selection of plasmid-containing clones was carried out on a medium containing 3 μg / ml chloramphenicol. In this way, the F. tularensis FSC24 strain was obtained.

Example 2. Creation of the recombinant sodA51 gene, consisting of the leader sequence of the F. tularensi fopA gene and the structural part of the modified sodA24M gene. tuberculosis.

For the secretion of the modified sodA24 gene product into the periplasmic space of the tularemia microbe, a recombinant sodA51 gene was created, consisting of the FopA-L leader sequence of the F. tularensis fopA gene and the structural part of the modified M. tuberculosis sodA24 gene.

An amplicon with the groE promoter region of the F. tularensis operon was synthesized by PCR using primers Xpr / F and Ppr / R1. The DNA of the plasmid pPMC1 was used as a template (Fig. 1, B).

Using the primer pair Eco47III-FopA-L / F and NdeI-FopA-L / R, the FopA-L amplicon was synthesized. The DNA of the F. tularensis 15 NIIEG strain was used as a template (Fig. 1, D).

An amplicon with the structural part of the modified sodATA gene was synthesized using a pair of primers Sod-Right / F and Sod-Left / R. The DNA of the plasmid pFSC24 was used as a template (Fig. 2).

After combining these three fragments at the NdeI and Eco47III restriction sites, a 1061 bp DNA fragment was obtained encoding the recombinant sodA51 operon flanked by the XhoI and BamHI restriction sites.

The created operon was inserted into the vector plasmid pPMC1 between the XhoI and BamHI sites. As a result, the pFSC51 plasmid was obtained, which was cryotransformed into the cells of the F. tularensis 15 NIIEG vaccine strain. The selection of plasmid-containing clones was carried out on a medium containing 3 μg / ml chloramphenicol. In this way, the F. tularensis FSC51 strain was obtained (Fig. 3).

Example 3. Creation of a suicidal vector for allele exchange

A cat amplicon with the cat gene of plasmid pC194 was synthesized by PCR using cat / Fcl and cat / Rcl primers. The pPMC1 plasmid DNA was used as a template.

A sacB amplicon with the sacB gene from B. subtilis was synthesized using a pair of primers sacB / F and sacB / R. The pPV plasmid DNA was used as a template [Golovliov et al., 2003].

After combining these amplicons at the SalI and XhoI sites, a DNA fragment of 2779 bp was obtained, flanked by the recognition sites for the BglII and BamHI restriction enzymes.

The created DNA fragment was inserted into the pBlueScriptIIKS / SK (+) vector plasmid at the BamHI site. As a result, a 5740 bp suicide vector pBCS was obtained, which was transferred by electroporation into E. coli DH5α cells. The selection of plasmid-containing clones was carried out on a medium containing 10 μg / ml chloramphenicol. The functional activity of the sacB gene was checked by the absence of bacterial growth on a medium with 5% sucrose. In this way, the E. coli BCS strain carrying the pBCS suicide vector was obtained (Fig. 4).

Example 4. Creation of a DNA fragment containing the recombinant sodA5 operon (groE-fopA-sodA), flanked by the nucleotide sequences of the F. tularensis genome, adjacent to the structural part of the iglC gene.

A 1583 bp SL amplicon was synthesized by PCR using primers 23 Sal-Left / F and Xhol-Left / R. with a fragment of the F. tularensis genome located to the left of the iglC gene; Using a pair of primers 23 Bam-Right / F and Sal I-Right / R, a 1565 bp SR amplicon with a fragment of the F. tularensis genome located to the right of the iglC gene was synthesized. In all cases, the DNA of the F. tularensis 15 NIIEG strain was used as a template. A fragment with the recombinant sodA51 operon was isolated from plasmid pFSC51 using restriction enzymes XhoI and BamHI.

After combining these three fragments at the XhoI and BamHI sites, a 4209 bp DNA fragment was obtained containing the recombinant sodASX operon inserted into the F. tularensis genome region instead of the iglC gene and flanked by the Sail restriction enzyme recognition sites (Fig. 5). DNA was inserted into the pBCS plasmid at the SaiI site of the polylinker. As a result, a suicidal plasmid pS51 / Δigl C with a size of 9949 bp was obtained, which was transformed into E. coli DH5α cells by transformation. The selection of plasmid-containing clones was carried out on a medium containing 10 μg / ml chloramphenicol. The functional activity of the sacB gene was checked by the absence of growth on a medium with 5% sucrose. Then the obtained clones were analyzed in polymerase chain reaction with two pairs of primers Xpr / F and Sod-Left for the presence of the modified sodA51 operon, with pairs of primers 23 Sal-Left / F and 23 XhoILeft / R, 23 Bam-Right / F and 23 Sall- Right / RHa the presence of fragments of the F. tularensis chromosome flanking the modified operon.

In this way, the E. coli S51 / ΔiglC strain was obtained, carrying the suicide plasmid pS51 / ΔiglC (Fig. 5).

Example 5. Integration of the modified sodA51 operon into the F. tularensis 15 NIIEG chromosome.

The DNA preparation of the suicide plasmid pS51 / ΔiglC was isolated from E. coli S51 / ΔiglC cells and used for plasmid electroporation of F. tularensis 15 NIIEG cells. Transformants with an integrated plasmid were selected on solid nutrient medium FT-agar; chloramphenicol 3 μg / ml was used as a selective agent. After 120 h of incubation at 37 ° C, the selected clones were verified by PCR with four variations of four primers: 5'SalI 2.5 F / 3'SODLeftR; 5 'SODLeftF / 3' SalI 2.5R; 5 'SODLeftF / 3' SalI 2.5R and 5'SalI 2.5 F / 3'SODLeftR (Fig. 5).

According to the analysis, four clones containing the suicide plasmid pS51 / ΔiglC were selected, two of which were integrated on the left "shoulder" of the iglC gene and two clones - on the right "shoulder" of the iglC gene (Fig. 6). For further selection of bacteria in which the allelic exchange of the iglC gene for the modified sodA51 gene occurred, the clones were plated on a solid nutrient medium based on erythritol agar with black albumin with 5% sucrose. The grown isolated clones were checked for the presence of the Cm ^S phenotype (140 clones tested) and the absence of the cat gene by PCR with Cat / Fcl and Cat / Rcl primers (Fig. 4).

Verification in polymerase chain reaction using primers Xpr / F and Sod-Left / R (Fig. 6) showed the presence of a modified sodA51 gene inserted into the chromosome of F. tularensis 15 NIIEG strain. Thus, the F. tularensis 15 CMSA strain was obtained. After cultivating the strain in a liquid nutrient medium for 18 h at 37 ° C, the bacteria were seeded on FT agar to isolated colonies. PCR analysis of 28 grown clones with primers Xpr / F and Sod-Left / R gave positive results.

Example 6. Obtaining serum specific to the rSodA protein of the tuberculosis microbe.

To obtain the recombinant SodA protein, an E. coli producer strain was created using the pET23b (+) plasmid vector (Novagen, USA).

6.1. Cloning of the sodA gene in an expression vector in E. coli cells

а праймер SA/R на 5'-конце содержал сайт рестрикции для фермента EcoRI -

The structural part of the sodA gene was obtained by PCR amplification using the template DNA of the M. tuberculosis H37Rv strain and a pair of primers SA / F and SA / R, calculated on the basis of the known nucleotide sequence of the M. tuberculosis H37Rv genome. Primer SA / F at the 5'-end contained a restriction site for the BamHI enzyme -

and primer SA / R at the 5'-end contained a restriction site for the EcoRI enzyme -

The amplicon was digested with restriction enzymes BamHI and EcoRI and inserted into the pET23b (+) plasmid between the BamHI and EcoRI sites. The resulting recombinant plasmid pET23b (+) SodA was transformed into E. coli BL21 cells. Selective selection of plasmid-containing clones of E. coli was carried out on LA medium supplemented with ampicillin up to 100 μg / ml. Among the transformants, the E. coli BL21 clone (pET23b (+) SodA) was selected by PCR using primers SA / F and SA / R. When the rSodA protein gene is cloned into the pET23b vector as a result of genetically engineered manipulations from the N-terminal part of the protein, an additional 14 amino acids appear, and a sequence of 13 additional amino acids and six histidines appears in the C-terminal part of the protein, allowing the protein to be isolated using an affinity chromatography. Table 1 shows the calculated parameters of the native and recombinant rSodA proteins.

The appearance of additional amino acid sequences in the gene of the recombinant rSodA protein led to an increase in the molecular weight of the recombinant rSodA protein from 23.0 kDa to 26.5 kDa.

The resulting recombinant E. coli strain BL21 (pET23b (+) SodA) synthesized the recombinant protein rSodA with an apparent molecular weight of 27.6 kDa, close to the calculated one (Table 1), the level of which increased upon the addition of the inducer isopropyl-beta-D-galactopyranoside (IPTG ) (Fig. 7). Apparent molecular weights of proteins were calculated from an electrophoretogram using the CaptMW program.

6.2. Isolation and purification of the recombinant rSodA protein

The bacterial mass of cells of the E. coli BL21 (pET23b (+) SodA strain was obtained according to the manufacturer's instructions (Novagen, USA). The induction of protein synthesis was carried out for 2.5 h at 37 ° C and a concentration of 2 mM IPTG. The main part of the target protein was The washed inclusion bodies were dissolved in the presence of 8 M urea and 6 M guanidine chloride in an ice bath with stirring for 1-2 hours. Insoluble components were separated by centrifugation at 23,000 rpm for 1 hour. The supernatant was passed through a membrane filter with a pore diameter of 0.22 μm and applied to a column with 2 ml of Ni ²⁺ -chelating Sepharose (Pharmacia Biotech, Sweden) equilibrated with a buffer for binding with 8 M urea. Then the protein from the column was eluted with an elution buffer with a gradient of imidazole concentrations 0 , 02-0.5 M. The recombinant rSodA protein weakly bound to the sorbent and eluted at an imidazole concentration of 0.06 M. Renaturation of the eluted recombinant rSodA protein was performed by dialysis against 20 mM sodium phosphate buffer pH 7.2. An electrophoretogram of the renatured recombinant rSodA protein is shown in FIG. 8.

According to the data presented, the purity of the purified preparation of the rSodA protein was at least 90%.

6.3 Preparation of rabbit polyclonal antiserum against recombinant rSodA protein

A rabbit weighing 2 kg was immunized three times with an increasing amount of the renatured recombinant protein rSodA at doses of 150, 200, and 250 μg / animal with an interval of 14 days in the presence of first complete and then incomplete Freund's adjuvant in a 1: 1 ratio. The drug was injected subcutaneously at several points in the back, and 7 days after the last immunization, blood was taken from the marginal ear vein. The titer of specific antibodies to the rSodA protein was determined by ELISA with a preparation of purified SodA protein as an adsorbed antigen. The homology of the native and cloned SodAn proteins, the specificity of antibodies in the antiserum was assessed by immunoblotting (Fig. 9). After renaturation, the enzymatic activity in the purified recombinant protein rSodA, assessed by the method [Edwards et al., 2001], was absent (Fig. 10, line 2), in contrast from the native protein SodA in the cellular ultrasonic disintegrate of mycobacteria (Fig. 10, line 1).

Example 7. Expression of the recombinant sodA24 gene as part of a plasmid in F. tularensis FSC24 bacterial cells

Expression of the recombinant sodA24 gene from M. tuberculosis in the cells of the tularemia microbe strain was assessed by immunoblotting with rabbit polyclonal antiserum to the recombinant rSodA protein (Fig. 11). All selected clones carrying the recombinant plasmid with the gene for the chimeric protein FopL expressed with rabbit monospecific polyclonal serum to rSodA protein. The product of the modified sodA24 gene did not exhibit enzymatic activity in the reaction with tetrazolium blue.

Example 8. Expression of the recombinant sodA51 operon as part of a plasmid in bacterial cells of the F. tularensis FSC51 strain

Mutant clones were tested in immunoblot with rabbit monospecific polyclonal serum to the recombinant protein rSodA, as a result of which one clone of F. tularensis FSC51 (Fig. 12) was selected, expressing the recombinant oper sodA51, synthesizing the chimeric protein in the composition of the plasmid at a level significantly higher than the level synthesis by the F. tularensis FSC24 strain.

All randomly selected clones carrying the recombinant sodA51 operon with the gene for the chimeric FopL-SodA protein in the plasmid in F. tularensis FSC51 cells expressed the gene and synthesized an immunologically active protein that reacted with rabbit monospecific polyclonal serum to the rSodA protein.

Example 9. Expression of the recombinant sodA51 operon inserted into the chromosome of cells of the F. tularensis 15 CMSA strain

Four mutant clones were tested by immunoblotting with rabbit monospecific polyclonal serum against the recombinant rSodA protein. The lysate of the recipient strain F. tularensis 15 NIIEG was used as a negative control. As a positive control, recombinant M. tuberculosis rSodA protein isolated from E. coli BL21 (pET23b (+) SodA) (Fig. 13).

All selected clones carrying the recombinant sodA51 operon with the gene for the chimeric FopL-SodA protein in the chromosome in F. tularensis 15 CMSA cells expressed the gene and synthesized an immunologically active protein that reacted with rabbit monospecific polyclonal serum to the rSodA protein.

F. tularensis 15 CMSA clones # 1-3 were tested for the enzymatic activity of the cloned SodA protein [Edwards et al., 2001]. The corresponding amount of lysate of the recipient strain F. tularensis 15 NIIEG was used as a negative control. An ultrasonic lysate of M. tuberculosis strain H37Rv was used as a positive control (Fig. 14). It was shown that the cloned SodA protein in tested recombinant clones of F. tularensis 15 CMSA does not possess enzymatic activity.

Example 10. Evaluation of the survival rate and stability of the F. tularensis 15 CMSA strain

The F. tularensis strain 15 CMSA was passaged in the organisms of outbred white mice (the dose of culture introduction was 1 × 10 ³ CFU / mouse subcutaneously). After 6 days, the animals were sacrificed, the spleens were removed and homogenized in 5 ml of PBS, (pH 7.6), the resulting organ suspensions were titrated to 7 dilutions with a step of 1:10 and 0.1 ml of dilutions were sown on a solid nutrient medium with the addition of polymyxin 100 μg / ml. According to the results of the seeding, it turned out that the average seeding of the spleens of animals was 3.2 × 10 ⁶ CFU / organ.

11 randomly selected isolated clones obtained from the spleens of infected mice were tested by immunoblotting with rabbit monospecific polyclonal serum against the recombinant rSodA protein (Fig. 15). As a result, it was shown that all 11 clones retained the ability to synthesize the rSodA protein.

Sources of information used in the examination:

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A. Nucleotide sequence of the gene

sodA (NCBIGenBank: AL123456.3_gene_4033) [locus_tag = Rv3846]

[location = 4320704..4321327]

1 GTG GCCGAATACACCTTGCCAGACCTGGACTGGGACTACGGAGCACTGGAACCGCACATC

61 TCGGGTCAGATCAACGAGCTTCACCACAGCAAGCACCACGCCACCTACGTAAAGGGCGCC

121 AATGACGCCGTCGCCAAACTCGAAGAGGCGCGCGCCAAGGAAGATCACTCAGCGATCTTG

181 CTGAACGAAAAGAATCTAGCTTTCAACCTCGCCGGCCACGTCAATCACACCATCTGGTGG

241 AAGAACCTGTCGCCTAACGGTGGTGACAAGCCCACCGGCGAACTCGCCGCAGCCATCGCC

301 GACGCGTTCGGTTCGTTCGACAAGTTCCGTGCGCAGTTCCACGCGGCCGCTACCACCGTG

361 CAGGGGTCGGGCTGGGCGGCACTGGGCTGGGACACACTCGGCAACAAGCTGCTGATATTC

421 CAGGTTTACGACCACCAGACGAACTTCCCGCTAGGCATTGTTCCGCTGCTGCTGCTCGAC

481 ATGTGGGAACACGCCTTCTACCTGCAGTACAAGAACGTCAAAGTCGACTTTGCCAAGGCG

541 TTTTGGAACGTCGTGAACTGGGCCGATGTGCAGTCACGGTATGCGGCCGCGACCTCGCAG

601 ACCAAGGGG TTGATATTCGG CTGA

B. Nucleotide sequence of the SodA 24 gene

Sod-Left / F AAA CAT ATG GCT GAA TAC ACA TTG C CA GAC

Met Ala Glu Tyr Thr Leu Pro Asp Leu Asp Trp Asp Tyr Gly Ala Leu Glu

1 ATG GCT GAA TAC ACA TTG CCA GAC CTG GAC TGG GAC TAC GGA GCA CTG GAA

TAC CGA CTT ATG TGT AAC GGT CTG GAC CTG ACC CTG ATG CCT CGT GAC CTT

Pro His Ile Ser Gly Gln Ile Asn Glu Leu His His Ser Lys His His Ala

52 CCG CAC ATC TCG GGT CAG ATC AAC GAG CTT CAC CAC AGC AAG CAC CAC GCC

GGC GTG TAG AGC CCA GTC TAG TTG CTC GAA GTG GTG TCG TTC GTG GTG CGG

Thr Tyr Val Lys Gly Ala Asn Asp Ala Val Ala Lys Leu Glu Glu Ala Arg

103 ACC TAC GTA AAG GGC GCC AAT GAC GCC GTC GCC AAA CTC GAA GAG GCG CGC

TGG ATG CAT TTC CCG CGG TTA CTG CGG CAG C GG TTT GAG CTT CTC CGC GCG

BglII-Left / R 5'-TATTCT AGATCT GCGCGCCTCTTCGAGTTTGG-3 '

BglII

~~~~~~~~

Arg Ser Lys Glu Asp His Ser Ala Ile Leu Leu Asn Glu Lys Asn Leu Ala

154 AGA TCT AAG GAA GAT CAC TCA GCG ATC TTG CTG AAC GAA AAG AAT CTA GCT

TCT AGA TTC CTT CTA GTG AGT CGC TAG AAC GAC TTG CTT TTC TTA GAT CGA

BglII-Right / F 5'-CGGTCT AGATCT AAGGAAGATCACTCAGCG-3 '

Phe Asn Leu Ala Gly His Val Asn His Thr Ile Trp Trp Lys Asn Leu Ser

205 TTC AAC CTC GCC GGC CAC GTC AAT CAC ACC ATC TGG TGG AAA AAC CTG TCG

AAG TTG GAG CGG CCG GTG CAG TTA GTG TGG TAG ACC ACC TTT TTG GAC AGC

Pro Asn Gly Gly Asp Lys Pro Thr Gly Glu LeuAlaAlaAlaIleAla Asp

256 CCT AAC GGT GGT GAC AAG CCT ACT GGC GAA CTA GCT GCA GCT ATC GCT GAC

GGA TTG CCA CCA CTG TTC GGA TGA CCG CTT GAT CGA CGT CGA TAG CGA CTG

Ala Phe Gly Ser Phe Asp Lys Phe ArgAla Gln Phe His AlaAlaAla Thr

307 GCG TTC GGA AGC TTC GAC AAG TTC CGT GCG CAG TTC CAC GCG GCT GCT ACT

CGC AAG CCT TCG AAG CTG TTC AAG GCA CGC GTC AAG GTG CGC CGA CGA TGA

Thr Val Gln Gly Ser Gly Trp AlaAla Leu Gly Trp Asp Thr Leu Gly Asn

358 ACT GTG CAG GGA TCA GGC TGG GCG GCA CTG GGC TGG GAC ACA CTC GGC AAC

TGA CAC GTC CCT AGT CCG ACC CGC CGT GAC CCG ACC CTG TGT GAG CCG TTG

Lys Leu Leu Ile Phe Gln Val Tyr Asp His Gln Thr Asn Phe Pro Leu Gly

409 AAG CTG CTG ATA TTC CAG GTT TAC GAC CAC CAG ACG AAC TTC CCG CTA GGC

TTC GAC GAC TAT AAG GTC CAA ATG CTG GTG GTC TGC TTG AAG GGC GAT CCG

Ile Val Pro Leu Leu Leu Leu Asp Met Trp Glu His Ala Phe Tyr Leu Gln

Sod-Right-Pst / F AC CTGCAG TAC AAGAACGTC AAA GTC GAC

Pst I

------

460 ATT GTT CCG CTA CTA CTA CTA GAC ATG TGG GAA CAC GCT TTC TAC CTG CAG

TAA CAA G GC GAT GAT GAT GAT CTG TAC ACC CTT GTG CGA AAG ATG GAC GTC

Sod-Right-Pst / R GG CTGCAG GTAGAAAGCGTGTTCCCACATGTCTAGTAGTAGTAGCG

Tyr Lys Asn Val Lys Val Asp Phe Ala Lys Ala Phe Trp Asn Val Val Asn

511 TAC AAG AAC GTC AAA GTC GAC TTT GCC AAG GCG TTT TGG AAC GTC GTG AAC

ATG TTC TTG CAG TTT CAG CTG AAA CGG TTC CGC AAA ACC TTG CAG CAC TTG

TrpAla Asp Val Gln Ser Arg Tyr AlaAlaAla Thr Ser Gln Thr Lys Gly

562 TGG GCC GAT GTG CAG TCA CGT TAT GCG GCT GCG ACT TCT CAG ACT AAG GGA

ACC CGG CTA CAC GTC AGT GCA ATA CGC CGA CGC TGA AGA GTC T GA TTCCCT

Leu Ile Phe Gly ***

613 TTG ATA TTC GGC TAA

AAC TAT AAG CCG ATT

Sod-Right / RAAA GGA TCC TTA GCC GAA TAT CAA T CCSTT AG

B. Fragment of the nucleotide sequence of the plasmid vector pPMC1 c

promoter region of the groE gene

XhoI

~~~~~~~~

NheI

~~~~~~~~

1 GCTAGCTCGAGAATAACTTAAGGGTAACTAGCCTCGCCGGCAATAGTTACC

CGA TCG AGC TCT TAT TGA ATT CCC ATT GAT CGG AGC GGC CGT TAT CAA TGG

52 CTT ATT ATC AAG ATA AGA AAG AAA AGG ATT TTT CGC TAC GCT CAA ATC CTT

GAA TAA TAG TTC TAT TCT TTC TTT TCC TAA AAA GCG ATG CGA GTT TAG GAA

103 TAA AAA AAC ACA AAA GAC CAC ATT TTT TAA TGT GGT CTT TAT TCT TCA ACT

ATT TTT TTG TGT TTT CTG GTG TAA AAA ATT ACA CCA GAA ATA AGA AGT TGA

154 AAA GCA CCC ATT AGT TCA ACA AAC GAA AAT TGG ATA AAG TGG GAT ATT TTT

TTT CGT GGG TAA TCA AGT TGT TTG CTT TTA ACC TAT TTC ACC CTA TAA AAA

205 AAA ATA TAT ATT TAT GTT ACA GTA ATA TTG ACT TTT AAA AAA GGA TTG ATT

TTT TAT ATA TAA ATA CAA TGT CAT TAT AAC TGA AAA TTT TTT CCT AAC TAA

256 CTA ATG AAG AAA GCA GAC AAG TAA GCC TCC TAA ATT CAC TTT AGA TAA AAA

GAT TAC TTC TTT CGT CTG TTC ATT CGG AGG ATT TAA GTG AAA TCT ATT TTT

307 TTT AGG AGG CAT ATC AAA TGA ACT TTA ATA AAA TTG ATT TAG ACA ATT GGA

AAA TCC TCC GTA TAG TTT ACT TGA AAT TAT TTT AAC TAA ATC TGT TAA CCT

358 AGA GAA AAG AGA TAT TTA ATC ATT ATT TGA ACC AAC AAA CGA CTT TTA GTA

TCT CTT TTC TCT ATA AAT TAG TAA TAA ACT TGG TTG TTT GCT GAA AAT CAT

409 TAA CCA CAG AAA TTG ATA TTA GTG TTT TAT ACC GAA ACA TAA AAC AAG AAG

ATT GGT GTC TTT AAC TAT AAT CAC AAA ATA TGG CTT TGT ATT TTG TTC TTC

460 GAT ATA AAT TTT ACC CTG CAT TTA TTT TCT TAG TGA CAA GGG TGA TAA ACT

CTA TAT TTA AAA TGG GAC GTA AAT AAA AGA ATC ACT GTT CCC ACT ATT TGA

511 CAA ATA CAG CTT TTA GAA CTG GTT ACA ATA GCG ACG GAG AGT TAG GTT ATT

GTT TAT GTC GAA AAT CTT GAC CAA TGT TAT CGC TGC CTC TCA ATC CAA TAA

562 GGG ATA AGT TAG AGC CAC TTT ATA CAA TTT TTG ATG GTG TAT CTA AAA CAT

CCC TAT TCA ATC TCG GTG AAA TAT GTT AAA AAC TAC CAC ATA GAT TTT GTA

613 TCT CTG GTA TTT GGA CTC CTG TAA AGA ATG ACT TCA AAG AGT TTT ATG ATT

AGA GAC CAT AAA CCT GAG GAC ATT TCT TAC TGA AGT TTC TCA AAA TAC TAA

664 TAT ACC TTT CTG ATG TAG AGA AAT ATA ATG GTT CGG GGA AAT TGT TTC CCA

ATA TGG AAA GAC TAC ATC TCT TTA TAT TAC CAA GCC CCT TTA ACA AAG GGT

715 AAA CAC CTA TAC CTG AAA ATG CTT TTT CTC TTT CTA TTA TTC CAT GGA CTT

TTT GTG GAT ATG GAC TTT TAC GAA AAA GAG AAA GAT AAT AAG GTA CCT GAA

766 CAT TTA CTG GGT TTA ACT TAA ATA TCA ATA ATA ATA GTA ATT ACC TTC TAC

GTA AAT GAC CCA AAT TGA ATT TAT AGT TAT TAT TAT CAT TAA TGG AAG ATG

817 CCA TTA TTA CAG CAG GAA AAT TCA TTA ATA AAG GTA ATT CAA TAT ATT TAC

GGT AAT AAT GTC GTC CTT TTA AGT AAT TAT TTC CAT TAA GTT ATA TAA ATG

868 CGC TAT CTT TAC AGG TAC ATC ATT CTG TTT GTG ATG GTT ATC ATG CAG GAT

GCG ATA GAA ATG TCC ATG TAG TAA GAC AAA CAC TAC CAA TAG TAC GTC CTA

Xpr / F- AGA TAG GCC TAA TGA CTG G

919 TGT TTA TGA ACT CTA TTC AGG AAT TGT C AG ATA GGC CTA ATG ACT GG CTTT

ACA AAT ACT TGA GAT A AG TCC TTA ACA GTC TAT CCG GAT TAC TGA CCG AAA

XhoI

~~~~~~~~

970 TAT AAT ATG AGA TAA TGC CGA CTG TAC TTT CTC GAG TGT ATG GAT TAG TCG

ATA TTA TAC TCT ATT ACG GCT GAC ATG AAA GAG CTC ACA TAC CTA ATC AGC

HindIII

~~~~~~~~

1021 AGC TAA AAA GCT CAT ATT TTT TAT ATT CAA ACT ATA TCC CTT CAA GCT TTG

TCG ATT TTT CGA GTA TAA AAA ATA TAA GTT TGA TAT AGG GAA GTT CGA AAC

1072 AAA AAT AAA CTT AAT TAT TAT ATA TGT TAT TTA GCT AGT TTT TTT AAT TAA

TTT TTA TTT GAA TTA ATA ATA TAT ACA ATA AAT CGA TCA AAA AAA TTA ATT

-35 signal groES

~~~~~~~~

1123 AGT TAA AAT CGA GAG CTT GTT TGA CAA AAA AAC AAA AAA ATT TCT TGA AAA

TCA ATT TTA GCT CTC GAA CAA ACT GTT TTT TTG TTT TTT TAA AGA ACT TTT

-10 signal groES

~~~~~~~~

XbaI

~~~~~~~~

1174 TTT TTT TTT TGA CTC AAT ATC TAG ACT TGC AAG AGC TTG GAA CTT TGA

GAT AAA AAA AAA ACT GAG TTA TAG ATC TGA ACG TTC TCG AAC CTT GAA ACT

CTA

1225 TGT TCT AAG ATG CAT ACA AAT TCA AAA TGC TTA AAC AAA AAT AAT TTA ACA

ACA AGA TTC TAC GTA TGT TTA AGT TTT ACG AAT TTG TTT TTA TTA AAT TGT

SD groES-overlappon

~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~

1276 AAG GAG TAA GAT TGT TAT GAA CAT TCG TCC ATT ACA AGA TAG AGT ATT AGT

TTC CTC ATT CTA ACA ATA CTT GTA AGC AGG TAA TGT TCT ATC TCA TAA TCA

groES-overlappon

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

SD (inner)

~~~~~

TCG TCG TGC AGA AGG AGA AAA ATG ATG

AG C AGC ACG TCT TCC TCT TTT TACTAC

PprR1- AAA CAT ATG A TTT TCT CCT TCT GCA CGA C

<---

Claims (1)

The method of obtaining from the vaccine strain F. tularensis 15 NIIEG an attenuated plasmid-free strain of F. tularensis 15 CMSA, which synthesizes the mycobacterial antigen superoxide dismutase A, consists in the allelic exchange of one of two copies of the iglC gene in the chromosome of F. tularensis 15 NIIEG by the recombinant plasmid p a DNA fragment consisting of a fragment of the F. tularensis genome located to the left of the iglC gene, the groE promoter region of the F. tularensis operon, the fopL leader sequence of the fopA gene of F. tularensis, the structural part of the modified M. tuberculosis sodA gene encoding the enzymatically inactive protein SOD A M tuberculosis, and a fragment of the F. tularensis genome located to the right of the iglC gene, and the sodA gene was modified by introducing additional AGATCT nucleotides, leading to the replacement of the Ala amino acid codon at position 52 in the native soda gene by two codons for the Arg and Ser amino acids, and at the end of the sodA gene were replaced codons rarely found in messenger RNAs of F. tularensis were found, which led to the appearance of a PstI site in the recombinant sodA gene at position 504.

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