RU2321628C1 - RECOMBINANT STRAIN Bacillus anthracis STIΔTPA-1 (pUB110PA-1) AS PRODUCER OF ANTHRAX MICROORGANISM ANTIGEN - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes the recombinant strain B. anthrax STIΔTPA-1 (pUB110PA-1) that produces the anthrax microorganism protective antigen. The strain is obtained by transformation of the strain B. anthrax STIΔT with hybrid plasmid pUB110PA-1 DNA and deposited in GK of pathogenic microorganisms "Mikrob" at number KM-96. The strain is characterized by stability of hybrid replicon and high level of production of the protective antigen representing the main component of chemical anthrax vaccines, and safety in using.

EFFECT: valuable biological properties of strain.

8 ex

Description

The invention relates to biotechnology and can be used in microbiology, genetics, biochemistry and production to obtain the protective antigen Bacillus anthracis, which is the main component of chemical anthrax vaccines and diagnostic drugs.

The strains producing anthrax antigens are known: in Russia - the B. anthracis STI-1 strain, in the USA - B. anthracis V770-NP1-R, in England - B. anthracis Sterne 34F ₂ [1]. These attenuated strains do not contain a capsule production plasmid (pXO2), which determines the virulence of the strain, but contain a toxin-forming plasmid (pXO1) encoding a binary action exotoxin synthesis consisting of protective antigen, edematous and lethal factors. The protective antigen of the anthrax microbe has the main immunogenic potential and is used in the production of chemical vaccines. Edematous and lethal factors, interacting with a proteolytically activated protective antigen, form toxic complexes that trigger the pathogenetic mechanisms of the infectious process and cause specific damage to the tissues of the macroorganism. All components of anthrax toxin have very close molecular weight values (83-89 kDa), which is an obstacle to their complete chromatographic separation. Impurities of edematous and lethal factors in the preparation of a protective antigen isolated from attenuated strains are reflected in the reactogenicity of a chemical vaccine made on its basis. When using such drugs for specific prophylaxis of people, side reactions often occur in the form of an allergy or even necrotic reactions at the injection site.

With the development of genetic engineering technologies, it became possible to obtain recombinant strains that produce only the protective component of anthrax toxin.

Known strains of Escherichia coli HB101 containing the cloned gene for the synthesis of protective antigen (pag) as part of the hybrid plasmids pSE24 and pSE36 [2] and strains of E. coli JM103 containing the cloned pag gene as part of pP1, pP2 and pP4 [3]. Hybrid replicons were constructed by inserting at the BamH1 restriction sites a region of the pXO1 plasmid containing the pag gene into the pBR322 or pUC19 vectors (for pPA2 and pPA4). However, these recombinant plasmids were unstable when functioning in E. coli cells and provided extremely low levels of synthesis of protective antigen. The amount of biologically active protein product isolated from the corresponding strains did not exceed 5-10 ng / ml of culture or 1-2 μg / ml in the case of using pPA2. For comparison, the production of protective antigen by an attenuated strain of B. anthracis Sterne 34F ₂ is 15-20 μg / ml.

A known E. coli strain in which the structural gene for the synthesis of protective antigen is cloned under transcriptional regulation of the T5 phage promoter [4]. This approach allowed us to significantly increase the level of synthesis of protective antigen, however, even in this case, the amount of protective antigen obtained from a recombinant strain did not exceed 2 μg / ml.

Known E. coli strain DH5α with the cloned pag gene synthesizing a protective antigen [5]. An increase in the yield of the purified preparation of protective antigen to 125 μg / ml was achieved by optimizing the cultivation process in the fermenter. However, E. coli is not an optimal model for cloning the protective antigen of the anthrax microbe, which is associated with the intracellular secretion mechanism of this protein. The advantage of representatives of the genus Bacillus as bacterial hosts of cloned DNA lies in their ability to secrete the synthesis of protein antigen. The procedure for isolating a protective antigen from a culture medium of a microorganism is much less laborious.

B. subtilis IS53 strains are known that carry the hybrid plasmids pPA101 and pPA102 with the pag gene included and produce protective antigen in the culture medium in an amount of 20.5–41.9 μg / ml [6]. The authors used a BamH1 pXO1 DNA fragment containing the gene for protective antigen synthesis from the hybrid plasmid pSE36 constructed by M. Vodkin and S. Leppla, inserting it into the multicopy bacillary vector pUB110. Restriction analysis of plasmids pPA101 (7.8 kbp) and pP102 (6.1 kbp) isolated from recombinant cultures revealed the presence of extensive deletions in the field of negative regulation of pagR. However, the strains were obtained on the basis of a recipient with high activity of proteolytic enzymes. The protective antigen of the anthrax microbe synthesized by recombinant strains under the action of bacillary proteases partially degraded, which led to significant losses of the protein product during its isolation and purification. In addition, the hybrid plasmids pPA101 and pPA102 in this expression system were unstable.

A known B. subtilis strain WB600 containing the plasmid derivative pRA101-pPA101-1 [7, 8]. In the replicon pPA101-1, in contrast to the initial variant, a 1.7 kbp region was delegated, as a result of which the plasmid acquired similarities with the obtained B. Ivins and S. Welkos pP102. Comparison of the nucleotide sequences of pPA101-1 and pPA102 revealed a difference in only one codon. In the B. subtilis WB600 recipient strain defective in the synthesis of 6 proteases, the authors induced mutations in the B. subtilis regulatory system — ccpA and abrB genes. This led to an increase in the production of protective antigen by 3 and 8 times, respectively. The yield of purified PA from the constructed recombinant strain was 7 μg / ml. In 2001, the authors obtained a patent for a method for producing protective antigen from B. subtilis strain WB600 pRA101-1 [9]. However, the improvement of the expression system in this case was due to multiple mutations in the chromosomal genes. This does not always favorably affect the viability and stability of the microorganism, intended for use in conditions of scalable production.

Known strains of B. subtilis DB104 containing the hybrid plasmids pRA3 and pRA10 [3]. The authors inserted BamH1 fragments of plasmids pPA2 and pPA4 containing the pag gene into the bireplicon vector pOLYA15. Hybrid replicons, when functioning in B. subtilis DB104 cells, provided the production of protective antigen at a level of 20 μg / ml. However, the maximum yield of protective antigen was achieved only at a certain, relatively short-term stage of the stationary phase of culture growth, after which rapid protein breakdown by accumulating proteases was observed.

B. anthracis ΔSterne and B. anthracis ΔANR strains with hybrid plasmids pJB1, pJB2 and pJB3 capable of replicating in gram + and gram bacteria are known [10]. Bireplicon (pBLKSPPA + pUB110 or pC194) vectors contained sequences encoding the protective antigen of the anthrax microbe, however, its expression in B. anthracis cells was low and, depending on the orientation of the pag gene, varied from 2.7 to 21 μg / ml

The creation of stable genetic constructs with high production of the protective antigen of the anthrax microbe is inevitably confronted with overcoming the difficulties arising from the limitation of pag gene expression due to the weak intrinsic promoter, the presence of positive (atxA) and negative (pagR) regulators in the pXO1 plasmid.

The B. anthracis MASC-10 and B. subtilis WB600 strains with the cloned pagA structural gene under the strong α-amylase promoter are known [11]. They are designed on the basis of recipients B. anthracis M770-NP1-R and B. subtilis WB600, which have low activity of proteolytic enzymes. The levels of production of protective antigen by recombinant strains reached values of 100-120 μg / ml. The authors report the stability of these genetically engineered strains. However, attempts to further improve them through the use of even more powerful promoters were unsuccessful [13]. Hypersecretion of protective antigen, on the one hand, was accompanied by instability of hybrid plasmids, on the other hand, it led to “secreted stress”, which manifested itself in a significant decrease in the production of antibodies to the protective antigen during immunization of laboratory animals.

The objective of the invention is the construction of a safe recombinant bacillary strain that retains properties during storage and cultivation and has a high level of production of protective antigen of anthrax microbe.

The essence of the invention is to obtain a stable recombinant strain producing a protective antigen of anthrax microbe.

The inventive strain was created on the basis of the plasmid-free derivative B. anthracis STIΔT (number in the State collection of pathogenic bacteria "Microbe", Saratov - KM56 (1), authors: Bolotnikova MF, Stepanov AS), obtained as a result of the temperature elimination of the plasmid pXO1 from the vaccine strain B. anthracis STI-1. The hybrid plasmid pUB110PA-1, containing the gene for the synthesis of protective antigen, was introduced into the recipient strain using electrostimulated transformation. Plasmid pUB110PA-1 is a derivative of plasmid pUB110PA, which we constructed by inserting a region of pXO1 plasmid containing the pag gene into the multicopy vector pUB110 on BamH1 restriction saigas.

The constructed strain was designated B. anthracis STIΔTPA-1 (pUB110RA-1) and deposited in the State collection of pathogenic bacteria "Microbe" (Saratov) under the number KM96.

Morphological signs: motionless, gram-positive, spore-forming rods, capsules do not.

Cultural traits: the strain, when plated on solid nutrient media, forms colonies with a rough surface and an uneven edge (in the R-form); when plated in a nutrient broth - a conglomerate of cells in the form of a lump of cotton wool on a background of a transparent medium.

Antibiotic Resistance: The recombinant strain is resistant to kanamycin. A selective antibiotic concentration of 25 μg / ml.

Antigenic properties - the strain contains the main immunogenic antigen of the anthrax microbe - a protective antigen, as well as spore and surface antigens.

Biochemical activity of the strain and other properties: the strain is characterized by low activity of proteolytic enzymes. The strain synthesizes a protective antigen that causes the formation of specific immunological reactions in a living organism. Based on experimental data, it was found that the level of production of protective antigen by the constructed strain is 4-6 times higher than the production of protective antigen by the control vaccine strain B. anthracis STI-1.

Plasmid composition: the genome of the constructed strain lacks its own plasmids pXO1 and pXO2. The strain contains a hybrid plasmid pUB110PA-1 with the pag gene included in its composition, which determines the synthesis of the protective antigen of the anthrax microbe. The size of the hybrid replicon is 6.5 kb. Plasmid pUB110PA-1 contains one site for Bam HI, Hind III, and two for Eco RI. In the delegated area, there was one site for Bam HI and Hind III. A hybrid replicon also carries the determinant of kanamycin resistance.

Pathogenic and immunogenic properties: a recombinant strain due to the absence in its genome of the main determinants of pathogenicity: capsules, edematous and lethal factors, cannot cause the development of an infectious process in a susceptible macroorganism. In experiments on laboratory animals, LD ₅₀ values were established for BALB / c mice> 3 × 10 ⁸ , for guinea pigs> 5 × 10 ⁸ ; and values of immunity indices: for BALB / c mice - 100.0, for guinea pigs - 25117.4.

Distinctive properties of the strain B. anthracis STIΔTPA-1 (pUB110PA-1):

- the presence of a hybrid plasmid pUB110PA-1 with the pag gene included in its composition, which determines the synthesis of the main immunogen of the anthrax microbe - a protective antigen;

- lack of native plasmids B. anthracis: pXO1, which determines the synthesis of edematous and lethal toxins, and pXO2, which determines the synthesis of capsules - the main pathogenicity factors of the anthrax microbe;

- a high level of production (about 70-80 μg / ml) of the protective antigen of the anthrax microbe, which is a component of chemical vaccines;

- low activity of proteolytic enzymes that destroy the protective antigen in the process of isolation and purification;

- the stability of the hybrid plasmid during storage of the strain, cultivation on nutrient media and after passages through the body of laboratory animals.

The possibility of using the strain is illustrated by example 1.

Example 1 - isolation and purification of protective antigen from a recombinant strain of B. anthracis STIΔTPA-1 (pUB110PA-1).

The daily agar culture of strain B. anthracis STIΔTPA-1 (pUB110PA-1) is seeded in 5 L of L-broth with the addition of kanamycin - 25 μg / ml; grown at a temperature of 37 ° C with aeration of 18 hours. After incubation, a protease inhibitor (up to 1 mmol EDTA) is added and filtered through membrane filters (Millipore, USA) with a pore diameter of 0.22 μm. The cooled filtrate was concentrated 10 times on an Amicon installation (USA) with a cartridge (Millipore, USA) with a protein exclusion limit of 30 kDa and dialyzed for 18 hours against 10 volumes of a buffer containing 25 mmol of diethanolamine, 50 mmol of NaCl, 2 mmol EDTA, 30 mmol KCl (pH 8.9). After dialysis, ion exchange chromatography of the preparation is carried out on a Macro Prep 50Q column (Bio-Rad, USA). Then it is again concentrated 10 times on the Amikon installation, dialyzed under the same conditions and filtered through membrane filters. The resulting preparation was further purified using Sephacryl 300HR gel filtration (Sigma-Aldrich, USA). Samples stored at a temperature of minus 70 ° C. About 400 μg of purified preparation of antigen is obtained from 5 l of broth culture.

The main properties of the recombinant strain, the expression of the cloned pag gene and the stability of the hybrid plasmid pUB110PA-1 are illustrated by the following examples:

Example 2 - screening for plasmid DNA.

The culture of the recombinant strain B. anthracis STIΔTPA-1 (pUB110PA-1) is grown for 16 hours in BHI broth with the addition of 0.4% glycerol and kanamycin (25 μg / ml) with aeration at a temperature of 37 ° C. Cells are pelleted by centrifugation at a rotor speed of 10,000 rpm and suspended in 100 μl of E-buffer (0.04 M Tris-OH; 0.002 M EDTA; 15% sucrose, pH 7.9), mix well and add 200 μl of lysis buffer (0.05 M Tris-OH; 15% sucrose; SDS - 0.03 g / ml in 5N NaOH). The mixture is incubated in a water bath at 60 ° C for 30 minutes, then placed in an ice bath for 10 minutes and 50 μl of chilled 2M Trio-OH are added, then 600 μl of chilled phenol-chloroform (1: 1) are carefully turned at least 40 times. Transfer all contents to centrifuge tubes and centrifuge for 30 minutes at a rotor speed of 10,000 rpm and a temperature of 5 ° C. The upper liquid phase is taken and stored at 4 ° C.

Plasmid DNA preparations and molecular weight markers (MBI Fermentas) were added to the wells of 0.7% agarose gel and electrophoresis was performed in 0.089 M Tris-borate buffer at a current strength of 80 mA for 90 minutes. At the end of electrophoresis, the gel is stained with a solution of ethidium bromide at a concentration of 0.5 μg / ml for 20 minutes. DNA is visualized using an ultraviolet light source and recorded using the ViTran gel documentation system (DNA technology, Russia).

On the electrophoregram of a DNA sample isolated from the tested recombinant strain B. anthracis KM96, the absence of their own plasmids of the anthrax microbe pXO1 and pXO2 and the presence of the hybrid plasmid pUB110PA-1 at the level of molecular marker migration of about 6.5 kb were noted.

Example 3 - determination of the proteolytic activity of a recombinant strain.

The culture of the recombinant - B. anthracis STIΔTPA-1 (pUB110PA-1) and the control - B. anthracis Sterne 34F ₂ strains are seeded by injection on the surface of a special medium containing (g / l): bacto-agar - 15.0; yeast extract - 4.0; casein - 2.0; Tris-HCl - 1.21; K ₂ HPO ₄ - 0.5; MnSO ₄ · H ₂ O - 0.03; MgSO ₄ · 7H ₂ O — 0.4; CaCl ₂ - 0.08; ZnSO ₄ · 7H ₂ O — 0.005; CuSO ₄ · 5H ₂ O - 0.005; (NH ₄ ) ₂ SO ₄ - 2.0; and grown in a thermostat at a temperature of 37 ° C for 24 hours. Estimate the width of the enlightenment zone around the colonies.

Around the colonies of the recombinant strain B. anthracis STIΔTPA-1 (pUB110PA-1), the zones of proteolytic degradation of protein substrates do not exceed 1 mm. Around the colonies of the control strain, with high activity of proteolytic enzymes, enlightenment zones up to 10 mm wide are formed against a cloudy background of the medium.

Example 4 - determination of the expression of the cloned pag gene based on the precipitation reaction on a medium with anthrax γ-globulin or antibodies to a protective antigen.

Isolated colonies from daily agar cultures of the recombinant and control vaccine strains were transferred by injection to a semisynthetic medium containing (mg / L): CaCl ₂ · 2H ₂ O - 14.7; MgSO ₄ · 7H ₂ O — 9.8; MnSO ₄ · H ₂ O - 0.85; KH ₂ PO ₄ - 680.0; K ₂ HPO ₄ - 871.0; adenine - 2.1; uracil - 1.4; L-tryptophan - 52.0; L-cystine - 12.0; glycine - 15.0; thiamine hydrochloride - 10.0; casamic acids - 3.6 (pH 6.9); to which add (g / l): agarose - 7.5; glucose - 20.0; sodium bicarbonate - 72.0 and kanamycin (for the recombinant strain) - 25 μg / ml. Directly during the filling of Petri dishes make (ml / 25 ml of medium): normal horse serum - 0.6; anthrax γ-globulin - 1.0 or rabbit antibodies to a protective antigen - 2.0. Incubated in an atmosphere of CO ₂ for 18 hours at a temperature of 37 ° C. The results are recorded in transmitted light, noting the width of the precipitation zones around the crops by injection.

The width of the precipitation zone in the recombinant strain of B. anthracis STIΔTPA-1 (pUB110PA-1) is about 5.0 mm, in the control vaccine strain B. anthracis STI-1 - 1.5 mm.

Example 5 - determination of the expression of the cloned pag gene based on the study of the protein composition of the culture filtrates.

Daily agar cultures of the test and control strains were seeded in 5 ml of broth containing (g / l): tryptone - 33.0; yeast extract - 20.0; L-histidine 2.0; NaCl - 7.4; Na ₂ HPO ₄ - 8.0; KH ₂ PO ₄ - 4.0 (pH 7.4). Sodium bicarbonate - 7.2 g / l and kanamycin (for the recombinant strain) - 25 μg / ml are added to the finished medium. The cultures are grown on a rotary shaker at 37 ° C for 18 hours, then centrifuged at a rotor speed of 10,000 rpm and a temperature of 5 ° C for 30 minutes. The supernatant is filtered through membrane filters (Millipore, USA) with a pore diameter of 0.22 μm and concentrated using centricones-30. After centrifugation at a rotor speed of 13000 rpm for 10 minutes, the supernatant is removed and the precipitate is diluted with electrophoresis buffer containing 0.125 mol of Tris-HCl, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, 0.03 mmol bromphenol blue (pH 6.8). Samples of 15 μl contribute to the wells of 10% polyacrylamide gel. To determine the electrophoretic mobility, commercial molecular weight markers are used (Sigma, USA). Electrophoresis is carried out in a vertical chamber (Amersham, USA) at a current of 70 mA for 5 hours. Polyacrylamide gels are stained according to Amersham's instructions and scanned.

The concentrated culture filtrates of B. anthracis STIΔTPA-1 (pUB110PA-1) and B. anthracis STI-1 strains (control) contain a protein with electrophoretic mobility established for the protective antigen (83 kDa). Based on the intensity of the bands corresponding to the protective antigen, the production of this protein by B. anthracis strain STIΔTPA-1 (pUB110PA-1) is several times higher than the production of protective antigen by the vaccine strain B. anthracis STI-1.

Example 6 - determination of the expression of the cloned pag gene based on the results of an immunoblot with antibodies to a protective antigen.

Preparation of culture filtrates of the tested strains and protein electrophoresis is carried out as described above (see example 5). The transfer of proteins to the nitrocellulose membrane (Hybond-C, Amersham, USA) is carried out at a current strength of 0.4 A and a voltage of 100 V for 1 hour. After transfer, the membrane is washed for 10 minutes in buffer (TBS) containing 20 mM Tris-OH, 500 mM NaCl (pH 7.5) and incubated for 1 hour in a blocking solution containing 1% bovine serum albumin (BSA) in TBS. Then the membrane is kept twice for 10 minutes in buffer (TTBS) containing 0.05% tween-20 in TBS (pH 7.5). Incubation of the membrane with specific antibodies to the protective antigen at a dilution of 1: 2000 is carried out for 1.5 hours, after which it is washed twice in TTBS. Interaction with peroxidase-labeled immunoglobulins diagnostic against rabbit immunoglobulins (Sigma, USA) at a 1: 2000 dilution is carried out for 2 hours. After incubation, the membrane was incubated twice for 10 minutes in TTBS and once in TBS. Staining is carried out with a solution of diaminobenzidine for 30 minutes.

As a result, at a level corresponding to electrophoretic mobility for a protein with a molecular weight of 83 kDa, a specific interaction with antibodies to a protective antigen is noted. The interaction of proteins of culture filtrates with specific antibodies to a protective antigen occurs more intensively in a recombinant strain.

Example 7 - comparison of production levels of protective antigen by recombinant and control strains using dot analysis.

Cultural filtrate preparations were prepared as described above (see Example 5) and applied 3 μl in double dilutions on a Hybond-C nitrocellulose membrane (Amersham, USA), incubated for 1 hour in a blocking solution containing 1% BSA in TBS. The membrane is washed in TTBS buffer and incubated for 1 hour with rabbit antibodies to the protective antigen at a dilution of 1: 100, then for the same period of time with peroxidase-labeled antibodies diagnostic against rabbit immunoglobulins (Sigma, USA) at a dilution of 1: 2000. After 15 minutes, washing in TTBS buffer was stained with diaminobenzidine solution for 30 minutes.

According to the results of dot analysis, the recombinant strain B. anthracis STIΔTPA-1 (pUB110RA-1) produces 4-6 times more protective antigen than the control vaccine strain B. anthracis STI-1.

Example 8 - determination of the stability of a hybrid plasmid in vitro.

The culture of the recombinant strain is grown in broth under selective pressure (10 μg / ml kanamycin) until the middle of the logarithmic phase. Then diluted 1: 100 medium without antibiotics and continue to grow until the end of the logarithmic phase. After 10-12 such alternating passages, the cell suspension is diluted with antibiotic-free sporulation medium and incubated for 3 days until spores form. Spore suspension is heated at a temperature of 65 ° C, seeded on agar medium to isolated colonies. 100 clones are transferred by replica method in parallel onto media with the addition of an antibiotic and without selective pressure. Count the percentage of clones grown on plates with selective medium.

In the process of alternating passages, 100% of the tested clones of the B. anthracis strain STIΔTPA-1 (pUB110RA-1) retain resistance to kanamipine. After spore formation, 90% of the tested clones remain resistant to kanamipine at a concentration of 25 μg / ml.

Thus, a safe, stable and effective producer of protective antigen, which is the main component of chemical anthrax vaccines, was obtained. The strain contains a hybrid plasmid pUB110PA-1 with the cloned pag gene, which provides the production of the protective antigen of the anthrax microbe that is 4-6 times higher than the level of production of this antigen by the control vaccine strain B. anthracis STI-1. The strain B. anthracis STIΔTPA-1 (pUB110PA-1) does not contain its own plasmids pXO1 and pXO2 and, therefore, does not synthesize the main virulence factors of the causative agent of anthrax - capsule, edematous and lethal factors. The strain B. anthracis STIΔTPA-1 (pUB110PA-1) has a low activity of its own proteases, leading to the degradation of protein antigens during isolation and purification. The indicated properties of the obtained strain indicate that it can be used to obtain the protective antigen Bacillus anthracis, which is a component of chemical anthrax vaccines and diagnostic drugs.

Information sources:

1. Onishchenko G. G., Vasiliev N. T., Litusov N. V., Kharechko A. T., Vasiliev P. G., Sadovoy N. V., Kozhukhov V. V. Anthrax: current aspects of microbiology, epidemiology, clinic, diagnosis, treatment and prevention - M .: VUNMTS Ministry of Health of the Russian Federation, 1999. - 447 p.

2. Vodkin M., Leppla S. Cloning of the protective antigen gene of Bacillus anthracis // Cell. - 1983. - Vol. 34. - P.693-697.

3. Tedikov V.M., Dobritsa A.P. Cloning and expression of the determinants of the protective antigen Bacillus anthracis in cells of Escherichia coli, Bacillus subtilis and Bacillus anthracis // Molecular Genetics, Microbiology and Virology. - 1993. - No. 2. - S.13-16.

4. Gupta P., Waheed S., Bhatnagar R. Expression and purification of the recombinant protective antigen of Bacillus anthracis // Protein Expr. Purif - 1999 .-- Vol.16. - P.369-376.

5. Chauhan V., Singh A., Waheed S. M., Singh S., Bhatnagar R. Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli // Biochem. Biophys. Res. Commun. - 2001. - Vol. 283. - No. 2. - P.308-315.

6. Ivins B., Welkos S. Cloning and expression of the Bacillus anthracis protective antigen gene in Bacillus subtilis // Infect. Immun. - 1986. - Vol. 54. - P.537-542.

7. Baillie L., Moir A., Manchee R. The expression of the protective antigen of Bacillus anthracis in Bacillus subtilis // J. Applied Microbiol. - 1998 .-- Vol.84. - P.741-746.

8. Miller J., McBride B., Manchee R., Moore P., Baillie L. Production and purification of recombinant protective antigen and protective efficacy against Bacillus anthracis // Letters in Applied Microbiol. - 1998 .-- Vol.26. - P.56-60.

9. United States Patent: 6267966. Vaccine production of the Bacillus anthracis protective antigen // Baillie L., Leslie W.J. - No. 242846. - 02.25.99. - 07/31/01.

10. Barnard J., Friedlander A. Vaccination against anthrax with attenuated recombinant strains of Bacillus anthracis that produce protective antigen // Infect. Immun. - 1999. - Vol. 67. - P.562-567.

11. Cohen S., Mendelson I., Altboum Z., Kobiler D., Elhanany E., Bino T., Leitner M., Inbar I., Rosenberg H., Gozes Y., Barak R., Fisher M., Kronman C., Velan B., Shafferman A. Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax // Infect. Immun. - 2000. - Vol. 68. - No. 8. - P. 4549-4558.

12. Gat O., Ihbar I., Aloni-Grinstein R., Zahavy E., Kronman C., Mendelson I., Cohen S., Velan B., Shafferman A. Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines // Infect. Immun. - 2003. - Vol. 71. - P.801-813.

Claims (1)

The recombinant strain Bacillus anthracis STIΔTPA-1 (pUB110RA-1), deposited in the State collection of pathogenic bacteria "Microbe" under the number KM96 - producer of the protective antigen of anthrax microbe.