RU2180349C1 - Strain bacillus anthracis km89 as producer of anthrax antigens - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention proposes the strain B. anthracis retaining properties in storage and culturing on nutrient media that can be used as producer of different anthrax antigens (antigens of cellular wall, protective antigen, edematous and lethal factors, proteins of S-layer and others). Invention can be used in microbiology, genetics, biochemistry, in production of Bacillus anthracis antigens and other biologically active substances that are components of chemical anthrax vaccines and diagnostic preparations. EFFECT: strain indicated above, valuable properties.

Description

 The invention relates to biotechnology and can be used in microbiology, genetics, biochemistry, in the production of Bacillus anthracis antigens and other biologically active substances, which are components of chemical anthrax vaccines and diagnostic preparations.

 Known strains producing anthrax antigens, currently used in the production of chemical vaccines and diagnostic drugs. In Russia it is a strain of B. anthracis STI-1, in the USA it is B. anthracis V770-NR-R, in England it is B. anthracis Sterne 34F2 (Onishchenko G. et al., "Anthrax: topical aspects of microbiology, epidemiology, clinic, diagnosis, treatment and prevention". -M., 1999). These strains do not contain a plasmid capsule production (pXO2), causing the virulence of the strain, but contain a plasmid toxin formation (pXO1), encoding the synthesis of a three-component toxin. All three components of the anthrax toxin - the protective antigen, edematous and lethal factors, are antigens, of which the protective antigen is the main component of the anthrax vaccines.

However, the above production strains, like all representatives of the genus Bacillus, are spore-forming. The ability of the anthrax microbe to form spores during cultivation in laboratory or industrial conditions requires the observance of strict work safety rules, the neglect of which can lead to contamination of the premises and equipment with spores of the causative agent of a dangerous infectious disease. Spores are the main form of conservation of B. anthracis in environmental objects. In water, they persist up to 18 years, in soil - up to 300 years or more. At a temperature of 120-140 ^o With spores die only after 2-3 hours. Conventional disinfectants used to work with non-forming spores species do not act on them. When exposed to favorable conditions, spores germinate and form full-fledged vegetative cells. Therefore, the use of a spore-free (asporogenic) strain of B. anthracis incapable of industrial or experimental isolation of anthrax antigens is more appropriate.

 Isolated cases of the isolation of asporogenic strains of B. anthracis are known.

 An asporogenic strain of B. anthracis Davis is described (Uchida 1., Sekizaki T., Hashimoto K. // J. Gen. Microbiol. - 1985. - V. 131. - P. 363-367).

 However, the strain B. anthracis Davis does not contain the main anthrax antigens that are part of the chemical vaccines and are necessary for most diagnostic preparations. The genome of B. anthracis Davis contains a capsule production plasmid, which determines the virulence of the strain, but there is no toxin-forming plasmid encoding the synthesis of a three-component toxin.

A similar asporogenic monoplasmid capsule-containing strain of B. anthracis M29Spo ^is known ^- (Eremin S., Nikiforov A., Kostyukova T., Mikshis N. // Journal of Microbiol. - 1999. - 4. - P. 91-92).

 However, this strain isolated from a naturally highly virulent B. anthracis M29 strain also does not contain the main anthrax antigens and, therefore, cannot be used in the production of anthrax vaccines and diagnostic preparations.

 The strains obtained by Farchaus J. et al. (Farchaus J., Ribot W., Jendrek S., Little S. // Appl. Environ Microbiol. - 1998. - V. 64. - P are closest to the claimed strain of B. anthracis KM92 . 982-991) and Worsham P., Sowers M. (Worsham P., Sowers M. // Canadian Journal of Microbiol. - 1999. - V. 45. - P. 1-8). Both strains are producers of one of the anthrax antigens - a protective antigen. The first of them is constructed on the basis of an asporogenous plasmid-free B. anthracis strain by cloning a gene of protective antigen. The second one was selected as a spontaneous asporogenic mutant in a population of a recombinant non-plasmid strain of B. anthracis with a cloned gene for protective antigen.

 However, the described strains are inferior in stability to natural strains. In addition, the cloning of a single gene encoding the synthesis of a single antigenic product reduces the possibility of using a recombinant strain.

 The objective of the invention is to obtain an asporogenic strain of B. anthracis, which retains properties during storage and cultivation on nutrient media and is a producer of various anthrax antigens (antigens of the cell wall, protective antigen, edematous and lethal factors, S-layer proteins, etc.).

 The essence of the invention is to obtain a stable asporogenous producer strain of anthrax antigens.

 The inventive strain of the causative agent of anthrax was obtained on the basis of the vaccine strain B. anthracis Sterne 34F2 using our developed method for producing an asporogenic strain of B. anthracis.

 The strain is deposited in the State collection of pathogenic bacteria "Microbe" (Saratov) under the number KM89.

The main properties of the strain B. anthracis KM89:
- Asporogenicity - the asporogenicity of the strain gives an advantage when it is used as producers of anthrax antigens and other biologically active substances - components of anthrax vaccines and diagnostic preparations, since there is no likelihood of contamination of workrooms, laboratory and industrial equipment with anthrax pathogens;
- avirulence - like the vaccine strain B. anthracis STI1 is monoplasmid, i.e. does not contain plasmid capsule production;
- immunogenicity - is a source of anthrax antigens:
- antigens of the cell wall,
- protective antigen,
- edematous and lethal factors,
- S-layer proteins. The inventive strain, unlike the production of vaccine strains of B. anthracis STI1 and B. anthracis 55, contains an S-layer in the surface cell structure, which plays an exceptional role in interaction with the environment and has antigenic properties;
- stability - retains its properties during storage, cultivation on nutrient media and after passages through the body of laboratory animals.

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

 Cultural traits: on solid nutrient media, the cells of the strain form colonies with a smooth surface and even edge (in S-shape). When grown in liquid culture media - uniform turbidity.

The biochemical activity of the strain and other properties: it has hemolytic and proteolytic activities, sorb Congo red, synthesizes and excretes yellow pigment in the culture medium, synthesizes S-layer proteins and anthrax toxin. The optimum growth temperature is 37 ^o C, the optimum pH is 7.2.

 Nutritional needs of the strain: is auxotroph, growth dependent on methionine, threonine, phenylalanine and thiamine.

Plasmid composition: contains a toxin-forming plasmid (pXO1 ⁺ pXO2 ^- ).

 Example 1 - determination of the ability to spore formation.

Colonies from crops incubated at a temperature of 37 ^o C for 5 days on Hottinger agar, suspended in physiological saline. As a control, a spore-forming strain of B. anthracis Sterne 34F2 is used. Test tubes with culture are placed in a water bath and heated at a temperature of 65 ^o C for 30 minutes or at 80 ^o C - 10 minutes The presence of growth before heating and the absence of growth after heating when plating 0.1 ml of culture on Hottinger agar indicates the inability of the anthrax microbe cells to form full-fledged spores.

 Additionally, the ability to form spores is checked by microscopic examination of smears stained with fuchsin Ziel. As a control, a spore-forming strain of B. anthracis Sterne 34F2 is used. In the experimental smear, the absence of characteristically colored spores is noted.

 Example 2 - determination of the stability properties of asporogenicity.

The strain is passaged several times (at least 3) in a nutrient medium (L-agar) at a temperature of 37 ^o C. After the last passage, seeding on L-agar is done and the ability to spore formation in 20 isolated clones is checked. The absence of spore formation in all tested clones indicates the stability of the trait. After 3 passages on a nutrient medium, 3 passages are conducted through the body of laboratory animals. For this, a white mouse is injected subcutaneously with 0.2 ml of a cell suspension of the strain at a concentration of 10 million spores. In the fallen specimen, they are seeded from the brain and a pure culture of B. anthracis is isolated. Highlighted by a pure culture of the causative agent of anthrax in the same concentration, the next mouse is infected and repeated 3 times. Then check the sign of spore formation according to the above scheme. The lack of ability to form spores indicates the stability of the sign.

 Example 3 - determination of protein production of the S-layer.

Determination of the production of S-layer proteins is carried out using protein analysis. To do this, 1 loop of strain B. anthracis KM90 is seeded in liquid nutrient medium (BHI) and grown with aeration at 37 ^o C for 16 hours. Then, 20 ml of culture are centrifuged at 100,000 rpm, 4 ^{° C.} , 30 minutes. The supernatant was removed, sterilized by filtration through membrane filters with a pore diameter of 22 μm (Millipore) and trichloroacetic acid was added to a final concentration of 10%. Protein is precipitated by centrifugation at 100,000 rpm, 4 ^{° C.} , 30 minutes. The drug is resuspended in 50 μl of 0.1 M Tris-Hcl buffer solution (pH-6.7) containing 10% glycerol. After which the samples are added to the wells of 7.5% polyacrylamide gel. Electrophoresis is carried out at a current of 90 mA for 5 hours. The S-layer protein has a molecular weight of 94 kDA.

 Thus, a stable asporogenic strain of B. anthracis KM89 was obtained, which is recommended for use in the preparation of anthrax antigens and other biologically active substances - components of anthrax vaccines and diagnostic preparations.

Claims (1)

 Strain Bacillus anthracis KM89 - producer of anthrax antigens.