Avirulent strain of microorganism vibrio cholerae km 169 of serogroup o139 as producer of capsule antigen. RU patent 2245363.

FIELD: biotechnology, microbiology.

SUBSTANCE: avirulent strain as a producer of capsule antigen is obtained on the base of the natural strain V. cholerae of serogroup O139 by method of step-by-step selection of cells with enhanced production of capsule. Prepared strain forms antigen by 3-4-fold more as compared with other strains of this serogroup. The strain shows high and stable level of capsule antigen production that is one of components of chemical choleraic vaccine against cholera pathogen of serogroup O139.

EFFECT: improved preparing method, enhanced yield of antigen.

7 ex

The invention relates to the field of biotechnology, namely, to construct a strain with increased production of capsular antigen, and can be used in the production of chemical cholera vaccines against the causative agent of cholera O serogroup.

The past decade has been marked by the emergence of Vibrio cholerae new O serogroup - V.cholerae O. The lack of immunity in the population, previously dealt only with Vibrio cholerae serogroup O1 - V.cholerae O1, caused large epidemics of cholera in South-East Asia and led to the removal of the infection on the territory of other countries and continents, including the USA and the CIS. A large number of cases of cholera caused by pathogenic vibrios O serogroup (17%), and the recent tendency to increase necessitates the creation of effective means of prevention of this pathogen.

Chemical vaccines are an effective means of preventing infectious diseases, including cholera. They include drugs protective bacterial antigens that trigger the development of immunity to the pathogen infection in vaccinated people. Emergence of Vibrio cholerae new O serogroup - V.cholerae O with new surface antigenemia - O - polysaccharide antigen and capsule makes necessary the introduction of the chemical vaccines against diarrheal infections these antigens.

Currently developed and used in the production of cholera preventive means of strains - producers with a high level of biosynthesis O antigen. At the same time, there are no strains - producers of capsular antigen serogroup O that can be used to produce capsular polysaccharide.

A known strain R serogroup O obtained from clinical sources, which produces a polysaccharide capsule. However, the high virulence of this strain limits its application in the production environment, and a small number formed by the capsular substance makes the ineffective strain when using it as a producer of this antigen (Smirnova N.I., Chekhov, GV, Davydova N, etc. Phylogenetic analysis of two morphologically different types of colonies V.cholerae O // Molecular genetics, Microbiology and Virology-1995. No. 3. - P.30-34).

Also known avirulent strains of V.cholerae KM115 that do not contain genes oxygenate determining the synthesis of cholera enterotoxin, and producing capsular antigen. However, the level of production of the capsules in this strain also not high enough (Ganin B.C., Urbanovich L.Y. and other Vibrio.cholerae O (“Bengal”) in Siberia //Materials of scientific conference devoted to the 100 anniversary of the anti-plague service of Russia. - Saratov, 1997 - T.1. - P.30).

To obtain drug capsular antigen, a component of cholera chemical vaccine against Vibrio cholerae serogroup A needed strain, producing a significant amount of this antigen.

The task of the invention to provide avirulent strains of V.cholerae O with high and stable level of production of capsular antigen.

The invention consists in that on the basis of the avirulent nature of V.cholerae strains serogroup A method stepwise selection of cells with increased production of capsules obtained avirulent producing strains of capsular antigen, stably forming 3-4 times more antigen compared with other strains of this serogroup.

The proposed strain derived from avirulent strains of V.cholerae 62 serogroup O isolated from the external environment. The strains of V.cholerae 62 were grown in liquid nutrient broth at 37°C for 18 hours and subcultured on solid nutrient medium until isolated colonies. Visually selected colonies with good growth characteristics and increased turbidity, indicating a significant production of capsules. A high level of capsulorraphy was confirmed using electron microscopy. In the 4-fold repetition of this phase of the received avirulent strain, whose products capsular antigen is 3-4 times higher than the level of education in other strains of this serogroup.

The strain deposited in the Public collections of pathogenic bacteria at antiplague research Institute “Microbe” (, Saratov), number 169 KM.

Cultural-morphological, biochemical and serological properties of the strain.

Gram-negative, slightly curved rods, motile with a single polar located flagellum.

On solid nutrient media forms of medium size, round, with smooth edges, muddy colony bluish-gray color. With the growth in broth is uniform clouding of the environment.

Optional gone anaerobic. Fermented to acid without gas mannose, sucrose, lures, maltose. Not ferments lactose and arabinose. Biochemical activity refers to the I group named " heuberg". Analyzes sheep erythrocytes. Prototroph.

Resistant to diagnostic by cholera phages and El tor circulation.

Not growing on alkaline agar with the addition of 50 μg/ml polymyxin b. Resistant to the antibiotic ampicillin (50 μg/ml).

Strain agglutinated serum to O139-antigen in the diagnostic titer and not agglutinated sera Oh, Inaba, Ogawa and RO.

The special properties of the strain.

1. Producer polysaccharide capsule. The strain produces a polysaccharide capsule in quantities at 3-4 times the production of this antigen from other strains of serogroup O139.

2. Avirulence. The strain does not contain genes oxygenate and not virulent for laboratory animals.

3. The stable. Strain stably produces a polysaccharide capsule. Summary of the invention the following examples.

Example 1. Determination of the virulence of the strain by PCR. The avirulence of the strain caused by the absence of the gene composition of toxigenicity. Their absence is to be installed using a polymerase chain reaction with primers on the ctxA gene. For this cell strain grown in liquid nutrient medium at 37°C for 18 hours, are lysed by boiling and used at a concentration of 10 7 cells/ml for research in PCR. The absence of a specific amplificata indicates the absence of the cholera toxin genes in the studied strain and, consequently, about the lack of virulence of the strain KM 169.

Example 2. Determination of the virulence of the strain in laboratory animals. Infection with a strain at a dose of 10 7 cells/ml small rabbit-suckers does not lead to the development of choleragenic effect, which indicates its avirulence for laboratory animals.

Example 3. Product definition capsules on the morphology of the colonies.

When grown on solid nutrient media of cells producing the polysaccharide capsule form turbid colonies, whereas cells not forming a capsule, colony transparent. The inventive strain forms a dense

environments muddy colonies, which indicates its ability to produce a polysaccharide capsule.

Example 4. Product definition capsules using electronic microscopy negative-contrast.

Cell culture strain CM and strains R and CM suspended in saline to a concentration of 2×10 9 cells/ml On polyvinylformamide the grid-substrate put drops of the suspension of cells of strains, which are fixed with 2% osmium oxide, treated with phosphate buffer and stained with 1.5% phosphomevalonate acid or uranylacetate. Browsing in the electron microscope at an instrumental magnification of 8.3×10 3 shows that all cells of strain 169 KM is surrounded by a thick layer of polysaccharide capsules. Its size is 3-4 times the size of the capsule strains analogues (R and CM).

Example 5. Product definition capsules sensitivity of Vibrio cholerae to the bactericidal action of normal human serum (SNP).

Cells of strain KM 169 grown in broth for 4 hours and incubated with 65% of the SNP. The resistance of cells of strains to the bactericidal action of the SNP is indicative of their polysaccharide capsule.

Example 6. The determination of the stability of the formation of capsules in a population of cells of strain.

The strain is cultivated in a liquid nutrient medium at 37°C for 2-3 days and scatter on a dense nutrient medium prior to the formation of isolated colonies. The resulting colonies are screened for the production of capsules. The study of the morphology of 500 colonies of strain shows that they all produce capsular substance and, therefore, strain is a stable producer capsular antigen.

Example 7. The determination of the stability of the formation of capsules after long-term storage in nutrient media.

The stability characteristic of capsulorraphy checked in strain CM kept during the year in semisolid nutrient agar. For this culture strain of semisolid nutrient agar rasshivaetsya on solid nutrient medium to obtain isolated colonies. The study of the morphology of 500 colonies of strain shows that they all produce capsular substance and, therefore, the strain does not lose the ability to stable capsular antigen after longterm storage in nutrient media.

Thus, the avirulent strain CM is the producer of the polysaccharide capsule. It can be successfully applied in the production of cholera chemical vaccine against Vibrio cholerae O139 serogroups as producer capsular antigen. The advantage strains of V.cholerae KM169 over the known analogues is that it is stable, avirulence and has a high level of production of polysaccharide capsules, 3-4 times higher than the education level of this antigen in other strains.

Avirulent bacterial strain Vibrio cholerae serogroup A, collection antiplague research Institute "Germ" No. 169 KM, producer capsular antigen.