Strain of bacteria vibrio cholerae - producer of choleraic toxin of ii type

FIELD: technological processes.

SUBSTANCE: strain of bacteria Vibrio cholerae, which is deposited in State collection of pathogenic bacteria of "ФГУЗ РосНИПЧИ" "Microbe" under number "КМ234" produces choleraic toxin of II type. Strain of V.cholerae possesses high production of choleraic toxin of II type, which forms antitoxic immunity in case of cholera.

EFFECT: preparation of choleraic chemical vaccines for prophylactics of cholera.

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The invention relates to the field of biotechnology, namely, to construct a strain with increased production of cholera toxin type II, and can be used to create chemical vaccines of new generation, as well as when designing immunoassay diagnostic test kits. In addition, this invention can be used in molecular genetic studies aimed at learning a new mechanism of gene expression regulation of virulence and immunogenicity in pathogenic bacteria.

Cholera toxin type II, produced holonymy vibrios biovars El tor circulation and different antigenic properties from the cholera toxin type I, which synthesize V. cholerae classical biovars, is a necessary component of multivalent chemical cholera vaccines forming antitoxic immunity in cholera. This immunogenic protein is also a member of immunodiagnostics test systems designed to differentiate between toxigenic and nontoxigenic strains of V. cholerae biovars El tor circulation.

A known strain of Vibrio cholerae 2414 classical biovars of serovar Ogawa - producer of the cholera toxin and the toxin-coregulatory pilej adhesion (RF patent No. 2169187, 12N 1/20), effectively producing a growing cholera toxin type I and II - 48-60 mg/ml, according to enzyme-linked immunosorbent assay is GM ₁ELISA.

Natural V.cholerae strains of biovars El tor circulation, used in the production of cholera vaccine in vitro to form a small number of cholera toxin type II is less than 0.1 μg/ml (Mekalanos J.J., Duplikation and amplifikation of toxin genes in Vibrio cholerae // Cell. - 1983. - V. - 253-263), therefore their use in an industrial environment is unlikely.

Among the experimentally obtained strains known V.cholerae strains of biovars El tor circulation of serovar Ogawa KM/RCO-2, described in the author's certificate of the USSR No. 1412306, 12N 15/00 and synthesizing 5 μg/ml cholera toxin. The relatively high level of production of this protein due to the presence in the cells of recombinant plasmids RSA-2, carrying the cloned genes for cholera toxin (ctxAB) and the gene for resistance to kanamycin (kan). At the same time, in the absence of selective pressure (kanamycin in the medium of cultivation), the stability of inheritance of recombinant plasmids is 70-80%, which leads to lower production XT. The need to add in an environment of growing antibiotic complicates the technology of cultivation of the strain in a production environment.

Closest to the claimed strain is a strain of V.cholerae KM195 producing cholera toxin type II (patent RF №2172776, MKI: 12N 1/20, 1/21, AT 39/106). The strain obtained experimentally, the infection of cells V.cholerae biovars El tor circulation moderate x is lernen phage 139. Received lysogeny cells were produced in the environment of the cultivation of 7.0 μg/ml (according to GM₁ELISA). However, this strain contains the chromosome temperate phage 139, able to induce secondary genetic changes in the genome, which can lead to the emergence of nontoxigenic options.

The invention consists in the construction of strain V. cholerae biovars El tor circulation with high and stable production of cholera toxin type II.

To create a strain with the desired properties was used transposon mutagenesis of propaga CTXϕcontaining genes ACE, zot and ctxAB, resulting in increased expression of structural genes for cholera toxin (ctxAB). Strain constructed by introducing into the cells of the natural strains of V.cholerae MAK757 biovars El tor circulation conjugative plasmids pSUP5011 (Ap^RCm^R)carrying the transposon Tn5-Mob (Km^R). Subsequent cultivation plasmid strain at 4°in the broth with the addition of kanamycin to select transconjugants Km^RAp^SCm^Sthat have lost the plasmid, but kept transposon embedded in the zot gene of propaga CTXϕ. As a result of reorganization of the genome of propaga in the resulting strain MAK757 zot::TnMob (Km^RTox⁺) there is a significant increase in gene expression of cholera toxin.

The strain deposited in the Public collections of pathogenic the bacteria "Microbe" in Russian research anti-plague Institute "Microbe" (Saratov) number KM.

The claimed strain is characterized by the following features:

- has a high level of production of the cholera toxin type II - 45,0 µg/ml (according to enzyme immunoassay GM₁ELISA);

- genetically labelled due to the introduction of transposon Tn5-mob in gene zot;

- stable - maintains these properties during storage and cultivation on nutrient media.

Cultural and morphological characteristics.

Laboudigue, slightly curved sticks that do not form capsules and spores. On solid nutrient media after 18-24 hour growth at 37°To form a round grayish-blue translucent colonies with smooth edges; in broth form a uniform turbidity. The strain grows on simple nutrient media and on media with addition of kanamycin (50 mg/ml). Prototroph.

Physiological properties.

Not analyzes sheep erythrocytes, agglutinate chicken erythrocytes, grows on alkaline agar with the addition of 50 μg/ml polymyxin b. Not ferments lactose and arabinose, ferments mannose, sucrose, mannitol, maltose to acid without gas. Agglutinated to titers of specific diagnostic sera and Ogawa. Sensitive to cholerea diagnostic phage "El tor circulation". The strain stored in a lyophilized state at least one year.

Example 1, indicating the high productivity of the strain.

About what the definition of products of the cholera toxin. The products of the cholera toxin is determined using radial passive hemolysis (BIG) on a dense environment and by ELISA GM₁ELISA.

BIG, whose sensitivity is 0.2 µg/ml, bet method Bramucei M.G., Holmes R.K. (1978), modified by Shahinian, I.A., Markusa BM (Shahinian I.A., Markusa BM // Ukr. Microbiol. - 1983. No. 7. - Pp.92-96). Colonies of the studied strains by the method of the replicas carry the Cup with 1.0% sensaslim agar containing 1.0% of laundered in shinkasen broth sheep erythrocytes. Crops incubated for 18 h at 30°, pour a layer of 0.5% sinkang agar containing sodium azide, in the presence of complement Guinea pigs and anoreceptive antitoxic serum (ATS) and placed in the incubator for 1-2 h at 37°C. Upon expiration of the specified period around microcolony created strain CM formed a clear zone of immune hemolysis size 2-2 .5 mm, whereas around microcolony original strain MAC this zone is absent.

For the quantitative determination of the cholera toxin used immunoassay method GM₁ELISA (Svennerholm, A.-M., Wikland G., 1983). As a control and to determine the sensitivity of the method using purified product cholera toxin. The original culture of the strain KM grown in Kazarinova broth (Kazarinova acid 30 g/l, yeast extract 5 g/l, pH 7,6) for 16-18 hours in the conditions of the intensive aeration. Cells precipitated by centrifugation at 4000 g for 15 minutes and the supernatant determine the content of the cholera toxin as follows. Samples incubated for two hours at 37°With holes in the blade, pre-blocking nonspecific sorption of inert protein (bovine serum albumin). Incubation with rabbit anti-toxic cholera serum, diluted 1:200, hold for 1 h at 37°C. After washing the wells 0.05 M phosphate buffer pH 7,2-7,4 add working dilution enzyme conjugates, which are a peroxidase labeled goat diagnostic antibodies against rabbit immunoglobulins. The reaction account for 15 minutes after addition of the substrate and 0.03% hydrogen peroxide in citrate buffer pH 4.0 with 0.1% ABTS (2.2 azino-di-(3-ethylbenzthiazoline sulphonate). The sensitivity of ELISA method is in this series of experiments 1 ng/ml Quantity produced cholera toxin in the tested strain CM calculated in accordance with the required sensitivity and counting the number of grown microbial cells.

According to the immunoassay method GM₁ELISA stated the strain produces at 2250 times the cholera toxin (45 µg/ml) compared to baseline (0.02 μg/ml). In comparison with the known strain producer of the cholera toxin type II V.cholerae eitor KM195 on Putin is the Russian Federation No. 2172776 (7 ág/ml) level of production of the cholera toxin has declared strain 6.4 times higher.

Example 2, indicating the presence in the genome of propaga CTXϕ transposon TP5-mob.

Polymerase chain reaction (PCR) with specific primers for the genes ctxA, ctxB, ACE, zot, included in propaga CTXϕ. Cells 18-hour culture grown on LB agar, resuspension in 1 ml of saline to a concentration of 10⁹cells in 1 ml, followed by titration in distilled water adjusted to a final concentration of 10⁷cells in 1 ml of the cells are lysed by boiling for 30 minutes and after centrifugation at 12000 g for 5 min the supernatant in an amount of 10 μl is used for research in PCR. To the sample add solutions of oligonucleotide primers synthesized based on the nucleotide sequences of genes ctxA, ctxB, ACE, zot - 0.4 µl of each, and 2.5 µl of 10x PCR buffer, and 2.5 μl of a solution dNTP, 1 μl of 50 mm solution of magnesium chloride, and 0.25 μl of Tag polymerase and 7,95 ál of deionized water.

As a positive control, using DNA from a typical toxigenic strain 0139-serogroup V. cholerae PI 6064, as well as the original strain of V. cholerae MAK. In the negative control was made 10 ál of deionized water.

PCR is carried out at the following temperature conditions: 1 cycle of 95°C - 5 min; 2-35-th cycles including denaturation at 95°C - 30 sec, annealing at 60°C - 30 sec, and synthesis at 72�B0; C - 30 sec; 36th cycle - a synthesis for 7 minutes at 72°C.

The results take into account PCR by electrophoresis at 80 V for 35 minutes in 1.5% agarose gel containing 0.5 µl/ml ethidium bromide, followed by viewing the gel under UV light. According to PCR analysis in the genome of the original strain MAC are all tested genes propaga ACE, zot, ctxA and ctxB. In the generated strain CM in the presence of genes ctxA, ctxB, ACE specific amplificat zot gene is absent, indicating that the introduction of transposon Tn5-mob.

Example 3, indicating the stability of the products of the cholera toxin.

Strain V. cholerae KM234 cultured for 18 hours in broth without antibiotics, and then scatter on dense medium to obtain isolated colonies. Subsequent checking 700 colonies on the production of toxin by the method of BIG shows that all studied clones were Km^Rand this protein is synthesized in a considerable amount of the area of immune hemolysis around microcolony was 2-2,5 mm These data confirm the stability of the products of the toxin and the stability of inheritance of the transposon Tn5-mob strain KM234. ELISA analysis of 100 randomly selected clones confirmed the high level of production XT (45-48 µg/ml). During production testing of the generated strains were also characterized by stable production of toxin and stable inheritance of the genetic marker Km ^R.

Thus, the claimed strain of Vibrio cholerae KM234 has a high and stable level of production of cholera toxin type II. The strain can be used in production for chemical vaccines of new generation, which contains a more complete set of protective antigens and used for the prevention of cholera caused by Vibrio cholerae classical and El tor circulation vibrios. In addition, the strain can be used for separation of purified cholera toxin type II, used to obtain antitoxic sera included in immunodiagnostics test systems, which are designed to detect natural toxigenic strains of Vibrio cholerae biovars El tor circulation. Moreover, the strain is recommended when conducting genetic research to study a new mechanism of gene expression regulation of virulence and immunogenicity of V. cholerae.

Bacterial strain Vibrio cholerae, deposited in the Public collections of pathogenic bacteria FGUZ antiplague research Institute "Microbe" number CM producing cholera toxin type II.