RU2326941C1 - Strain of bacteria vibrio cholerae - producer of choleraic toxin of ii type - Google Patents

Abstract

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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Description

The invention relates to the field of biotechnology, namely to the construction of a strain with increased production of type II cholera toxin, and can be used to create a new generation of chemical vaccines, as well as in the design of enzyme-linked immunosorbent diagnostic test systems. In addition, this invention can be used in molecular genetic studies aimed at studying a new mechanism for regulating the expression of virulence and immunogenicity genes in pathogenic bacteria.

Type II cholera toxin, produced by the biovar eltor cholera vibrios and differing in antigenic properties from type I cholera toxin, which is synthesized by V. cholerae of the classical biovar, is an essential component of polyvalent chemical cholera vaccines that form antitoxic immunity in cholera. This immunogenic protein is also part of the immunodiagnostic test systems designed to differentiate toxigenic and non-toxigenic strains of cholera vibrio biovar eltor.

A known strain of Vibrio cholerae 2414 of the classic biovar of Serovar Ogawa is a producer of cholera toxin and toxin-regulated adhesion pyla (RF patent No. 2169187, C12N 1/20), which effectively produces cholera toxin I and II types - 48-60 μg / ml, according to the enzyme immunoassay GM ₁ ELISA.

Natural V. cholerae biovar eltor strains used in the production of cholera vaccines, in vitro form a small amount of type II cholera toxin - less than 0.1 μg / ml (Mekalanos JJ, Duplikation and amplifikation of toxin genes in Vibrio cholerae // Cell. - 1983. - V. - 253-263), because of which their use in an industrial environment is unlikely.

Among the experimentally obtained strains, the V. cholerae biovar eltor serovar Ogawa strain KM1446 / pCO107-2 is described, described in USSR author's certificate No. 1412306, C12N 15/00 and synthesizing 5 μg / ml cholera toxin. A rather high level of production of this protein is due to the presence in the cells of the recombinant plasmid pCO107-2 carrying the cloned cholera toxin genes (ctxAB) and the kanamycin resistance gene (kan). At the same time, in the absence of selective pressure (kanamycin in the growth medium), the inheritance stability of the recombinant plasmid is 70-80%, which leads to a decrease in XT production. The need to add an antibiotic to the growing medium complicates the technology of culturing the strain under production conditions.

Closest to the claimed strain is strain V. cholerae KM195 - producer of cholera toxin type II (RF patent No. 2172776, MKI: C12N 1/20, 1/21, AK61/106). The strain was obtained experimentally by infection of V. cholerae biovar eltor cells with a moderate cholera phage 139. The obtained lysogenic cells were produced in the growth medium 7.0 μg / ml (according to GM ₁ ELISA). However, this strain contains a moderate phage 139 on the chromosome, capable of inducing secondary genetic rearrangements in the genome, which can lead to the emergence of nontoxigenic variants.

The essence of the invention consists in the construction of a strain of cholera vibrio biovar eltor with high and stable production of cholera toxin type II.

To create a strain with the desired properties, transpositional mutagenesis of CTXφ prophage containing ace, zot, and ctxAB genes was used, as a result of which the expression of structural cholera toxin genes (ctxAB) was increased. The strain was constructed by introducing into the cells a natural strain V. cholerae MAK757 biovar eltor conjugative plasmid pSUP5011 (Ap ^R Cm ^R ) carrying the transposon Tn5-Mob (Km ^R ). During subsequent cultivation of the plasmid strain at 4 ° C in the broth with the addition of kanamycin, Km ^R Ap ^S Cm ^S transconjugants were selected that lost the plasmid but retained the transposon introduced into the zot gene of the CTXφ prophage. As a result of the reorganization of the prophage genome in the obtained strain MAK757 zot :: TnMob (Km ^R Tox ⁺ ), a significant increase in the expression of cholera toxin genes occurs.

The strain was deposited in the State collection of pathogenic bacteria "Microbe" in the Russian research anti-plague institute "Microbe" (Saratov) under the number KM234.

The claimed strain is characterized by the following features:

- has a high level of production of type II cholera toxin - 45.0 μg / ml (according to the enzyme immunoassay GM ₁ ELISA);

- genetically marked due to the introduction of the Tn5-mob transposon into the zot gene;

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

Cultural and morphological characters.

Weak, slightly curved sticks that do not form capsules and spores. On solid nutrient media, after 18-24 hours of growth at 37 ° C, round grayish-blue translucent colonies with even edges are formed; in the broth form a uniform turbidity. The strain grows on simple nutrient media and on media supplemented with kanamycin (50 mg / ml). Prototroph.

Physiological properties.

It does not lyse sheep red blood cells, agglutinates chicken red blood cells, grows on alkaline agar with the addition of 50 μg / ml polymyxin. Does not ferment lactose and arabinose, ferment mannose, sucrose, mannitol, maltose to acid without gas. Agglutinized to titer with specific diagnostic sera O and Ogawa. Sensitive to cholera diagnostic phage "Eltor". The strain is stored in a freeze-dried state for at least one year.

Example 1, indicating high productivity of the strain.

Determination of cholera toxin production. The production of cholera toxin is determined using radial passive hemolysis (RPIG) in a dense medium and the enzyme immunoassay GM ₁ ELISA.

RPIG, the sensitivity of which is 0.2 μg / ml, set according to the method of Bramucei M.G., Holmes R.K. (1978), modified by Shaginyan I.A., Marakusha B.M. (Shaginyan I.A., Marakusha B.M. // Journal. Microbiol. - 1983. - No. 7. - S.92-96). Colonies of the studied strains using the replica method are transferred to plates with 1.0% syncase agar containing 1.0% sheep erythrocytes washed in the syncase broth. Crops were incubated for 18 hours at 30 ° C, poured with a layer of 0.5% syncase agar containing sodium azide, guinea pig complement and monoreceptor antitoxic serum (ATS) and placed in a thermostat for 1-2 hours at 37 ° C. After this period, a clear zone of immune hemolysis of 2-2.5 mm in size is formed around the macrocolonies of the created strain KM234, while this zone is absent around the macrocolonies of the original strain MAK757.

For the quantitative determination of cholera toxin, the enzyme immunoassay GM ₁ ELISA is used (Svennerholm A.-M., Wikland G., 1983). As a control and to determine the sensitivity of the method, a purified cholera toxin preparation is used. The initial culture of strain KM234 is grown in casamine broth (casamic acids - 30 g / l, yeast extract - 5 g / l, pH 7.6) for 16-18 hours under intensive aeration. Cells are pelleted by centrifugation at 4000 g for 15 minutes and the cholera toxin content in the supernatant is determined as follows. Samples are incubated for two hours at 37 ° C in microplate wells, after having previously blocked non-specific sorption with an inert protein (bovine serum albumin). Incubation with rabbit antitoxic anti-cholera serum diluted 1: 200 is carried out for 1 h at 37 ° C. After washing the wells with 0.05 M phosphate buffer pH 7.2-7.4, working dilution of enzyme-linked immunosorbent conjugates, which are goat peroxidase-labeled diagnostic antibodies against rabbit immunoglobulins, is added. The reaction is taken into account 15 minutes after adding the substrate - 0.03% hydrogen peroxide in citrate buffer pH 4.0 with 0.1% ABTS (2.2 azino-di- (3-ethylbenzthiazoline sulphonate). The sensitivity of the ELISA method in this series of experiments 1 ng / ml the Number of produced cholera toxin in the studied strain KM234 is calculated in accordance with the established sensitivity and counting the number of grown microbial cells.

According to the immunoenzyme method GM ₁ ELISA, the claimed strain produces 2250 times more cholera toxin (45 μg / ml) compared to the original (0.02 μg / ml). Compared with the known strain producing cholera toxin type II V. cholerae eitor KM195 according to the patent of the Russian Federation No. 2172776 (7 μg / ml) the level of cholera toxin production in the claimed strain is 6.4 times higher.

Example 2, indicating the presence in the genome of the CTXφ prophage transposon Tn5-mob.

Use the polymerase chain reaction (PCR) with specific primers for the genes ctxA, ctxB, ace, zot, which are part of the CTXφ prophage. 18-hour culture cells grown on LB agar are resuspended in 1 ml of physiological saline to a concentration of 10 ⁹ cells in 1 ml, then titrated in distilled water to a final concentration of 10 ⁷ cells in 1 ml. Next, the cells are lysed by boiling for 30 minutes and after centrifugation at 12000 g for 5 minutes, the supernatant in an amount of 10 μl is used for PCR testing. Solutions of oligonucleotide primers synthesized based on the nucleotide sequences of the ctxA, ctxB, ace, zot genes — 0.4 μl each, 2.5 μl 10-fold PCR buffer, 2.5 μl dNTP solution, 1 μl 50 mM are added to the sample a solution of magnesium chloride, 0.25 μl of Tag polymerase and 7.95 μl of deionized water.

As a positive control, DNA is used from a typical toxigenic strain 0139-serogroup V. cholerae PI 6064, as well as the original strain V. cholerae MAK757. 10 μl of deionized water was added to the negative control.

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

PCR results are taken into account by electrophoresis at 80 V for 35 minutes in a 1.5% agarose gel containing 0.5 μl / ml ethidium bromide, followed by viewing the gel in UV light. According to PCR analysis, all the tested genes for the ACE, zot, ctxA, and ctxB prophage are present in the genome of the original MAK757 strain. In the created strain KM234 in the presence of ctxA, ctxB, and ACe genes, there is no specific amplification of the zot gene, which indicates the introduction of the Tn5-mob transposon into it.

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

Strain V. cholerae KM234 is cultured for 18 hours in a broth without antibiotics, and then seeded into solid media to obtain isolated colonies. A subsequent check of 700 colonies for the production of toxin by the RPIG method shows that all the studied clones were Km ^R and synthesized this protein in significant quantities - the zone of immune hemolysis around the macrocolonies was 2-2.5 mm. These data confirm the stability of the toxin production and the stability of the inheritance of the transposon Tn5-mob strain KM234. Enzyme-linked immunosorbent assay of 100 randomly selected clones confirmed a high level of XT production (45-48 μg / ml). During production tests, the created strain was also characterized by stable toxin production 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 type II cholera toxin. The strain can be used in production for the production of a new generation of chemical vaccines containing a more complete set of protective antigens and used for the prevention of cholera caused by cholera vibrio of both the classical and eltor vibrios. In addition, the strain can be used to isolate purified type II cholera toxin, used to produce antitoxic sera that are part of the immunodiagnostic test systems, which are designed to detect natural toxigenic strains of Vibrio cholerae biovar eltor. Moreover, the strain is recommended for use in genetic studies to study a new mechanism for regulating the expression of virulence and immunogenicity genes of cholera vibrios.

Claims (1)

The strain of bacteria Vibrio cholerae, deposited in the State collection of pathogenic bacteria of the Federal State Research Institution RosNIPCHI "Microbe" under the number KM234 - producer of cholera toxin type II.