RU2222595C1 - Strain of bacterium vibrio cholerae km 207 of classic biovar serovar inaba as producer of protective antigens - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: invention relates to preparing the strain Vibrio cholerae KM 207 of classic biovar serovar Inaba as a producer of protective antigens (subunit B as component of choleraic toxin, the main colonizing factor of choleraic vibrio, toxin-coregulated adhesion pili, protective and immunogenic protein OmpU of external membrane). Method involves conjugative incorporation to cells the natural toxigenic strain Vibrio cholerae 569B of classic biovar serovar Inaba recombinant plasmid pCT105 (Km^rTc^r) with cloned genes of choleraic toxin and the following elimination of plasmid. The level of choleraic toxin production in the strain is increased by 2.6 times (23.4 mcg/ml), synthesis of toxin- -coregulated adhesion pili is elevated also and protein OmpU appeared. In culturing the strain addition of antibiotics to medium is not required as in the case of plasmid strains. Using the strain allows the enhancement of choleraic toxin production level, toxin-coregulated adhesion pili (this causes self-agglutination of cells in broth) and synthesizes protein OmpU of external membrane. Invention can be used for preparing vaccines and diagnostic test-systems. EFFECT: valuable properties of strain. 2 ex

Description

 The invention relates to biotechnology, namely, to obtain a strain of cholera vibrio of the classic biovar Serovar Inaba, effectively producing the main protective antigens - cholera toxin, toxin-regulated adhesion pills and the outer membrane protein OmpU, and can be used in production to create highly effective chemical cholera vaccines of the new generation, obtaining purified preparations of these antigens, intended for further preparation of diagnostic test systems, as well as for gene studies on the systems of regulation of the expression of virulence and immunogenicity genes of cholera vibrio.

The known strain V. cholerae 2414 of Serovar Ogawa (RF patent 2169187 MKI: C 12 N 1/20), effectively producing cholera toxin (XT) in the growth medium, the B subunit of which is the main immunogen (according to GM ₁ ELISA 48-60 μg / ml), synthesizes on the cell surface the main colonizing factor of cholera vibrio toxin-regulated adhesion pills (TKPA) and having an outer membrane protein OmpU.

 However, this strain belongs to another serovar, contains a plasmid carrying the antibiotic resistance genes, and for the effective expression of protective antigens when growing this strain, antibiotics must be added to the growing medium.

Closest to the claimed strain is a strain of V. cholerae 569B Inaba, which is used in foreign and domestic medicine as a production for vaccines (RF patent 2076734, A 61 K 39/00, 35/76; Ketley J. et all. Infect. Immun., 1990, v. 141, p. 893-899). However, this strain according to GM ₁ ELISA produces only 9 μg / ml of cholera toxin in the growth medium and a small amount of toxin-regulated adhesion piles detected only by the highly sensitive immunoblot method, but not in the self-agglutination reaction, and also does not produce a powerful adhesion factor of cholera vibrio, OmpU protein located on the surface.

 To create more effective chemical vaccines, as well as cheap diagnostic test systems, strains of cholera vibrio with high production of the main protective antigens are needed: cholera toxin, toxin-regulated adhesion pili and OmpU outer membrane protein.

 The objective of the invention is to obtain a strain of cholera vibrio classic biovar Serovar Inaba with high production of the main protective antigens.

 The essence of the invention lies in the fact that a strain of the classical biovar V. cholerae of Serovar Inaba was obtained, which is the producer of the main protective antigens: cholera toxin, toxin-regulated adhesion pili, and the outer membrane protein - OmpU.

The inventive strain was constructed by conjugative introduction into the cells of the natural strain V. cholerae 569B Inaba, recombinant plasmid pST105 (Km ^r Tc ^r ), with cloned cholera toxin genes, and subsequent elimination of the plasmid from the cells. With the introduction of the plasmid, there is a simultaneous increase in the production of three main protective antigens (XT, TKPA, OmpU protein), apparently due to an increase in the activity of the toxR gene due to plasmid-chromosomal relationships, which is a common regulatory gene for the structural genes ctxAB, tcp, ompU, which does not decrease upon elimination of the plasmid.

 As a result, a strain of the classical biovar Serovar Inaba was obtained, in which the level of cholera toxin production increased by 2.6 times, the synthesis of toxin-corrected adhesion patches increased significantly and the OmpU protein appeared. For the cultivation of the indicated strain, the addition of antibiotics to the growing medium is not required, as in the case of plasmid strains, which simplifies the process of growing the culture, reduces production costs, and also does not contaminate the finished product with antibiotics.

 The strain was deposited in the State collection of pathogenic bacteria RosNIPCHI "Microbe" (Saratov) under the number KM207.

The main properties of the strain V. cholerae KM207:
- a high level of production in the growing medium (23.4 μg / ml), cholera toxin, the B-subunit of which is the main immunogen;
- the formation of toxin-regulated pili adhesion, the main colonizing factor of cholera vibrio;
- OmpU outer membrane protein synthesis with protective and immunogenic properties.

 Cultural and morphological characters.

 Movable, slightly curved sticks that do not form capsules and spores. On solid nutrient media it grows in the form of round grayish-bluish translucent colonies; when sown in broth, it causes uniform turbidity of this medium. The strain grows on simple environments. Prototroph.

Pathogenic properties - virulent for sucker rabbits at a dose of 10 ⁵ mt / ml

 Physiological properties.

 Does not lyse sheep erythrocytes, does not agglutinate chicken red blood cells, does not grow on alkaline agar with the addition of 50 μg / ml polymyxin. Agglutinated to titer with cholera diagnostic sera O and Inaba. Sensitive to cholera diagnostic phage C. Does not ferment lactose and arabinose. To acid without gas, it ferments mannose, sucrose, mannitol, maltose.

 The strain is stored in a freeze-dried state for at least one year.

 Example 1 illustrating the high productivity of the strain.

The dried ampoule culture of strain V. cholerae KM207 is seeded on Hottingen agar pH 7.6 and incubated for 18 hours at 37 ^° C. The grown culture is seeded on LB agar to obtain isolated colonies and then check for the presence of toxin-regulated adhesion piles by culture self-agglutination broth [Chiang SL et al. Molecular Microbiology, 1995, v.17, p.1133-1142]. After 18 hours, one grown colony is resuspended in 1 ml of LB broth and 7 µl of this suspension is added as an inoculum to 10 ml of casamine broth containing 3% casamino acids and 0.5% yeast extract, pH 7.6. The culture is grown for 17-18 hours in a shaker at 30 ^o C. The degree of agglutination is taken into account visually. Strain KM207 causes visible self-agglutination of bacterial cells in the broth due to the synthesis of a significant amount of toxin-corrected adhesion pylons located on the surface.

Cells after self-agglutination are used to determine the synthesis of OmpU protein by electrophoresis, and cholera toxin is determined by the enzyme immunoassay in the broth [Svennerholm AM et al. J. Clin. Microbiol., 1983, v. 17, p. 596-601]. Samples of the broth are incubated for two hours at 37 ^o C in the wells of microplates, then block 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 ^o C. After washing the wells with 0.05 M phosphate buffer pH 7.2-7.4 add working dilution of enzyme-linked immunosorbent conjugates, which are labeled with peroxidase goat diagnostic antibodies against rabbit immunoglobulins. 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-thylbenzthiazoline sulphonate)]. The sensitivity of the ELISA method in this series of experiments is 1 μg / ml. The amount of cholera toxin produced in the studied strain is calculated in accordance with the established sensitivity and counting the number of grown microbial cells. Strain V. cholerae KM207 produces 23.4 μg / ml of toxin.

 Example 2 Confirming OmpU Outer Membrane Protein Synthesis

 The culture of cholera vibrio strain KM207 after self-agglutination is studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to study the protein composition [Laemmli U.K. Nature, 1970, v. 227, p. 680-685]. Cells are resuspended in distilled water, an equal volume of “sample buffer” is added, boiled in a water bath for 5-7 minutes. Electrophoresis is carried out before the sample enters the gel at a current load of 10 mA and then at 20 mA. An electrode buffer was used containing 0.195 M glycine / 0.025 M Tris / 0.1% sodium dodecyl sulfate, pH 8.3. Protein fixation in polyacrylamide gel after electrophoresis is carried out for one hour or night in a solution containing isopropanol, fixed Coomassie R-250 proteins are stained for one hour. Excess dye from the gel is washed out by diffusion in a solution of 7% acetic acid. The presence of OmpU protein in the claimed strain is detected by comparing the protein profile of cell lysates of strain KM207 with the original natural strain Inaba V. cholerae 569B that does not contain this protein and with known molecular weight markers.

 Thus, the inventive strain Vibrio cholerae KM207 has a high level of production of cholera toxin, toxin-regulated pili adhesion, protein OmpU and for its growth in the environment does not require the addition of antibiotics. The strain can be used in production to produce a more effective cholerae vaccine - an toxoid containing new protective antigens, which is used to prevent cholera; preparation of cheap purified antigens used in the development of diagnostic test systems for the detection of natural strains with the production of cholera toxin and toxin-regulated adhesion pili; for conducting genetic studies on the systems of regulation of the expression of virulence and immunogenicity genes of cholera vibrio.

Claims (1)

The bacterial strain Vibrio cholerae KM207 of the classic biovar Serovar Inaba is a producer of protective antigens.