Bacterial strain vibrio cholerae km 200 - producer of the cholera toxin and the toxin - coregulatory pilej adhesion

 

(57) Abstract:

The invention relates to the field of biotechnology and can be used in production for vaccines and diagnostic test kits. The task of the invention to provide strains of V. cholerae classical biovars of serovar Ogawa with high production of cholera toxin and toxin-coregulatory pilej adhesion, not synthesizing soluble hemagglutinin/protease). The resulting strain of Vibrio cholerae 200 KM, not synthesizing the hemagglutinin/protease - producing cholera toxin and toxin-coregulatory pilej adhesion. The strain obtained by the method of selection of natural toxigenic strain M29, not synthesizing soluble hemagglutinin/protease. The strain does not synthesize soluble hemagglutinin/protease, has a high level of production (14 µg/ml), cholera toxin and toxin-coregulatory pilej adhesion in excess of 2-3 times the level of production of this antigen in known industrial strains, and can be used in production for more effective cholera vaccine choleragen-toxoid by enriching it with new protective antigens (TCP), the preparation of purified antigens of the cholera toxin and the toxin-corehole the spruce identify natural strains of V. cholerae with the products of ART and TCP, to carry out genetic studies of systems of gene expression regulation of virulence and immunogenicity V. cholerae.

The invention relates to biotechnology, namely to receive the strain of V. cholerae classical biovars of serovar Ogawa, effectively producing major protective antigens - cholera toxin, toxin-coregulatory drank adhesion and not synthesizing Wednesday cultivation soluble hemagglutinin/protease. The strain can be used in production to create a highly effective chemical cholera vaccines of new generation, to obtain purified preparations of the cholera toxin and the toxin-coregulatory pilej adhesion intended for further manufacture of diagnostic test systems, and to conduct genetic studies of systems of gene expression regulation of virulence and immunogenicity V. cholerae.

A known strain of classical biovars Vibrio cholerae 569(B) of serovar Inaba, which is used in foreign and domestic medicine as a production to get 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 esto toxin-coregulatory pilej adhesion (TCP), detectable only by highly sensitive method immunoblot, but not in the reaction of coagglutination. This strain, like most of the strains of V. cholerae, produces soluble hemagglutinin/protease with the presence of toxic properties, and ability to the destruction of cellular receptors for the attachment of V. cholerae associated residual reactogenicity and lower survival vaccines.

Closest to the claimed strain is a strain of Vibrio cholerae 2414 of serovar Ogawa (RF patent N2169187 C12 N1/20), effectively producing a growing cholera toxin B-subunit which is the main immunogen (according to GM1ELISA 48-60 mg/ml), synthesized on the surface of cells toxin-coregulatory drank adhesion.

However, this strain contains a plasmid carrying resistance genes to antibiotics, and effective expression of protective antigens growing this strain must be made on media with addition of tetracycline or kanamycin. Strain V. cholerae 2414 produces soluble hemagglutinin/protease.

To create a more effective chemical vaccines need exemption from non-immunogenic, deprotecting antione with high production major protective antigens - the cholera toxin and the toxin-coregulatory pilej adhesion

The objective of the invention is the obtaining of a strain of V. cholerae with high production of cholera toxin and toxin-coregulatory pilej adhesion and absence of soluble hemagglutinin/protease.

Strain V. cholerae 200 KM is characterized by the following features.

Cultural and morphological characteristics.

Motile, slightly curved sticks that do not form capsules and spores. On solid nutrient media grows in the form of a circular grayish-bluish translucent colonies, while sowing in the broth is uniform opacity of this environment. The strain is auxotrophic, carrying a mutation in the gene Pur-.

Pathogenic properties - slability, for rabbit-suckers virulent dose of 107MT/ml.

Physiological properties

Not analyzes sheep erythrocytes, not agglutinate chicken erythrocytes, does not grow on alkaline agar with the addition of 50 μg/ml polymyxin b. Agglutinated to title holonymy diagnostic sera and Ogawa. Sensitive to cholerea diagnostic phage With. Not ferments lactose and arabinose. To acid without gas ferments mannose, sucrose, mannitol, maltose.

In chromosome registered gene hap, but the synthesis of the product encoded by this gene is a hemagglutinin/protease, is missing.

The strain stored in a lyophilized state at least one year.

The strain of Vibrio cholerae KM 200 serovar Ogawa classical biovars obtained by selection of clones with high production TCP from natural strains of V. cholerae M29, producing only cholera toxin and not synthesizing soluble hemagglutinin/protease. Strain V. cholerae KM 200 serovar Ogawa deposited in the Public collections of pathogenic bacteria antiplague research Institute "Microbe" number 200 KM.

Example 1 illustrating the high productivity of the strain

Dried ampoule culture of a strain of V. cholerae 200 KM seeded on the agar Hottingen pH 7.6 and incubated 18 h at 37oC. Grown culture scatter on LB-agar to obtain isolated colonies and further validate the presence of toxin-coregulatory pilej adhesion method coagglutination culture broth [Chiang, S. L. et al. Molecular Microbiology, 1995, v.17, p. 1133-1142] . After 18 h resuspending one grown colony in 1 ml LB broth and as the inoculate contribute 7 μl of this cusp grow 17-18 h in a shaker at 30oC. the Degree of agglutination consider visually. Strain V. cholerae KM 200 causes a visible coagglutination all bacterial cells in broth, indicating that the synthesis of a significant number located on the surface of the toxin-coregulatory pilej adhesion. When compared with the strain of V. cholerae 569B used in the production of chemical cholera vaccine choleragen-toxoid, a strain of V. cholerae KM200 exceeds the rate of protein synthesis TCP 2-3 times.

The broth after coagglutination cells are used to determine the cholera toxin by ELISA [A. M. Svennerholm et al. J. Clin. Environ. , 1983, v.17, R. 596-601]. Samples incubated for two hours at 37oWith holes in the blade, block nonspecific sorption of inert protein (bovine serum albumin). Incubation with rabbit anti-toxic cholera serum, diluted 1:200, hold for 1 h at 37oC. After washing the wells 0.05 M phosphate buffer pH 7,2-7,4 add working dilution enzyme conjugates, which are labeled with peroxidase 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 R. the number produced cholera toxin in the tested strain calculated in accordance with the required sensitivity and counting the number of grown microbial cells. The strain of Vibrio cholerae KM 200 produces 14 μg/ml cholera toxin.

Example 2, confirming the absence of soluble hemagglutinin/protease

To determine the production of soluble hemagglutinin/protease culture grown on agar containing 10% skim milk. Bacteria synthesizing the specified enzyme form a zone of lysis around the colony on the background of the cloudy agar. The strain of Vibrio cholerae KM 200 does not synthesize soluble hemagglutinin/protease and does not form zones of enlightenment around microcolony in contrast, the positive control, which confirms the absence of soluble hemagglutinin/protease.

Thus, the strain of Vibrio cholerae KM 200 does not synthesize soluble hemagglutinin/protease and has high level of production of cholera toxin and toxin-coregulatory pilej adhesion in excess of 2-3 times the level of production of these antigens have known industrial strains. The strain can be used in production for more effective cholera vaccine choleragen-toxoid by enriching it with new protective antigens (TCP); preparation of purified antigens of the cholera toxin and the toxin-coregulatory pilej adhesion, which can be used for time is of the NT and TCP; to carry out genetic studies of systems of gene expression regulation of virulence and immunogenicity V. cholerae.

Bacterial strain Vibrio cholerae KM 200 - producer of the cholera toxin and the toxin-coregulatory pilej adhesion.

 

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SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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