Bacterial strain vibrio cholerae eltor km 195-producer of the cholera toxin type ii, the method of obtaining strain

 

(57) Abstract:

The invention relates to biotechnology and medical Microbiology, and can be used in the production of cholera vaccine. This invention concerns a strain of bacteria Vibrio cholerae eltor 195 KM and the method of obtaining this strain. The method comprises the stages: the growing culture of Vibrio cholerae eltor to a concentration of 109cells/ml, the introduction of genetic information by infection of cells of the strain of Vibrio cholerae eltor moderate by phage 139 to obtain lysogenic cells, selected cells of Vibrio cholerae eltor, lysogenic on phage 139 and containing in the structure of chromosomes, selection lysogenic cells with significant production of cholera toxin. The method allows to obtain a strain of Vibrio cholerae eltor with high and stable production of cholera toxin type II - 7 µg/ml 2 C. C.p. f-crystals.

The invention relates to biotechnology and medical Microbiology, and may find application in the production of cholera vaccine.

Known strains used to obtain cholera vaccines, the latter must contain, in addition to the antigens of the cholera toxin type I formed holonymy vibrios classical biovars, antigens cholera toxin type II formed holonymy VI the local natural strains of Vibrio cholerae eltor, used in the production of cholera vaccine. They have typical for V. cholerae properties and form required for the production of cholera vaccine antigens [Live oral vaccines against cholerae: an update. Levine M. M., G. Kaper // Vaccine. - 1993. - V. 11.- Issue 2. - P. 207-212].

However, natural strains of the El tor circulation form in vitro negligible amounts of cholera toxin type II (less than 0.1 μg/ml) [Mekalanos J. J. Duplication and amplfication of toxin genes in Vibrio cholerae // Cell. - 1983. - V. 35. - P .253-263], therefore their use in an industrial environment is ineffective. The use of special media and culturing conditions can only slightly increase the amount they formed toxin (0.2 ág/ml) [Differential expression of the tox R regulon in classical and El Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression. Dirita, V. G. et al. // Proc. Natl. Acad. Sci. USA. - 1996. - V. 93. - P. 7991-7995].

Closest to the claimed strain is a strain of V. cholerae eltor of serovar Ogawa 1446 KM/RDF 107-2, described in the author's certificate of the USSR N 1412306 C 12 N 15/00. This strain obtained experimentally and forms a significant amount of cholera toxin type II - 5 µg/ml.

However, it is characterized by insufficient stability of the products the cholera toxin, which complicates the technology of cultivation of strain.

However, the described methods of obtaining these strains laborious and inefficient, because it is still not possible to obtain a strain with a high production of cholera toxin type II.

Closest to the claimed method of producing producer strain of cholera toxin type II on the achieved result is a method that is described in the author's certificate of the USSR N 1412306 C12N 15/00, which consists in the introduction of genetic information, which causes the increase of oxygenate, in cells of strain Vibrio cholerae eltor method of the conjugation transfer plasmids RDF 107-2 containing cloned genes for cholera toxin. It includes the following stages:

1. Growing cultures of Vibrio cholerae eltor to a concentration of 109cells /ml.

2. The introduction of genetic information by crossing strains of Vibrio cholerae eltor strain of E. coli containing plasmid RDF 107-2 the genes for cholera toxin.

3. The selection of cells of Vibrio cholerae eltor containing plasmid RDF 107 - 2.

4. The selection of cells of strain Vibrio cholerae eltor containing plasmid RDF 107-2 and producing cholera toxin type II.

The application of this method allows to obtain produce is that introduced the cholera toxin genes are located on extrachromosomal replicon is a plasmid, which is often lost by the cell, which leads to a decrease in level formed by the strain of toxin.

The objective of the invention is to construct a highly efficient producer strain of cholera toxin type II, as well as the development method, which allows to obtain a strain with stable and high production of cholera toxin type II.

The invention consists in that the resulting strain of Vibrio cholerae eltor producing cholera toxin type II.

The inventive strain produces 7 μg/ml cholera toxin type II in the growth medium.

The strain of Vibrio cholerae eltor producing cholera toxin type II is typical for V. cholerae biovars El tor circulation properties [baroan O. Century Cholera El tor. - Moscow, 1971]

The strain deposited in the Public collections of pathogenic bacteria "Microbe" as Vibrio cholerae eltor KM 195.

The essence of the proposed method get called stemma is that in the method, which includes the cultivation of Vibrio cholerae eltor, the transmission of genetic information, which causes the increase of oxygenate, screening and selection of cells with a high content of products cholera toxin, ate by phage 139 to obtain lysogenic cells, containing the phage in the structure of chromosomes and the selection of cells select the cells that have improved the content of the production of cholera toxin.

The inventive method involves the following stages:

1. Growing cultures of Vibrio cholerae eltor to a concentration of 109cells /ml.

2. The introduction of genetic information by infection of cells of the strain of Vibrio cholerae eltor moderate by phage 139, consisting in the adsorption of the phage to the bacterial surface, the penetration of its DNA into the cell followed by integration into the chromosome and formation of lithogenous.

3. The selection of cells of Vibrio cholerae eltor, lysogenic on phage 139 and containing it within a chromosome.

4. The selection among them lysogenic cells with significant production of cholera toxin.

Detailed description of the method of producing strain of Vibrio cholerae eltor producer cholera toxin type II below.

The original strain MAK 757 grown for three hours at a temperature of 37oC under intensive aeration to a concentration of 109cells / ml, mixed with LB-agar in the ratio of 1:25 and layer on a plate of LB-agar. Then pour on it put a drop (0.05 ml) of the culture fluid strain of Vibrio cholerae 0139 P16064 grown within the. Lawn MAK 757 coated with phage grown at a temperature of 37oC for 18 hours until lysogenic colonies. Then from the middle of the spot lysis selected colonies grown lithogenes strain MAK 757(139). Their twice scatter on LB-agar. The resulting isolated colonies examined for sensitivity to phage 139 and on production of this phage. Colony MAK 757 resistant to re-infection by phages and producing it in the growth medium are tested for ability to form toxin. With a frequency of one percent among the received lithogenes MAK 757(139) identify cells producing 10-30 times more toxin compared with the parent strain. Among them select the clone stably forming a significant amount of cholera toxin. The amount of toxin is determined by the ELISA method.

The method of DNA-DNA hybridization shows that in a chromosome is a DNA sequence of phage 139.

The inventive method allows to obtain a strain of Vibrio cholerae eltor with high and stable production of cholera toxin type II - 7 µg/ ml.

Example 1. Product definition the cholera toxin type II in strain Vibrio cholerae eltor KM 195.

For the quantitative determination of the cholera toxin ISP purified product cholera toxin. The original culture of strain 195 KM grown in Kazarinova broth (Kazarinova acid 30 g/l, yeast extract 5 g/l, pH 7,6) for 16-18 hours under conditions of intensive aeration. Cells precipitated by centrifugation at 4000 g for 15 minutes. The supernatant determine the content of the cholera toxin. Samples incubated for two hours at 37oC in the wells of blades, 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 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 pH 4.0 with 0.1% of ABTS (2,2 azino-di-(3-thylbenzthiazoline sulphonate). The sensitivity of the ELISA method is in this series of experiments 1 ng/ml Quantity produced cholera toxin in the tested strain 195 KM calculated in accordance with the required sensitivity and counting the number of grown microbial CL is drop the cholera toxin type II.

Example 2. The determination of the stability of the production strain of Vibrio cholerae eltor KM.

Strain 195 KM scatter to isolated colonies that analyze the production of cholera toxin in the manner specified in example 1. Out of 100 tested cells in all products cholera toxin is 7 µg/ml, which confirms the stability of toxin production in strain 195 KM.

Thus, the strain of Vibrio cholerae eltor KM is hyperproduction the cholera toxin type II, and the new method allows to obtain a strain with stable and high production of cholera toxin type II. The strain can be successfully applied in the production of cholera vaccines used for the prevention of cholera. In addition, it can be used to obtain monospecific serum to cholerea toxin type II, as well as to conduct scientific studies on the virulence of V. cholerae.

1. Bacterial strain Vibrio cholerae eltor KM 195 - producer of the cholera toxin type II.

2. The method of producing strain of Vibrio cholerae eltor KM 195 - producer cholera toxin type II, which includes the cultivation of culture, the transmission of genetic information, which causes the increase of oxygenate, incubation, selection and allocation of producers, different is Olamim phage 139 to obtain lysogenic cells, containing the phage in the structure of chromosomes, and in the process of selection of the selected cells with increased production of cholera toxin type II.

 

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