The bacterial strain escherichia coli km 147 - producer - subunit of the cholera toxin

 

(57) Abstract:

The invention relates to biotechnology, namely, construction of Escherichia coli - producer cholera toxin, and can be used to create a diagnostic test systems, allowing to detect toxigenic clones of Vibrio cholerae and Escherichia coli, as well as for the design of live vaccine strains against diarrhoeal diseases caused by pathogenic strains of V. cholerae and E. coli. Avirulent strain of E. coli KM 147 - active producer of protective antigen produced by conjugating introducing into the cell a recombinant plasmids pIEM3 with the cloned gene In the-subunit of the cholera toxin. The invention allows to obtain non-pathogenic for humans and animals strain with a high products the main protective antigen of V. cholerae (- subunit of the cholera toxin).

The invention relates to biotechnology, namely, construction of Escherichia coli - producer cholera toxin, and can be used to highlight the purified immunogenic B-subunit of cholera toxin, which is part of diagnostic test systems, allowing to detect toxigenic clones of Vibrio cholerae and Escherichia coli. In addition, the strain can serve as the genetic strains of V. cholerae and E. coli.

Known strain of E. coli K. S3043 producing B-subunit of cholera toxin. However, the level of production of this protein is 0.85 µg/ml (N.In.Janiszewski, A. L. Ginzburg, Y. C. Vercieu, Y. M. Gemme, G. I. Karataev, G. B. Smirnov. Construction of recombinant plasmids encoding the biosynthesis of the B-subunit of cholera toxin. //Molecules. genetics. - 1987. No. 4). In addition, In the-subunit of the cholera toxin from this strain is in periplasm and not secreted into the medium of cultivation. To select it requires the destruction of cells by exposure to both physical and chemical factors.

Also known strain of V. cholerae KM93-II pathogenicity groups. who is the producer of B-subunit of cholera toxin released into the environment is growing. The number of b-subunit of this strain of V. cholerae is 8.5 µg/ml (USSR author's certificate No. 1505022, MKI C 12 N 15/00).

The objective of the invention is the construction of a non-pathogenic for human and animal strains with high production one of the major protective antigens In the-subunit of the cholera toxin.

The invention consists in that the avirulent strain of Escherichia coli producing B-subunit of cholera toxin (main Ave the M 17 (which is the basis of preventive medicine colibacterin) by conjugating the introduction into the cells of this strain recombinant plasmids: REM (KmrTcrwith cloned genes cholerae toxin (ctxB). This plasmid is conjugatively cointegrate consisting of two plasmids pIEM1 (a derivative of plasmid Roh containing the transposon mini-kan and IS1-sequence) and rst (a derivative of plasmid pBR322 carrying the gene for resistance to tetracycline (TCr), and the cloned fragment of the chromosome of strain biovars V. cholerae El tor circulation RV79 with ctxB gene, determining the synthesis of the B-subunit of cholera toxin).

The strain deposited in the Public collections of pathogenic bacteria antiplague research Institute “Microbe” (,Saratov) number KM 147.

Characterization of Escherichia coli KM147.

Morphological features.

Cells are straight, rod-shaped, motile with peritrichous flagella, gram-negative, nesporoobrazuth, oxidatively.

Pathogenic properties.

The strain is non-pathogenic for humans and animals.

Cultural properties.

Chemoorganotrophs, catalogoplantillas. Grows on nutrient media under optimal temperature C at pH 7.2, forming a smooth intense haze. Prototroph.

Plasmid composition.

Strain KM 147 contains plasmid RAM bearing structural ctxB gene, is icine. The basic properties of strain:

- high level of production In the-subunit of the cholera toxin in the environment of growing to 10 ug/ml;

- ability to develop antitoxic antibodies in immunized animals;

- stability - stable in storage and cultivation on nutrient media, as well as in the organism of laboratory animals;

- safe for laboratory animals.

Example 1. The determination of the stability of inheritance of recombinant plasmids in cells of strain.

The stability of plasmids REM was determined by growing the strain in non-selective conditions within 48-72 hours in LB broth at S. then subcultured to monocolonal on LB agar. The resulting transconjugants tested on markers of resistance plasmids, i.e., determined resistance to kanamycin (50 μg/ml) and tetracycline (10 μg/ml), encoded by plasmid RAM. Were selected monopedigree clones with 100% stable inheritance of the plasmid RAM for further research.

The stability of the introduced recombinant plasmids were also tested in vivo in the passages of the recombinant strain in the organism of laboratory animals white mice and rabbits-suckers. It is established that PI MT) and then seeding it on non-selective agar all grown clones were kept in their cells plasmid - REM (KmrTCr).

The small rabbit-suckers were infected with the indicated strain intragastrically (1104MT). 48 hours after infection of inoculated on complete agar, monoclone were tested for the presence of plasmid markers of antibiotic resistance found that after a 48-hour stay in the intestines of rabbits 100% of the studied clones kept replica - pIEM3(KmrTcr).

Example 2. Product definition protective antigen.

To assess gene expression In the-subunit of the cholera toxin constructed in the E. coli strain KM147 was used the reaction of passive immune hemolysis (BIG) on a dense environment and immunoenzymatic method (ELISA). But this BIG entered ctxB gene effectively expressively in cells of E. coli strain KM147. The area of immune hemolysis of erythrocytes around microcolony the studied strain was much greater than the size of the zone of hemolysis around microcolony high-toxigenic strains of V. cholerae 569B.

The products of the cholera toxin was determined also highly sensitive quantitative immunoassay using ELISA method. It was found that the results of this m is almost 12 times higher than the production in a previously created strain-producer of this antigen E. coli KS3043 of 0.85 μg/ml).

In addition, if the cells of recombinant E. coli containing the cloned gene In the-subunit of the cholera toxin, more than 98% produced In-subunit remains in periplasm and not secreted to the outside, we created a strain of E. coli KM147 produced antigen is secreted to the outside.

Example 3. The specific definition of the safety of recombinant strain.

Safety engineered strain was studied on the model of white mice and rabbits-suckers. White mice were infected intraperitoneally with 1 ml of the cell suspension at a dose of 110 MT During the observation period (7 days) death of the infected animals was not observed physiological parameters remained within the normal range. After this time the animals were killed and determined the presence of macroscopic changes in the intestine. All experimental animals there were no macroscopic changes in the colon - accumulation of fluid in the lumen of the cecum and adjacent departments. Rabbit-suckers infected vnutriserdechno and intragastrically at doses of 1109and 5109MT Animals were monitored for 4 days. It is established that the introduction of a recombinant strain of E. coli KM147 did not lead to the development of diarrhea or death. All sometimeswhen properties.

To study the immunogenic properties of the recombinant strain of E. coli KM147 was administered intragastrically to adult rabbits at a dose of 1010MT Antigenic activity of the studied strain was determined by identifying serum antitoxic antibodies. The accumulation of these antibodies was determined in the dynamics (the serum of animals received at 7, 14, 21, 28, 35, 42 and 49 days). Immunization was performed twice with an interval of 28 days. The resulting serum to remove heterologous antibodies barbirolli cells of the original strain. According to the results of a highly sensitive DOT-immunoassay (qualitative method) serum of immunized animals contained antibodies to the b-subunit of cholera toxin. Quantitative evaluation of education study of antibodies was performed using enzyme-linked immunosorbent ELISA method (determination of antibodies to the b-subunit of cholera toxin).

When determining the level of antibodies to the b-subunit of cholera toxin using immunoenzymatic ELISA method it was found that the maximum titer (1:3200) antitoxic antibodies were recorded on 35 day of immunization and persisted throughout the observation period.

Thus, the claimed strain has a high level of production In-subheading the ski immunity. The strain is safe for laboratory animals regardless of the method of introduction into the organism. The specified properties of the recombinant E. coli strain KM147 indicate that strain can be used to isolate purified immunogenic B-subunit of the cholera toxin and the subsequent preparation of antisera for detection of toxigenic strains; for subsequent creation of diagnostic test systems, allowing to detect toxigenic clones of V. cholerae as the basis for construction of vaccine strains against diarrhoeal diseases caused by pathogenic strains of V. cholerae and E. coli.

The bacterial strain Escherichia coli KM producing B-subunit of cholera toxin.

 

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