RU2238975C1 - Strain of bacterium escherichia coli km 147 - producer of choleraic toxin b-subunit - Google Patents

Abstract

FIELD: biotechnology, microbiology, genetic engineering, molecular biology.

SUBSTANCE: invention relates to constructing the strain of colon bacillus as a producer of choleraic toxin and can be used for creature of diagnostic test-system providing revealing toxicogenic clones of Vibrio cholerae and Escherichia coli and for constructing live vaccine strains against diarrheal diseases caused by pathogenic strains of V. cholerae and E. coli. The avirulent strain E. coli KM 147 is an active producer of protective antigen that is prepared by conjugative incorporation of recombinant plasmid pIEM3 with the cloned gene of B-subunit of choleraic toxin into cells. Invention provides preparing the non-pathogenic for human and animals strain with high production of basic protective antigen V. cholerae (choleraic toxin B-subunit).

EFFECT: valuable properties of strain and antigen.

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Description

The invention relates to biotechnology, in particular to the construction of a strain of Escherichia coli - a producer of cholera toxin, and can be used to isolate the purified immunogenic B-subunit of cholera toxin, which is part of the diagnostic test system that allows the detection of toxigenic clones of Vibrio cholerae and Escherichia coli. In addition, the created strain can serve as the basis for the construction of live vaccine strains against diarrheal diseases caused by pathogenic strains of V. cholerae and E. coli.

Known strain E.coli K.S3043 - producer of the b-subunit of cholera toxin. However, the level of production of this protein is 0.85 μg / ml (N.V. Yanishevsky, A.L. Gintsburg, Yu.V. Vertiev, Yu.M. Demme, G.I. Karataev, G. B. Smirnov. recombinant plasmids encoding the biosynthesis of the B-subunit of cholera toxin // Molecule genetics. - 1987. - No. 4). In addition, the b-subunit of cholera toxin in this strain is located in the periplasm and is not secreted into the growth medium. To isolate it, it is necessary to destroy cells with the help of both physical and chemical factors.

The V. cholerae KM93 strain II pathogenicity group is also known. which is a producer of the b-subunit of cholera toxin secreted into the growing medium. The number of b-subunit in this strain of cholera vibrio is 8.5 μg / ml (USSR copyright certificate No. 1505022, MKI C 12 N 15/00).

The objective of the invention is the construction of a strain non-pathogenic for humans and animals with high production of one of the main protective antigens of the B-subunit of cholera toxin.

The essence of the invention lies in the fact that an avirulent strain of Escherichia coli, a producer of the B-subunit of cholera toxin (the main protective antigen), is obtained.

The inventive strain was created on the basis of the well-studied avirulent E. coli M 17 strain (which is the basis of the prophylactic colibacterin drug) by conjugative introduction of recombinant plasmid pIEM3 (Km ^r Tc ^r ) with cloned cholera toxin (ctxB) genes into the cells of this strain. This plasmid is a conjugative cointegrate consisting of two plasmids pIEM1 (derivative of plasmid pOX38 containing transposon mini-kan and IS1 sequence) and pSTΔ27 (derivative of plasmid pBR322 carrying the tetracycline resistance gene (Tc ^r ), and a cloned fragment of the chromosome strain Eltor V. cholerae RV79 with the ctxB gene that determines the synthesis of the B subunit of cholera toxin).

The strain was deposited in the State collection of pathogenic bacteria RosNIPCHI "Microbe" (Saratov) under the number KM 147.

Characterization of the strain Escherichia coli KM147.

Morphological signs.

Cells are straight, rod-shaped, motile, with peritrichous flagella, gram-negative, non-spore-forming, oxidase-negative.

Pathogenic properties.

The strain is non-pathogenic for humans and animals.

Cultural properties.

Chemoorganotroph, catalase-positive. It grows on nutrient media at an optimum temperature of 37 ° C at pH 7.2, forming a smooth intense haze. Prototroph.

Plasmid composition.

Strain KM 147 contains the plasmid pIEM3 carrying the ctxB structural gene that determines the biosynthesis of the B-subunit of cholera toxin, as well as tetracycline and kanamycin resistance genes. The main properties of the strain:

- a high level of production of the B-subunit of cholera toxin in the growing medium - 10 μg / ml;

- the ability to produce antitoxic antibodies in immunized animals;

- stability - retains its properties during storage and cultivation on nutrient media, as well as in the body of laboratory animals;

- safe for laboratory animals.

Example 1. Determination of the stability of inheritance of a recombinant plasmid in the cells of the strain.

The stability of plasmid pIEM3 was determined by growing the strain under non-selective conditions for 48-72 hours on LB broth at 37 ° C. then scattered to monocolonies on LB agar. The resulting transconjugants were checked by plasmid resistance markers, i.e. resistance to kanamycin (50 μg / ml) and tetracycline (10 μg / ml), encoded by plasmid pIEM3, was determined. Monoplasmid clones with 100% inheritance stability of plasmid p1EM3 were selected for subsequent studies.

The stability of the introduced recombinant plasmid was also tested in vivo under passages of the recombinant strain in the body of laboratory animals - white mice and baby rabbits. It was established that when the E. coli strain KM147 was passaged for 48 hours in the body of white mice (intraperitoneal infection at a dose of 1 × 10 ⁷ bw) and its subsequent plating on non-selective agar, all grown clones contained the plasmid p1EM3 in their cells ( Km ^r Tc ^r ).

Sucker rabbits were infected with the indicated strain intragastrically (1 × 10 ⁴ mt). 48 hours after infection, monocolonies seeded on complete agar were checked for the presence of plasmid antibiotic resistance markers. It was found that after 48 hours in the intestine, rabbits were rabbit 100% of the studied clones were kept with the replica pIEM3 (Km ^r Tc ^r ).

Example 2. Determination of the production of protective antigen.

In order to evaluate the expression of the cholera toxin B subunit gene in the constructed E. coli KM147 strain, we used the passive immune hemolysis (RPIG) reaction in a solid medium and the enzyme-linked immunosorbent assay (ELISA). But with the RPIG data, the introduced ctxB gene was efficiently expressed in the cells of the E. coli strain KM147. The zone of immune hemolysis of erythrocytes around the macrocolonies of the studied strain significantly exceeded the size of the hemolysis zone around the macrocolonies of the highly toxic V.cholerae 569B strain.

Cholera toxin production was also determined by the highly sensitive quantitative enzyme-linked immunosorbent assay ELISA. It was found that, according to the results of this method, the constructed strain produces 10.0 μg / ml of the cholera toxin B subunit in the growth medium, which is almost 12 times higher than the production in the previously created producer strain of this antigen E. coli KS3043 (0.85 μg / ml) .

In addition, if in cells of recombinant E. coli strains containing the cloned cholera toxin B subunit gene, more than 98% of the produced B subunit remains in the periplasm and is not secreted outward, then in the E. coli KM147 strain we created, the antigen produced is secreted out .

Example 3. Determination of the specific harmlessness of the recombinant strain.

The safety of the constructed strain was studied on a model of white mice and sucker rabbits. White mice were infected intraperitoneally with 1 ml of a suspension of cells in a dose of 1 × 10 MT During the observation period (7 days), the death of infected animals was not observed, physiological parameters remained normal. After this time, the animals were killed and the presence of macroscopic changes in the intestine was determined. In all experimental animals, no macroscopic changes were noted in the large intestine - accumulation of fluid in the lumen of the cecum and adjacent sections. Sucker rabbits were infected intragastrically and intragastrically at doses of 1 × 10 ⁹ and 5 × 10 ⁹ mt Animals were observed for 4 days. It was found that the introduction of the recombinant E. coli strain KM147 did not lead to the development of diarrhea or death of animals. All slaughtered animals were not observed neither macroscopic nor microscopic changes.

Example 4, reflecting immunogenic properties.

To study the immunogenic properties, the recombinant strain of E. coli KM147 was intragastrically administered to adult rabbits at a dose of 5 × 10 ¹⁰ MT The antigenic effect of the studied strain was determined by the detection of antitoxic antibodies in the blood serum. The accumulation of these antibodies was determined in dynamics (blood serum from animals was obtained on days 7, 14, 21, 28, 35, 42 and 49). Immunization was carried out twice with an interval of 28 days. The obtained serum was removed by the cells of the original strain to remove heterologous antibodies. According to the results of a highly sensitive DOT immunoassay (qualitative method), the sera of immunized animals contained antibodies to the b-subunit of cholera toxin. A quantitative assessment of the formation of the studied antibodies was carried out using the 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 the ELISA method, it was found that the maximum titer (1: 3200) of antitoxic antibodies was recorded on the 35th day of immunization and remained throughout the observation period.

Thus, the claimed strain has a high level of production of the B-subunit of cholera toxin, which is the main protective antigen that provides the formation of antitoxic immunity. The strain is safe for laboratory animals, regardless of the route of administration. The indicated properties of the recombinant E. coli strain KM147 indicate that the strain can be used to isolate the purified immunogenic B-subunit of cholera toxin and subsequent preparation of antiserum to detect toxigenic strains; for the subsequent creation of a diagnostic test system that allows the detection of toxigenic clones of V. cholerae and as the basis for the construction of vaccine strains against diarrheal diseases caused by pathogenic strains of V. cholerae and E. coli.

Claims (1)

The bacterial strain Escherichia coli KM147 is a producer of the B-subunit of cholera toxin.