RU2188233C2 - Strain of bacterium bacillus subtilis pbco1e2 as producer of colicin e2 used for veterinary preparation production - Google Patents

Abstract

FIELD: microbiology, veterinary medicine, biotechnology. SUBSTANCE: invention relates to the developed transformatory strain Bacillus subtilis producing hybrid colicin E2 showing ability to penetrate through cellular membranes of microorganisms being both the matrix strain Escherichia coli and strains Pseudomonas, Salmonella, Haemophilus, Streptococcus and inducing the endonuclease degradation of bacterial DNA. The strain can be used for the development of the veterinary preparation showing antibacterial and antioxidant properties. EFFECT: bacterial strain indicated above, valuable medicinal properties. 3 dwg

Description

 Field of use: microbiology; Veterinary medicine and biotechnology.

 Antimicrobial compounds produced by E. coli and related bacteria are currently known as colicins, which have activity against these bacteria [Plasmids: methods / Ed. C. Hardy. - M.: Mir, 1990]. The main disadvantage of known colicins is their low antibacterial activity and their lack of antioxidant properties.

 Known strain of E. coli M17 / p Colap used to obtain a probiotic preparation (Pat. 2144954, RU). This strain produces colic, which is resistant to moderate doses of antibiotics. At the same time, the disadvantage of this strain is the production of colicin, which has low antibacterial activity and does not have antioxidant properties.

Known strain of E. coli pS ₅ - mutant universal indicator strain (application 94038721, RU). This strain allows you to identify in combination with known indicators of single colicin E1 and its combinations. However, this strain is intended for indication, and as a strain producing colicin, which has an antioxidant effect, is not suitable.

 The objective of the invention is to provide a transformant strain of Bacillus subtilis producing hybrid colicin E2, which has both antibacterial and antioxidant properties.

 The technical result consists in the construction of a strain of Bacillus subtilis introduced with E. coli plasmid pBCO1E2.

 This goal is achieved by the fact that using genetic engineering methods to construct a strain of Bacillus subtilis producing hybrid colicin E2, which has both antibacterial and antioxidant properties.

 The strain Bacillus subtilis pBColE2 is deposited in the All-Russian collection of microorganisms of the Research Institute of Agriculture of North-East Russia named after N.V. Rudnitsky under registration number B-771.

 The essence of the patented genetic engineering solution is illustrated by the following scheme, where Fig. 1 shows the construction scheme of the hybrid plasmid pBColE2, and Fig. 2 shows the preparation of a plasmid clone in Bacillus subtilis that contains a portion of E. coli genomic DNA.

 The patented strain design is based on the following provisions.

 At the first stage of the construction of the Bacillus subtilis strain based on pCo1E2-P9 and pBR 322 plasmids, the hybrid plasmid pBColE2 was constructed using known methods (Sihavy TI, Berman ML Experiments with Gene Fusichs, 1984, Corol Spring harbor Laboratory Press, New York, p. 78-126). (figure 1).

 The host plasmid pColE2-P9 is hosted by E. coli strain BZB 2125. Moreover, its nucleotide sequence, the functions of all its genetic elements, and the regulation of their activity are known for this plasmid. The plasmid determines the synthesis of colicin E2, which has a detrimental effect on the cells of related bacteria E.coli, splitting their DNA into low molecular weight oligonucleotides. Plasmid pBR322 is the starting material in the construction of new vectors, each of which has more than 10 unique restriction sites in pBR322.

 Restriction of the site of PStl, which determines the resistance to ampicillin, as one of the genetic markers of the constructed strain, is carried out by the enzyme EcoR1. Using the Bsplu11.2 restriction endonuclease, a Cna fragment was excised from the pCo1E2-P9 plasmid without affecting the other genetic elements of the plasmid. The Cna fragment is then ligated with the fragment of plasmid pBR322 of Bacillus subtilis lacking the Pst I.

 The result is a non-conjugative and non-mobilizable plasmid pBCo1E2, which determines the synthesis of colicin E2 by a strain of Bacillus subtilis.

 In the second step (Fig. 2), the plasmid clone Bacillus subtilis is obtained, which contains a portion of the genomic DNA of E. coli K12 adjacent to the Tn 917 insert. To do this, ligation of the cleavage products is carried out at a low concentration of E. coli DNA to transfer fragments into a ring form and further transformation into a strain of Bacillus subtilis. In this case, the EcoRI restriction enzyme is located inside the sequences of the plasmid pBR322, and the obtained clones contain sections of genomic DNA beyond the recognition site used by the EcoRI enzyme closest to the Tn 917 insert.

 When cloning sites containing the insert Tn 917, which carries the erm gene, use a strain of Bacillus subtilis pBCO1E2. The recombination integration of pBR322 sequences into the genomic insert of Tn 917 Bacillus subtilis was carried out according to the method of Youngman P., Zuber P., Perkins J.B. (1985, Science, 228, p. 284-285).

 Thus, a strain of Bacillus subtilis pBCO1E2, producing hybrid colicin E2, is obtained.

 The strain has the following characteristics.

 Morphology. Gram-positive short thin movable rods 3x5 microns, spores oval 0.6 • 0.9 microns located eccentrically.

 Cultural and physiological signs.

On meat and peptone agar or Hottinger agar after 36 ... 40 h of growth at 37 ^o C - dry, rough, fine-wrinkled, colorless colonies with a matte or whitish hue.

In meat and peptone broth with 5% NaCl or Hottinger broth after 20 ... 24 h of growth at 37 ^o C - a powerful folded surface film.

 Relation to temperature and pH.

It grows well at a temperature of 35-40 ^o C and at a pH of 6.8 ... 7.2 units of N.

 Biochemical properties.

 Ferment (dilutes) gelatin, hydrolyzes starch, forms acid from glucose and sucrose without gas, does not break down tyrosine, casein, arabinose, xylose, mannitol. Forms an alkali on citrate-salt agar.

 Attitude to antibiotics.

 It is resistant to ampicillin in concentrations up to 120 μg / ml and tetracycline up to 170 μg / ml. Resistant to diamond greens in concentrations up to 70 mg / ml.

 The obtained strain of Bacillus subtilis with a transformed Col plasmid when cultured in a liquid nutrient medium consisting of tryptone, yeast extract, glucose and potassium phosphate disubstituted [Methods of soil microbiology and biochemistry / Ed. prof. D.G. Zvyagintsev. - M., publishing house of Moscow State University, 1991] produces hybrid proteins containing sections of the colicin polypeptide. Moreover, during the cultivation process, hybrid proteins are released into the medium, facilitating the rapid purification of large quantities of colicin E2. The yield of hybrid colicin E2 is up to 10 mg / ml.

 Hybrid colicin E2 has both antibacterial and antioxidant effects, since it penetrates through the cell membranes of bacteria as a matrix strain of E. coli K12, as well as strains of Pseudomonas, Salmonella, Haemophilus, Pasteurella, Streptococcus and causes endonuclease degradation of bacterial DNA, i.e. it does not differ in species specificity with a general high bactericidal activity.

 It was experimentally established that hybrid colicin E2 has a molecular weight of 78 kDa and differs from colicin E2 (97 kDa) produced by E. coli strains (Fig. 3). It was shown that hybrid colicin E2 consists of two polypeptide subunits - bacteriocin proper with a molecular weight of 68 kDa and an immune protein with a molecular weight of 10 kDa.

 The invention is illustrated by examples that characterize hybrid colicin E2, as having antibacterial and antioxidant properties.

 Example 1. When studying the effect of hybrid E2 colitis on transplantable cell cultures of alveolar macrophages, data were obtained on macrophage production of the antioxidant enzyme superoxide dismutase in the amount of 1400 units. 18 hours after inoculation of hybrid colicin E2. This confirms that macrophages have specific cell surface receptors for binding to the hybrid E2 colony.

Example 2. Since the study of the toxicity of hybrid colicin E2 in white mice showed the absence of toxin formation in the created strain of Bacillus subtilis pBCO1E2 (LD ₅₀ = (12768.2 ± 291.6) mg / kg), a veterinary preparation was tested in a number of households based on it. In particular, with pasteurellosis of calves aged 2 ... 4 months. hybrid colicin E2 was used as an injectable solution, at a dose of 20 mg per 10 kg of weight once a day, for three days. 60 animals were sick, calf mortality was observed (13 animals). 3 days after treatment with colicin in the experimental group, the case stopped, and the duration of the disease was reduced from 20 to 3 days. Over the next two months, cases of calf disease in the experimental group did not recur. After three injections of hybrid colicin E2, the level of class G1 immunoglobulins in the blood serum of calves increased on average from (9.0 ± 0.1) to (10.5 ± 0.5) g / l, class G2 - from (11, 6 ± 0.16) to (12.9 ± 0.1) g / l. The number of B-lymphocytes in the blood increased from (6.8 ± 0.7) to (10.2 + 0.6)% (P <0.05).

 The above description of the method of constructing a patentable strain is sufficient for its re-production and industrial use.

Claims (1)

 The bacterial strain Bacillus subtilis pBcolE2-B771 is a hybrid colicin E2 product used to obtain a veterinary preparation.

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