The method of obtaining anthrax bacteriophage gamma a-26

 

(57) Abstract:

The invention relates to Microbiology and can be used in biotechnology receiving the anthrax bio-medical drugs. To reduce the cost of drug - anthrax bacteriophage, increasing its stability, while maintaining the required biological properties of vegetative culture of Bacillus anthracis grown on solid agar of Hottinger for 14-18 h, specific lizirovania culture is carried out in a liquid synthetic medium by simultaneous suspension culture and uterine bacteriophage and incubating the suspension for 5-10 h followed by filtration and freeze drying of the finished product in a protective environment for 18-20 hours

The invention relates to Microbiology and can be used in biotechnology receiving the anthrax bio-medical drugs.

Technological scheme for obtaining phage includes adding in broth culture of a special production of phage reproduction at 37oC during the day, filtering and checking for cleanliness and activity (K. D. Pyatkin, Y. S. Krivoshein, Microbiology, M., "Host serial receive the vegetative culture and specific lizirovania culture in liquid nutrient broth of Hottinger with the addition of adjuvants (C. RF N 96123389/13, C 12 N 7/00//C 12 N 1/20).

The method is as follows. Prepare a nutrient medium from the broth of Hottinger, add yeast extract 2.5 g/l, 3 ml/l of 10% solution of calcium chloride, establish a pH in the range of 7.4. Nutrient medium is sown a suspension of spores of the anthrax vaccine strain 55 based 6-10 million spores per 1 ml of medium. The cultivation is carried out with shaking for 5-6 h, and then make the drug fallopian phage Gamma calculation of 1-2 ml per 100 ml of broth culture. Spend the incubation of cultures at 37oC for 24 h the product was filtered at the "Millipore" with the use of sterilizing filters. On Appelman determine the specificity and activity of the finished product. The phage has a high concentration of 109.

When sufficient simplicity and short process (30 hours) this method is disadvantageous to apply in biotechnology diagnostic drug because of its high cost due to the use of expensive animal products in the nutrient media. In addition, the phage in liquid form not to be stable, its validity period is 6-12 months.

The known method, the selected prototype obtain anthrax Gamma phage by the EPA, in which 1 l) was added 20 mg of tryptophan, 100 mg magnesium chloride and magnesium sulfate and 5 g of glucose. Used phage preparations and K initial concentration of 6 to 1081 108respectively. As a vegetative culture sensitive clone of strain B. anthracis N 303 N.A. Kuzmin, B. A. Komarov, Laboratory work N 12, 1967, S. 741-743).

The mode of cultivation includes seeding 12-14-hour agar culture, washed with jamb by perevorot of Hottinger on the mattress with a dense environment. After 6-7 hours of incubation on the lawn heats parivar of Hottinger, as amended by Royal phage with a titer of 108- 109M. K./ml. Process lizirovania vegetative culture is carried out in a thermostat at 37oC for 16-17 hours Then the lysate from the mattress centrifuged with a speed of 5-6 thousand rpm for 6 min and the supernatant filtered through asbestos records of the office of Seitz.

The method provides for obtaining a phage with a titer of not less than 1010. Under optimal indicator of the multiplicity of infection for phage strain on the N 303, 0.35 yield reaches 100-400 phage particles per bacterial cell.

The disadvantage of this method is a multistage and expensive animal raw material, which complicates egreene is to achieve a technological way, providing cost reduction of the drug in industrial production, increasing stability while maintaining the highest product yield, specific activity and biological properties.

This objective is achieved in that a method of obtaining anthrax bacteriophage includes the cultivation of vegetative culture on solid nutrient agar of Hottinger for 14-18 h, specific lizirovania culture in liquid synthetic medium by simultaneous suspension culture and uterine bacteriophage and incubating the suspension for 5-10 h, filtration and stabilization of the finished product by the method of freeze drying in a protective environment for 18-20 h

Medium SM for specific lizirovania culture contains 5.8 g/l NaCl, 2 g/l MgSO4; 7H2O, 5 ml/l 2% gelatin, 50 ml/l 1 M Tris buffer solution with pH 7.5 (T. Maniatis, E. Fritsch, J. Sambrook, Molecular cloning, M., Mir, 1984, S. 497).

Protective environment for freeze drying product contains 1.5 g of gelatin and 20 g of sucrose in 100 ml of distilled water.

Know the use of similar protective environment for freeze drying moderate cholera phages (A. C. USSR N 12 is entrusted freezing is carried out for 3-4 min, cooled to minus 55oC ethanol, and drying can be performed during 26-27 hours

The main feature of the proposed method are the following. Getting vegetative culture on plates with agar of Hottinger allows you to select typical colonies of Bacillus anthracis, which provides specific purity process lizirovania and saving costly environment. The use of synthetic environments for lizirovania vegetative culture allows to obtain a stable output of the bacteriophage in high titers and reduces the cost of the product. Liquid preparation obtained by this technology is cleaner and can be used to obtain antifirewall serum, as well as in genetic research. Stabilization of the finished product by the method of freeze drying in a protective environment for 18-20 h, increases the shelf life of the finished product from 6-12 months to 3 years.

The possibility of practical use of the invention is confirmed by the example of his particular run.

Example 1. Bacteriological loop seeded lawn suspension of spores of the anthrax vaccine strain N 55 on a Cup of agar Hottinger and incubated oraut in 100 ml of distilled water, adjusting the concentration of the HCl pH to 7.5. 100 mg of gelatin dissolved in 5 ml of water. Weighed 5.8 g NaCl, 2 g MgSO47H2O dissolved in 100 ml of water. 50 ml Tris pH 7.5 and the remaining solutions are combined, bring the total volume to 1 L. the Medium is sterilized by autoclaving at 1 ATM for 20 minutes or by filtration through membrane filters. In a flask with 100 ml of a liquid medium suspended 3 loops vegetative culture and add 10 ml of the bacteriophage Gamma A-26 with a titer of not less than 107. The mixture is stirred, the flask is placed in a thermostatted rocking chair and incubated at 37oC for 5-10 hours, depending on the enlightenment lysate.

The product is sterilized through membrane filters with a pore diameter of 0.25 to 0.45 µm at the "Millipore". Method Appelman in the modification of Grazia determine the lytic activity and specificity of the finished product. The titer of the bacteriophage is 109.

Prepare a protective environment for freeze drying of the drug. In 100 ml of distilled water dissolve 1.5 g of gelatin and 20 g of sucrose, autoclave at 1 ATM 30 minutes

To finish liquid bacteriophage add cooked protective environment in the ratio of 1:1 and directed to the lyophilic drying.

oC and 0oC for 5 h 0oC to 10oC 5 h 10oC to 25oC and 2 hours at a constant temperature. The working pressure in the chamber is 30 PA. Ampoules with the finished product is sealed in gas-oxygen burner.

Thus, the invention is practicable, its use in biotechnology will allow you to organize industrial production of highly concentrated anthrax diagnostic bacteriophages And Gamma-26 with a long shelf life.

The method of obtaining anthrax bacteriophage, including the cultivation of vegetative culture on solid nutrient medium, reseeding, incubation in the presence of phage, filtration and sterilization of the resulting lysate, wherein the vegetative culture is subcultured in liquid synthetic medium with simultaneous addition of Royal bacteriophage, incubated for 5-10 h, and the resulting sterilized filtrate lysate stabilized by the method of freeze drying in a protective environment for 18-20 hours

 

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FIELD: biotechnology, microbiology, medicine.

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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