SU1210278A1 - Escherichia coli hb 101 prd 39 strain - producer of cholera germ neuraminidase - Google Patents

Abstract

SHTAMM Escherichia coli HE 101 pRD 39, No. 124 (collection of the State Research Institute for Standardization and Control of Medical and Biological Preparations named after LA Tarasevich) is a producer of cholerae vibrio neuraminidae.

Description

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This invention relates to microbiology and can be used to produce a cholera vibrio neuraminidase.

The aim of the invention is to create a strain capable of producing large quantities of chibrio neuraminidase and ensuring job safety.

The strain was designed on the basis of E. coli strain HB 101 when carrying out genetic engineering works by introducing artificial genetic recombinant plasmid pRD 39 into it using genetic transformation. 5879) with genes for the synthesis of neuraminidase. The plasmid has a molecular weight of 7.7 megadaltons, is able to amplify in the presence of chloramphenicol and functions on the basis of the vector pBR 322. It has 4 recognition sites for restriction endonuclease Hind III, 2 for Eco RV 4 for Eco RI, 1 for for Sal GI, Bam H I, PSt I n O - for Ml ul.

The strain of Escherichia coli HB pRD 39 deposited in the collection of the State Research Institute of

stitute standardization and control of medical and biological products to them. L.A. Tarasevicha USSR Ministry of Health under the number 124.

Cultural and morphological features.

Short sticks with a size of 1.0-2.0x0.3-0.4 microns. Motionless. Gram-negative with moderate polymorphism. On LB and Hottinger agar, cloudy, greyish-white colonies form. They cause clouding of Hottinger broth with the formation of flocculent sludge.

In minimal medium, they depend on thiamine, leucine and proline. Antibiotic resistant penicillin group (more than 100 µg / ml) due to the formation of penicillinase. Fermented to form acid and gas

VNIIPI

Order 294/1

Random polygons pr-tie, Uzhgorod, st. Project, 4

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glucose, arabinose, mannose, galactose. Do not ferment lactose. They carry a mutation in the chromosome A gene, in connection with which they exhibit sensitivity to UV rays and methyl methane sulfate fans. Lyseum intestinal phages T and lmbda-sup.

Strain is virulent for mice and guinea pigs with subcutaneous, aerosol and intraconjugative infections at a dose of up to 10 cubic meters, which gives it advantages in regards to regimen.

The Escherichia coli 124 strain forms, under controlled conditions, large amounts of cholerae neuraminidase, 8-10 times higher than the level of production of the enzyme by the parent strain Vibrio cholerae eltor 5879.

Take p. For the cultivation of cells of the proposed strain, a wide variety of nutritious speds can be used, including Martin's broth (pH 7.6) and Hottenger broth (pH 7.4), yeast broth (pH 7, 3) and LB broth (pH 7.1) , various pentones and other media.

Cultivation of the strain cells was carried out under vigorous injection conditions on a shunt apparatus (175 rpm) in flasks with a 3% solution of bactopentone Difco (pH7.7-7.8), supplemented with 0.1% calcium chloride and 0 , 5% sodium chloride, at 30-37 ° C for 18-22 hours. Next, the flasks are cooled to 4 ° C, the cells are separated by centrifugation at 6000 rpm for 20 minutes, and the supernatant is used as the source of the target enzyme. Neuraminidase activity in fluid is-. follows Warren's thiobarbituric method. Usually, 300-400 units of the enzyme are contained in 1 ml of culture liquid.

The E.coli 124 strain stably forms a cholera vibrio neuraminidase on artificial media, which significantly simplifies the process of obtaining and purifying the target product.

Circulation 596

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Claims (1)

SHTAMM Ezsleginsky sON HB 101 pM> 39, No. 124 (collection of the State Research Institute for Standardization and Control of Medical and Biological Preparations named after LA Tarasevich) - blowing through a neurominidase of Vibrio cholerae. one 1210278 2