SU1280008A1 - Escherichia coli of qd 5003 t-1 strain used for producing protease of cholera germ - Google Patents

Abstract

This invention relates to microbiology and can be applied to produce a cholera vibrio protease. The Escherichia coli Qfl 5003 T-1 strain was obtained by cotransformation with the DNA of the vector plasmid pBR 322 transferred into the plasmid-free E. coli strain QD 5003 cells. The plasmid tells the strain the ability to grow on a modified diagnostic compound of the following composition, g / l:, 1; MgSO O, 1; ZnSQ. 0.05; NaCl 5.0; NaHCO O, 25, Difco 10 agar, gelatin 10, pH 7.27, 3. The yolk is mixed in a volume of 1: with saline, 70 ml of this emulsion is added to 1 l of prepared medium, cooled to 40-45. § The Escherichia doli strain RD 5003 T-1 is deposited in the State Collection (LAR Tarasevich Medical Research Institute for Standardization and Control of Medical and Biological Preparations under No. 128. 1 table.

Description

About o The invention relates to microbiology and can be used to produce a cholera vibrio protease. The purpose of the invention is the creation of a basopic strain 1-1a capable of producing the cholera vibrio protease, Escherichia coli Q, L1 5003 T-1, obtained by cotranssormation with DNA vector plasmid pBR 322, non-radiating into the plasmid-free strain of E. coli RD 5003. Strain carries the cholera plasmid pViB, which determines protease synthesis, the plasmid is stably inherited in the cells of the new host and informs it of the ability to grow on the modified active diagnostic medium of the following composition, g / l: CaCl, 0, lf MgSO4, 0.1; ZnSO. 0.05; NaCl5.0; NaHCO, 0.25: Difco 10 agar; gelatin 10; pH 7j2-7.3. The yolk is mixed in a 1: i volume with saline and 70 ml of this emulsin are added per liter of the prepared medium, cooled: d, etc. 40-45 C. Escherichia coli strain QII 5003 T-1 is deposited in the collection of the State Research Institute of Standardization and Control of Medical, and Biological Preparations to them. L.A. Tarasevich at number 128 and is characterized by the following signs. Cultural-morphological signs. The cells of strain 128 in smears from a day-old culture have the form of short rods 1.0–250 x 0 ,,, 4 microns. Non-moving. Gram-negative Polymor fizm expressed moderately. On LB agar, turbid colonies of a grayish-white color form. On the Hottinger agar (pH 7.6) they form turbid colonies, the grayish-white Brothon of Hottinger causes broth turbidity, diffusion growth, Resistant to ampicillin (more than 100 μg / ml). Depends on proline. Lysed with intestinal phages T-7 and vir. Fermented with the formation of acid and gas lactose, glucose, arabinose, mannose, galactose. I do not ferment sucrose, 05 V ase vibrocin, coagulase, fibrinolysin and lecithinase. PCT avirule. - 1ten for white mice and guinea pigs with subcutaneous, aerosol and intraconjunctival methods of infection, as well as with intraintestinal infection on a model of rabbits - from sunko z in ji to 0 m, cl. The strain carries a plasmid pBR 322 and rL / ib plasmids with a molecular weight of 2.8 and 2 megadalgon, respectively, the p Vib plasmid allows the strain to form a protease similar to the enzyme produced by the parent Vibrio eltor P-9898 strain under controlled conditions . The use of the strain is illustrated by the following examples. II p and me. The daily agarous culture of Escherichia coli 128 and a sterile wand or sterile tape was applied to the plate of the above-described diagnostic medium in the form of a macrocolony using a sterile wand or string. Crops are incubated at 37 ° C for 24-A8 h. Accounting for the production of protease is carried out according to the formation of circular enlightenment zones around the seeding. The controlless strain of Escherichia coli QD 5003, which is not capable of producing protease and does not grow on an elective medium, is used as a control. Example 2. Cultures of strain 28 and control gates were seeded in 1% -10% peptone water at a dose of 10 cells, / ml k. Grown on a rocking chair for 18 hours at 37 ° C. The cells were discarded by centrifugation, and determined protease activity using aseaseline as substrate. The results are expressed in terms of the standard units of activity of the ml of cultured fluid in terms of 1 optical unit of turbidity of the grown cultures. Compared with data on protease production. The strain of pro-number of units to conventional units E.coli dd 5003 (original) About E. coli dd 5003 -;: 28 V..eltor P-9898 (prototype)

 312800084

Claims (1)

The invention of the invention standardization and control of Escherichia coli OD 5003 them. L.A. Tarasevicha), used by T-1, No, 1.28 (Collection of the State for the production of the cholera thiosis of a vibrio.  Qing Biological Preparations