RU2046145C1 - Recombinant strain of salmonella minnesota - a producer of plague microbe capsule antigene - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: on the basis of Salmonella minnesota R595 with Re-form of lipopolysaccharide strain carrying plasmid pFS1 encoding synthesis of plague microbe F1 antigene is constructed. The use of recombinant strain S. minnesota R595 pFS1 ensures to obtain highly purified F1 antigene preparation showing expressed protectivity by a single stage of isolation and with high yield. Method is used for isolation of bioactive preparations. EFFECT: construction of strain indicated above. 2 tbl

Description

 The invention relates to biotechnology, and in particular to methods of using producer strains to isolate biologically active preparations.

Currently, the capsular protein FI of the plague microbe is isolated by special methods from the vaccine strain Yersinia pestis EB NIIEG. However FI synthesis occurs mainly at ³⁷ ° C when plague microbe has limited growth rate and increased nutritional needs. This leads to the need to use expensive nutrient media and to extend the cultivation of the strain to 2 days.

 A plague microbe is characterized by a violation of the assembly of lipopolysaccharide (LPS), at the level of attachment of O-chains to the cortex. The polymer structure of FI contributes to the fact that the protein is secreted together with O-chains and R-fragments of LPS. As a result, the degree of purity of the drug decreases.

 There are avirulent strains of microorganisms with reduced LPS. In particular, the LPS of Salmonella minnesota R595 cells (Re chemotype glycolipid) consists only of lipid A and ketodesoxy octonate. These structures are common to the vast majority of gram-negative bacteria. They are also present in the LPS of the plague microbe. Glycolipid Re has increased hydrophobicity and is solubilized using special extraction methods, which reduces the likelihood of FI contamination when using S. minnesota R595 as a producer.

 The aim of the invention is to obtain a producer strain of the capsule antigen of the plague microbe with a maximum yield, minimal impurities of LPS and high and high protective activity.

 The goal is achieved by the fact that the proposed strain (number N KMI, Museum of living cultures RNIPCHI "Microbe") S. minnesota R595 pFS1 obtained by cryotransformation.

From the E. coli strain HB101 pFSI, the pFS1 plasmid coding for the synthesis of FI antigen was isolated by alkaline method and transferred to the cells of strain S. minnesota R595 by cryotransformation. The obtained recombinants were grown 16-18 hours at ³⁷ ° C on Hottinger agar containing ampicillin at a concentration of 5.0 ug / ml. Cells were washed with agar with phosphate buffer, pelleted by centrifugation, FI was isolated from the supernatant, adjusting the pH to an isoelectric point. The resulting protein precipitate was separated by centrifugation and resuspended in the same buffer. The protein was stored frozen at -20 ° ^C.

 Strain S. minnesota R595 pFS1 is characterized by the following features.

 Morphological signs.

 Cells grow well on simple nutrient media. When grown on Hottinger agar, Difco nutrient agar, the colonies are rough, round, convex, shiny, silvery, cloudy, with smooth edges. When grown in liquid media (meat and peptone broth, LB broth) they form a uniform intense turbidity.

 Physiological and biochemical characteristics.

Cells grow at a temperature of 4-45 ^о С, at neutral pH values. Synthesis antigen and its secretion into the culture liquid is carried out by growing a culture S. minnesota R595 pFS1 in a buffered medium of pH 6.8-7.2 at 37 ° ^C. As the carbon source cells are used by many carbohydrates, alcohols, organic acid: in particular d-glucose, d-fructose, galactose.

 Mineral salts of ammonium and nitrate forms, as well as peptone and amino acids in organic form serve as a nitrogen source.

 Antibiotic resistance.

 Resistant to ampicillin and tetracycline, due to the used plasmid pFS1, the vector part of which is represented by cosmid pH7979.

EXAMPLE 1. Plasmid pFS1 was isolated by the alkaline method. The working concentration of plasmid DNA is 200 μg / ml. Cryotransformation was carried out in the following modification. An overnight culture agar with one petri dish were suspended in 5 ml of chilled ^to 4 ° C with distilled water and kept in the ice bath for 10 minutes. Then, 1 ml of 5% peptone water (pH 6.2) was added to 4 ml of the suspension and 0.2 ml were poured into thin-walled glass tubes. One microgram of plasmid DNA in 0.1 ml of distilled water was added to microbial cells. Samples were placed for 5 min in liquid nitrogen. Then the suspension of microbial cultures was incubated for 10 min at a temperature of 42 ^about ; Then to each tube was added 0.7 ml of chilled ^to 4 ° C nutrient broth, incubated at ³⁷ ° C for 1 h and plated onto plates containing 50 .mu.g / ml ampicillin. Transformants grew the next day.

 Immunochemical properties of the vaccine strain Y. pestis EB, the original and recombinant strains. S. minnesota are given in table. 1.

Recombinant S. minnesota clones containing the plasmid pFS1 did not morphologically differ from the cells of the original strain. The obtained recombinants were grown 16-18 hours at ³⁷ ° C on Hottinger agar containing ampicillin at a concentration of 50 ug / ml. At the end of cultivation, the cells were washed with 0.02 M phosphate buffer pH 7.2 and used to isolate FI. Immunochemical testing was carried out in a suspension of cells with a concentration of 10 ⁹ cells / ml. The cell concentration was determined turbidometrically. For comparison, we used cell cultures of Y. pestis UV NIIEG grown under similar conditions for 2 days. In the table. Figure 1 presents data on FI synthesis by cultures of S. minnesota R595 pFS1 and Y. pestis EB at different temperature conditions. From the data presented in table. 1, it can be seen that the production of FI in S. minnesota R595 pFS1 preserves the temperature dependence as in the strain Y. pestis EB. Activity in RPGA FI from S. minnesota R595 pFS1 in terms of 10 ⁹ cells / ml was 30–40 times higher than in Y. pestis.

PRI me R 2. Isolation of FI from S. minnesota R595 pFS1. Cells were cultured in mattresses on Hottinger agar at ³⁷ ° C for 16-18 hours. The microbial mass was washed with 0.02 M sodium phosphate buffer pH 7.2 with glass beads. The resulting cell suspension was pelleted by centrifugation at 10,000 rpm for 15 minutes. The supernatant was acidified by the addition of a 0.5 N HCl solution to a final pH of 4.1. Protein sediment was precipitated by centrifugation at 14,000 rpm for 10 min. Protein pellet was redissolved in the same buffer and stored at -20 ° ^C. The amount of protein was determined by Bradford. SDS-polyacrylamide gel electrophoresis was performed by Laemmli. Isoelectric focusing was carried out according to the instructions of the company LKB, attached to the plates. Data on the properties of FI are presented in table. 2. The yield of FI from S. minnesota R595 pFS1 strain from 1 liter of medium was 9.3 mg. The yield of FI from Y. pestis grown for 2 days under similar conditions was 0.25 ml, which is almost 40 times less. The immunochemical properties of the proteins did not differ; moreover, FI from the recombinant strain was more active in RPHA and RTPHA than the original FI from strain Y. pestis EB. In the work used commercial preparations of immunodiagnostics. Carbohydrate content as determined by Dubous. amounted to 2% in the protein FI from S. minnesota R595 pFS1 and 21.1% in the protein from strain Y. pestis EB. The content of LPS, determined by Karamian, amounted to 1.2 units in relative values. in FI from a recombinant strain, which is 10 times lower than in the preparation from Y. pestis EB.

The efficacy of capsular antigen preparations was determined by the immunity index, which is the ratio of LD ₅₀ for immune animals to LD ₅₀ for intact animals. White mice were immunized with capsular antigen preparations at a dose of 20 μg per animal. After 21 days, the animals were infected with a reference strain of Y. pestis 231. The results were taken into account for 3 weeks. The immunity index for FI from the strain S. minnesota R595 pFS1 is about 150 times higher than that of the drug from strain Y = pestis EB.

 The whole complex of the obtained characteristics indicates that the capsular antigen isolated from the S. minnesota R595 pFS1 strain is identical in its physicochemical characteristics to the initial preparation and exceeds its purity by many times. Using a recombinant strain of S. minnesota R595 pFS1 allows you to get a highly purified preparation of the FI antigen in one stage of isolation with high yield and pronounced protection.

 The use of a recombinant strain of S. minnesota R595 pFS1 to obtain the FI antigen allows to overcome a number of significant disadvantages inherent in known methods. Firstly, the constructed strain provides a 40-fold increase in the yield of the product (from 0.25 to 9.3 mg / L), the absence of the accompanying hardly separated component (polysaccharide chains of the LPS of the plague microbe). As a result, it becomes possible to single-step purify the product without the use of chromatographic equipment. Secondly, the use of S. minnesota R595 pFS1 as a producer of bacteria instead of Y. pestis makes it possible to use cheaper media (without adding hemolyzed blood) when culturing, to reduce the cultivation time by 2-3 times (from 2-3 days to 16-18 h). All of the above advantages allow you to speed up, simplify and reduce the cost of the method of obtaining FI antigen of the plague microbe.

Claims (1)

 Recombinant strain Salmonella minnesota R 595 p FSIN KM I producer of the capsule antigen of the plague microbe.

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