The strain of the yeast saccharomyces cerevisiae vkpm y 1678, the producer of the surface antigen of hepatitis b virus

 

(57) Abstract:

The invention is intended for the production of drugs for the diagnosis and prevention of hepatitis C. the Strain obtained by crossing strains of YF-135 and 5D P. To obtain a cell mass of strain-producer of HBsAg using two-stage scheme of cultivation. At the first stage, the recovery of cells and biomass accumulation. Strain Y-1678 has no latent phase of growth. Cells begin to share directly after planting. While the doubling time of 1.5 h and specific growth rate is set to = 0,48 including Exit on the stationary growth phase occurs after 8 to 10 h at a maximum yield of cells 2,410-8cells/ml In the process of cultivation known strains considerable time is phase synthesis of HBsAg. The use of the proposed strain allows for the increase in the number of cells and more intense synthesis of HBsAg to reduce the time of cultivation, on average, 10 h, which in General reduces the labor intensity, the intensity and the energy intensity of the cultivation process. table 1.

The invention relates to biotechnology and medicine and can be used to produce recombinant surface antigen of hepatitis B virus /HBsAg/ p who cerevisiae, used to obtain HBsAg: YNN 27/p2-S11 (J.-H. Hsieh, K.-Y. Shih. H.-F. Kung e.t. Biotech/Bioeng., V. 32, pp. 334-340, 1988): TLD 746/pSGL2 (C. 3. Capregen, I. P. Arman, N. N. Granovsky. Optimization of gene expression of the surface antigen of hepatitis B virus in yeast. Mol. genetics, Microbiology, Virology, 1990, 5, S. 17-20); GRF-18/pJ DW (O. F. Krupnova, N. And. Sizov, A. R. Smolyanitsky. Increase the yield of recombinant proteins in the yeast S. cerevisiae as a result of optimization of the conditions of their cultivation. Go active. biochemistry and Microbiology, 1995, T. 31, 1, N 3, S. 311-315) cannot be used for industrial cultivation due to low level of production.

The prototype strain LD-5 /Voropaeva L. A., 1992, abstract of Diss. on saisc. the degree of Cand. Biol. Sciences/ use for the synthesis of recombinant yeast HBsAg of hepatitis B. the Strain obtained by crossing the haploid strains with subsequent transformation of the plasmid pCGA7, in which HBsAg gene cloned under the PH05 promoter. A standard scheme for the cultivation of recombinant yeast strains with the regulation of expression of the cloned gene HBsAg system PH05 gene (acid phosphatase of yeast) includes two stages. First, in an environment with a high content of phosphorus to 1 g/l occurs only increase the biome is ment after migrating cell mass in an environment with a low content of inorganic phosphorus 0.03 g/l - is the actual synthesis of the antigen. Stage of the synthesis prototype has a length of 26-28 hours, 12-14 hours of growth, the number of antigen is in the credits of RNA (reaction of indirect haemagglutination) 1/1024 - 1/2048.

The disadvantage of the prototype is the long duration of the stage of synthesis of the antigen, making the process laborious cultivation.

The aim of the invention is to reduce the time of synthesis of the producer strain while maintaining the two-stage scheme of cultivation.

To ensure the specified Cedi offered the strain deposited in Russian national collection of industrial microorganisms (research Institute of Genetics of industrial microorganisms under the number Y-1678.

The strain has the following characteristics:

Pedigree strain: obtained by crossing strains of YF-135 and 5D-P; cells are diploid, heterozygotes on the type of mating, homozygotes for the mutation leu 2-3, 2-112, the need for leucine compensated marker Leu2 gene on the plasmid pCGA7, which transformed cells of strain.

Cultural and morphological characteristics of the strain: the cells are spherical, dense surface of the agar to form rounded colonies with a diameter of 1-5 mm, with rowney layer slightly turbid.

Method and storage conditions of strain (Bank, Working Cell Manufacturer). Cells are stored frozen at -70oC and -193oC in liquid nitrogen, the composition of the medium: 1 WARD/1 glycerol; a cell concentration of 109cells/ml.

The contamination. Cell culture sterile strain, and when the crops on the different types of environments undetected infection by bacteria and fungi.

Example. Haploid parental strains of the yeast S. cerevisiae YF-135 and 5D-P cross-strokes are seeded in a Petri dish on an organic environment WARD (2% glucose, peptone, agar-agar, 0.2% of yeast extract) and incubated for 18 h, then transferred to selective for diploids Wednesday YB ("Difco) and incubated under the same conditions and exposure. The resulting diploids transform vector pCGA7, in which the cloned gene HBsAg under the PH05 promoter, the expression of which occurs only in an environment with a low content of inorganic phosphorus (0.03 kg/l). Transformation of cells carry out the processing of diploid cells in 0.2 M aqueous solution of lithium chloride at 30oC for 2 h followed by addition of 70% polyethylene glycol -4000 and 40 µg DNA vector. The mixture is incubated at 30oC for 1 h, and then plated on YNB medium in the cups on the activity-lactamase (Chevallier, M. R., Aigle M., FEBS Lett., 1979, v. 108, p. 179-180). For this, they carry on the indicator nutrient medium containing, wt.%: glucose - 0.1, starch and 0.2, nourishing the basis of YB - 0,67, agar - 2,0, 0.1 M phosphate buffer pH 7.0 - rest. After 18 h incubation at 30oC formed colonies pour melted at 42oC in the same medium additionally containing 0.3 wt.% ampicillin and iodine and 1.5 wt.% potassium iodide and re-incubated at 30oC. Record the start time of the enlightenment indicator medium under the influence-lactamase. Select the clone in which the enlightenment environment under the effect of-lactamase was observed already after 1 h of incubation (vs. 3-9 h the rest of the clones) and analyze it for the duration of the stage of synthesis of the antigen.

To obtain a cell mass of strain-producer of HBsAg expressing the antigen, using two-stage scheme of cultivation. At the first stage, the recovery of cells and biomass accumulation. Cells from frozen thawed for 30 minutes at room temperature, after which they sow in the environment Hpi with a high content of inorganic phosphorus to 1 g/l and in g/l: MgSO4to 0.5, KCl And 1.0, NaCl -0,1, CaCl2- 0,1, L-asparagine - 2,0, D-glucose 20 g-alanine 510-4; bio 210-4, distilled water - the rest. The initial cell concentration is 1-510-6cells/ml, and cultivation occurs within 18-20 hours at a temperature of 30oC in a water shaker "Elpan". After concentrating the cells by centrifugation and a single washing with saline, the suspension is seeded in medium with Lpi, in comparison with the Hpi reduced concentration of inorganic phosphorus, up to 0.03 g/l, and in which there is a synthesis of the antigen in the cells due to derepression PH05 promoter. The initial number of cells is 2-510-6cells/ml Every 2 hours growth are selected aliquots of cells to calculate their concentration and determination of activity of the antigen. As can be seen from the table, the strain Y-1678 has no latent phase of growth, cells begin to divide immediately after planting, with a doubling time of 1.5 hours, and specific growth rate is set to = 0,48 including Exit on the stationary growth phase occurs after 8-10 hours at a maximum yield of cells 2,410-8cells/ml Prototype strain has a hidden phase of growth of about 4 hours, doubling the number of cells after 2 hours at = 0,14 hours To 8-10 hours of growth, the number of cells is 1,7107cells/ml. There is also a large ex is on antigen level 1/6144-1/8192, while the prototype only 1/512-1/1024. Expression of HBsAg in cells Y-1678 begins almost immediately after planting, and appreciable quantities of protein are determined in the culture after 4-6 hours of growth - 1/128 -1/512 in the credits of RNA. The prototype synthesizes the antigen is much slower, and by 8 o'clock the growth amount is only 1/64. To 14-16 hours of cultivation, the amount of antigen in the population Y-1678 has a maximum performance - 1/16384 - 1/32768, whereas the fermentation time for the prototype is 26-28 hours. The rapid growth of Y-1678 environment Lpi, accompanied by an intense synthesis of antigen, reduces the time of HBsAg synthesis and, consequently, the entire cultivation cycle, on average, 10 hours, which reduces the consumption of materials (environment and equipment, labour and energy cost.

The strain of the yeast Saccharomyces cerevisiae VKPM Y 1678, the producer of the surface antigen of hepatitis B.

 

Same patents:

The invention relates to means of protection of plants from diseases and relates to a new strain Pseudomonas chlororaphis to get this tool and to use it

-aminobutyric acid" target="_blank">

The invention relates to microbiological and medical industry and the receipt-aminobutyric acid from L-glutamic acid or its salts using biomass bacteria Escherichia coli

The invention relates to the microbiological industry, and can be used for cleaning oil-contaminated surfaces

The invention relates to the microbiological industry, and can be used for cleaning oil-contaminated surfaces

The invention relates to biotechnology, in particular Microbiology, and can be used for growing cultures of Escherichia simple and cheap nutrient medium without losing its effectiveness

The invention relates to biotechnology, in particular Microbiology, and can be used for growing cultures of Escherichia simple and cheap nutrient medium without losing its effectiveness

The invention relates to biotechnology, in particular Microbiology, and can be used for growing cultures of Escherichia simple and cheap structure without losing its effectiveness

The invention relates to biotechnology, immunology and Virology and can be used to produce immunogenic polypeptide compositions, cross-reactive with many isolates of hepatitis C virus (HCV)

The invention relates to biotechnology, protein engineering and medicine, specifically to obtain the crustal antigen of hepatitis B virus(HBcAg), allowing to expose on the surface of the alien epitopes

The invention relates to the field of biotechnology and concerns a method for obtaining viral antigens of hepatitis b in the yeast Saccharomyces cerevisiae

The invention relates to microbiological and medical industry, genetic and protein engineering, in particular to the preparation of recombinant plasmid DNApKNVc-33, containing the gene for the crustal antigen of hepatitis B virus (HBcAg) exposed on the surface of a major neutralizing epitope of the surface antigen of hepatitis B virus (137-147) (HBsAg) and the corresponding strain-producer

The invention relates to biotechnology, genetic engineering and immunology and is a set of recombinant proteins synthesized in bacterial cells and allowing serodiagnostic hepatitis C
Up!