RU1660388C - Recombinant plasmid dna pvgib encoding bovine gamma-interferon, method of its preparing and yeast strain saccharomyces cerevisiae - a producer of bovine gamma-interferon - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: plasmid containing yeast gene LEU2 and fragment of yeast two-micron DNA is prepared that provides its stable content in yeast at high copies level. Plasmid has also encoding gene site of bovine gamma-interferon flanked with promoter and transcription terminator of yeast gene PHO5, and also fragments of bacterial DNA providing plasmid replication in E. coli cells and carrying resistance gene to ampicillin. Yeast cells of strain GRE18 transformed with plasmid pYGIB produce bovine gamma-interferon with the yield 10 mg per 1 l yeast culture. EFFECT: construction of yeast strain - a producer of bovine gamma-interferon. 4 cl

Description

 The invention relates to biotechnology, in particular to genetic engineering.

 The aim of the invention is the creation of yeast producer of gamma interferon bovine.

Plasmid pYGIB, which provides synthesis of bovine gamma-interferon in yeast cells, consists of the following elements:
6.6 kb Hind III-BamHI plasmid DNA fragment of the bifunctional bacterial yeast vector pjDV 207, comprising the bacterial ampicillin resistance gene, the bacterial replication initiation region, the yeast two-micron plasmid fragment and the LEU2 yeast gene;
A BamHI-EcoRI yeast PH05 gene fragment containing the promoter of this gene, 0.52 kbp.

 XbaI-Hind III fragment of the recombinant phage mp 18 gamma-IFN 0.42 kb in length

 Sma I-BamHI fragment of the polylinker portion of plasmid pvC 19 with a length of 8 bp

 Sau3F-PstI of the yeast PH05 gene fragment containing the transcription terminator of this gene, 0.37 kbp

 PstI-Hind III fragment of the polylinker of the plasmid pvC 19 size 20 bp

 The total plasmid size is 8 kbp. (mol.m. 5.2 MD).

 The yeast strain Saccharomyces cerevisiae obtained by transformation of the strain with plasmid pYG 18 was obtained.

 The strain is characterized by the following features.

 Morphological signs. The cells are round, slightly oval in size, approximately 5-10 microns in size, and some of the cells have daughter cells or kidneys attached to their surface.

 Cultural signs. Cells grow well in complete organic media (PEP: 2% peptone, 2% glucose; PEPFO: 2% peptone, 2% glucose, 0.15% monosubstituted potassium phosphate; YEPD: 2% peptone, 1% yeast extract, 2% glucose )

 In addition to complete organic media, the cells grow well on a mineral composition medium: 0.67% Yeast nitrogen base ("Difco"), 2% glucose (SC medium) with the addition of 50 mg / l histidine, as well as on other synthetic media for yeast containing supplement of 50 mg / l histidine.

 When growing on solid media, they form smooth, round, cloudy colonies with a matte surface, light cream in color, the edge is even.

 When growing in liquid media, they form an intense, even haze. The culture has a characteristic smell of yeast.

Physiological and biochemical characteristics. The cells grow within 4-37 ^{° C,} with the optimum 30 ° ^C.

 With growth under aerobic conditions, the cells significantly acidify the culture medium. The optimum pH for growth is 3.5-5.5.

 As a carbon source, cells can use many simple organic compounds, in particular, glucose, sucrose, glycerin.

 As a source of nitrogen, provided histidine is added, cells can use both mineral salts in the ammonium form and simple organic compounds urea, amino acids.

 Cells are capable of both aerobic and anaerobic growth.

 The essential features of the strain are the need for histidine and prototrophy for leucine.

 The strain is deposited in the All-Union collection of industrial microorganisms under the number Y-1223.

Example 1. E. coli bacteria cells containing plasmid pMS 46 were grown overnight in 500 ml of LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride), to which ampicillin (50 mg / l). Cells are harvested by centrifugation (5000 rpm, 10 min, 4 ^° C), resuspended in 10 ml of lysis buffer (25 mM trishydrochloride buffer, pH 8, 10 mM EDTA and 50 mM glucose), 20 mg lysozyme are added and incubated for 10 min at 4 ° ^C. 10 ml of 0.2 M sodium hydroxide containing 1% sodium dodecyl sulfate. After gentle stirring for about 1 minute the solution was neutralized with 10 ml of 3M sodium acetate, pH 5.3, incubated at ⁴ ° C for 1 hour and centrifuged. 0.6 volumes of isopropyl alcohol are added to the supernatant. The precipitate was separated by centrifugation, dried in vacuo, dissolved in 3.5 ml of water, 3.5 g of cesium chloride and 100 μl of ethidium bromide solution (10 mg / ml) were added and centrifuged at 50,000 rpm for 12-16 hours. After centrifugation, they were taken a strip of plasmid DNA, extracted twice with an equal volume of isoamyl alcohol, diluted with a solution of cesium chloride 2 times with water and precipitated DNA with two volumes of ethyl alcohol. The precipitate, separated by centrifugation, was washed with 70% ethanol, dried in vacuo and dissolved in water (500 μl). The DNA concentration is determined by the optical density of the solution at 260 nm (optical density 1 corresponds to a concentration of 50 μg / ml), the purity of the drug is controlled by electrophoresis in 0.7% agarose gel in TBE buffer (0.1 M trisborate, 1 mm EDTA and 1 mg / l ethidium bromide).

Hydrolysis of plasmid pMS 46 with SmaI restriction enzyme is carried out in 10 mM Tris-hydrochloride buffer containing 10 mM magnesium chloride, 20 mM potassium chloride, 1 mM dithiothreitol. 30 units are added to 20 μg of DNA in a volume of 50 μl. restrictase and carry out the reaction at 30 ^about 2 hours. Control the completeness of hydrolysis is carried out using electrophoresis. Next, the DNA solution was diluted to 200 μl with a buffer of 50 mM trishydrochloride, 10 mM magnesium chloride, 100 mM sodium chloride, 1 mM dithiothreitol, 100 μg / ml bovine serum albumin (BC buffer) and treated with 50 units. restriction enzyme EcoRI for 2 hours at 37 ^C. The reaction mixture was poured into a well of an agarose gel, prepared as described and electrophoresed at 5 V / cm for 2-3 hours. Immediately prior to strip the linear form of plasmid cut hole (3h60 mm), a dialysis membrane of the appropriate size is placed in this well and filled with TBE buffer. Electrophoresis was continued for another 10-15 minutes, after which the buffer was removed from the well, extracted once with phenol and once or twice with butanol, 1/10 part of 3M sodium acetate buffer, pH 5.3, and three volumes of ethanol were added. DNA was separated by centrifugation, the precipitate was washed with ethanol, dried in vacuo and dissolved in 100 μl of water.

The isolation of the double-stranded DNA form of the phage mp 18-gamma-IEN phage is similar to the isolation of the plasmid pMS 46 with the difference that bacterial cells of strain TGI are grown on LB medium without antibiotics to a density of 0.4, infected with phage and grown to a culture density of 0.8-1, 0 at 600 nm, after which the cells are harvested by centrifugation and phage DNA is isolated. The hydrolysis of 10 μg of double-stranded form of phage DNA is carried out using 30 units. Hind III restriction enzymes. To the DNA solution, add 80 μl of 100 mm Trishydrochloride buffer, pH 7.4, containing 50 mm magnesium chloride, a mixture of four deoxynucleoside triphosphates to a final concentration of each of them 250 μm and 10 units. Klenovskoy fragment of DNA polymerase I. After incubation for 4 hours at room temperature, the mixture is heated at 65 ^about 10 minutes and carry out the hydrolysis of plasmid DNA 30 units restriction enzymes EcoRI.

 Cleaning a 420 bp fragment carried out for the vector fragment of plasmid pMS 46 with the addition that a hole (5x3 mm) is cut out next to the hole for the purified DNA preparation to apply molar standards. (DNA of lamb phage hydrolyzed by PstI): the desired band before elution is identified by comparing its mobility with the mobility of standard DNA fragments.

To obtain a plasmid designated as pMSGIB, DNA fragments containing the promoter and terminator of the PH05 gene are ligated with a DNA fragment carrying the bovine gamma interferon gene. To do this, DNA fragments from plasmid pMS 46 and phage DNA are mixed with 0.1 μg in 10 μl of 70 mm Tris-hydrochloride buffer, pH 7.6, containing 5 mm dithiothreitol, 5 mm magnesium chloride, 1 mm ATP, add 10 units DNA T4 ligase and incubated overnight at 4 ° ^C.

Ligation mixture was transformed E. coli cells HB 101. For this purpose E. coli cells were grown in 100 ml LB medium at ³⁷ ° C until the optical density at 600 nm of 0.4-0.5, cooled on ice, centrifuged at 3000 rev / min 10 min at ⁰ ° C, resuspended in 50 ml of 0.1 M calcium chloride, incubated on ice bath for 40 min, centrifuged again, resuspended in 5 ml of calcium chloride solution was added glycerol to 20% for transformation to a suspension of competent cells was added a mixture of ligase reaction products and incubated in an ice bath for 40 minutes. Cells were heat-shocked for 2 minutes at 42 ^{° C,} incubated in 1.5 ml LB medium at ³⁷ ° C for 1 hour. The cell suspension was concentrated by centrifugation and triturated on the surface of the cup with the same medium containing 2% agar and 50 mg / l ampicillin.

Plasmid DNA is isolated from the individual clones obtained. In this case, instead of the last stage of purification (centrifugation in a solution of cesium chloride), treatment with pancreatic RNAase is used. To this precipitate nucleic acids from the precipitation with isopropanol, dissolved in 100 .mu.l of restriction buffer and treated with 10 .mu.l of a solution of RNAse (1 mg / ml) for 30 min at 37 ^C. The drug is used for restriction analysis of the plasmids in individual buildings clones.

 In the case of the plasmid pMSGIB analysis is carried out as follows. To portions of 3 μl of the plasmid preparation, 7 μl of water are added and then individual portions are treated with the following combinations of restriction endonucleases (10 units each): SalI + Hind III (the desired plasmid gives fragments 520 and 1100 bp), EcoRI + Hind III (fragment 820 bp), BamHI + SalI (fragments 275, 520 and 430 bp). DNA was isolated from the selected clone containing the pMSGIB plasmid.

 The senseless DNA fragment between the restriction sites EcoRI and XbaI is removed as follows. The plasmid pMSGIB is hydrolyzed with a mixture of restriction enzymes EcoRI and HbaI, purified by agarose gel electrophoresis, treated with a maple fragment of DNA polymerase I and cyclized by ligation. After bacterial transformation of such DNA, clones containing the pMSGIBI plasmid are isolated (such clones are easily distinguished from clones with the original plasmid pMSGIB by restriction analysis with BamHI endonuclease.

 To obtain the shuttle plasmid pYGIB, the ASalI-Hind III fragment of the pMSGIBI plasmid of 1.62 kbp and the vector fragment of the pjDB 207 plasmid hydrolyzed by the same restriction enzymes are purified.

The restriction enzyme hydrolysis is carried out as follows. 20 μg of plasmid DNA in 200 μl of the buffer is digested 100 units Hind III restriction enzyme for 3 hours at ³⁷ ° C, the reaction mixture was added 10 units. restriction enzymes SalI and incubated at 37 ^about 10 minutes, placed in an ice bath and an aliquot of the hydrolyzate is analyzed by agarose gel electrophoresis, controlling the relative number of fragments of 250, 1100 and 1620 bp. Such cycles (incubation at ³⁷ ° C for 10-20 minutes, transferred to an ice bath and electrophoretic analysis) are repeated until the number of long fragments of 1100 and 1620 bp in the reaction mixture does not become approximately equal. Purification of a 1620 bp DNA fragment carried out by agarose gel electrophoresis. E. coli cells were transformed with a ligase mixture and restriction analysis of transformer clones with restriction enzymes EcoRI, SalI + Hind III, BamHI + SalI was performed, and a clone containing the plasmid pYGIB was selected. Plasmid DNA is isolated from the cells of this clone and used to transform yeast cells.

PRI me R 2. The yeast strain of the recipient is grown on YEPD medium until the culture reaches optical density at 600 nm 2-4. Cells were washed twice with water and once with 0.1 M sodium citrate buffer containing 1 M sorbitol, resuspended in the same buffer and treated first with 159 ul of mercaptoethanol for 15 minutes at 30 ^{° C,} and then 150 .mu.l glucuronidase for 30-60 minutes at the same temperature. The obtained spheroplasts were washed three times with 1 M sorbitol, suspended in 0.01 M Tris-Hcl buffer containing 0.01 M calcium chloride and 1 M sorbitol, and after adding 10 μg of the pjDB DNA plasmid (MSIL), they were incubated for 30 min at room temperature. To the cell suspension was added 44% solution of polyethylene glycol 4000 solution, incubated for 30 min at ³⁰ ° C, followed by 2 minutes at 42 ^{° C} and seeded seed depth on a medium containing 1% agar and histidine at a concentration of 50 mg / l.

To analyze the production of transformer gamma-interferon bulls by cells, they are grown on PEP medium until a stationary growth phase is reached. Cells are harvested by centrifugation, washed with water and destroyed. For this, the cells are suspended in an equal volume of PB buffer (0.15 M sodium chloride, 10 mM sodium phosphate, pH 7.4) containing 5 mM phenylmethanesulfonyl fluoride equal to the volume of the cells, a third of the volume of the glass bead suspension is added and shaken in a homogenizer at maximum power for 1 min, centrifuged at 10,000 rpm for 10 min at room temperature. The precipitate is washed with a 10-fold volume of PB buffer and suspended in the amount of the same buffer four times with respect to the precipitate volume. Each 1 ml of the suspension was added 20 mg of sodium dodecyl sulfate and 10 mg of dithiothreitol, the suspension was incubated 10 min at 80 ° ^C and separated by centrifugation. The presence of bovine gamma interferon is determined in the supernatant.

To analyze the production of gamma interferon of a bull, the enzyme immunoassay is diluted in ratios from 1:10 to 1: 200 with 50 mM water and placed in the wells of plastic plates for enzyme immunoassay. The samples were dried overnight at room temperature, incubated with a 1% solution of bovine serum albumin at ³⁷ ° C for 30 min and washed with PB buffer. A solution of monoclonal mouse antibodies to human gamma-interferon (2 μg per well) is added to the wells, and then, after another washing, a diluted 1: 200 conjugate of species-specific antibodies specific for mouse gamma globulins and peroxidase are added. After incubation of the formed complexes with a substrate solution (0.0003% hydrogen peroxide, 4 mg / ml orthophenylenediamine in 0.1 M sodium acetate buffer, pH 4.6) and isolation of the reaction, absorbance was measured by adding an equal volume of 2 M sulfuric acid at 486 nm.

 The yeast cells of the obtained strain synthesize a polypeptide that gives a clear immunological signal with antibodies to human gamma interferon.

 To control the production of interferon by strain cells, the method of protein electrophoresis in the presence of sodium dodecyl sulfate in a 15% acrylamide gel in a standard buffer system is also used (electrode buffer: 0.2 M glycine, pH 8.3, gel buffer: 0.375 M Tris-HCl, pH 8.9). The gels are stained with Coomassie R and washed. Bovine serum albumin, ovalbumin, chymotrypsinogen and cytochrome C are used as molecular weight standards.

For electrophoretic analysis of yeast cells are opened, as described, after which the material is insoluble in PB buffer was treated with 2% sodium dodecyl sulfate containing 1% mercaptoethanol at ⁹⁵ ° C for 2 min. The suspension is separated by centrifugation, 10,000 rpm, 10 min) and the supernatant is applied to a polyacrylamide gel.

 Electrophoresis detects a protein strip with a mol.m. about 15 kD, which corresponds to the molecular weight of the gamma interferon of the bull. Scanning electrophoretograms allows you to measure the content of interferon in the protein mixture. It reaches several percent of all proteins extracted from yeast cells in the manner described.

 The obtained yeast strain VKPM Y-1223 synthesizes bull gamma interferon of about 10 mg per 1 liter of yeast culture.

Claims (3)

 1. Recombinant plasmid DNA pYGIB encoding gamma interferon bovine, size 8.0 TPO and mol.m. 5.2 MD containing 6.6 kb Hind III-BamHI fragment of plasmid DNA of bifunctional bacterial-yeast vector pjDB 207 BamHI-EcoRI DNA fragment of plasmid pMS 46 with a size of 0.52 T. p. XbaI Hind III / BamHI DNA fragment of the recombinant phage mp 18-gamma IFN, the nucleotide sequence of which encodes a bovine gamma interferon, 0.428 kb in size. BamHI / Sau 3A-PstI / Hind III DNA fragment of pMS 46 plasmid 0.39 kbp genes and regulatory sites; bla gene providing ampicillin resistance; the LEU 2 gene, allowing growth on selective media without leucine; a bacterial ori locus that provides plasmid replication in E. coli cells; a yeast two-micron DNA fragment providing autonomous replication of the plasmid in yeast; yeast PH05 gene promoter providing transcription initiation; PH05 gene transcription terminator for terminating transcription; a portion of the bovine gamma interferon gene encoding the structure of this protein; restriction sites - three sites EcoRI and RstI, two sites SalI and BamHI, one site Hind III and XbaI.  2. The method of obtaining the plasmid pYGB, namely, that the DNA of the recombinant phage mp 18-gamma IFN is digested with restriction enzymes EcoRI and HindIII, ligated with the DNA of plasmid pMS 46, hydrolyzed by restriction enzymes EciRI and SmaI, the resulting plasmid is hydrolyzed with a mixture of restriction enzymes EcoRI and XbaI, -polymerase and DNA ligase from the obtained plasmid with restriction enzymes SalI and Hind III cut a DNA fragment containing the yeast gene promoter PH05, the coding part of the interferon gene and the transcriptional terminator of PH05, are ligated with plasmid pjD 207, hydrolyzed by restriction enzymes SalI and Hind II I and select clones according to restriction analysis.  3. The yeast strain Saccharomyces cerevisial VKPMU-1223-producer of gamma interferon bovine.