RU2231545C1 - Strain of yeast pichia pastoris ps105(pbig) as producer of bovine intracellular immune interferon, recombinant plasmid dna pbig providing biosynthesis of bovine intracellular immune interferon and method for constructing recombinant plasmid dna pbig - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, veterinary science, molecular biology.

SUBSTANCE: invention can be used in microbiological industry and agriculture. Plasmid DNA pBIG providing the biosynthesis of bovine intracellular gamma-interferon is constructed by excision of yeast Saccharomyces cerevisiae gene MFα prepro-region from bacterial-yeast vector pPIC9 with restriction endonucleases Bam HI and Xho I. The yeast strain Pichia pastoris PS105(pBIG) as a producer of bovine intracellular immune interferon is obtained and deposited in All-Russian collection of industrial microorganisms at number VKPM-Y-2990. Application of invention provides preparing the bovine intracellular gamma-interferon with the enhanced yield.

EFFECT: improved preparing method, valuable biological properties of strain.

4 cl

Description

The invention relates to biotechnology, genetic engineering, the microbiological industry, veterinary medicine and agriculture, and is a yeast strain producing a bovine immune interferon containing a plasmid constructed in vitro that provides synthesis of the bovine intracellular immune interferon.

Immune interferon, gamma-interferon, belongs to a large group of evolutionarily related proteins, called interferons. The formation of gamma interferon occurs in T-lymphocytes and EC cells (natural killers) under the influence of viruses and tumor cells, respectively. Interferon gamma activates specific cellular immunity, stimulates macrophage cytotoxicity, and increases the body's resistance to various infections. It has been shown that bovine, porcine immune interferons increase the bactericidal and fungicidal activity of macrophages and are effective drugs for treating various animal diseases (Kishko IH, Vasilenko MI Mikrobiol. Z., 1998, v.60, p. 65-75; Zhang ZD et al ., Arch. Virol., 2002, v. 147, p. 2157-2167). The use of interferons for the treatment of diseases of various etiologies has significant advantages compared to traditional antibiotics and chemotherapeutic drugs due to the wide spectrum of action due to the activation of the immune system and due to the absence of side effects. According to experts of the World Health Organization, cytokine therapy is especially relevant in connection with the danger of the appearance of pathogenic microorganisms resistant to antibiotics in food products (Lowenthal J.W. et al., Vet. Immun. Immunopathol, 1999, v.15, p. 183-188). In addition, gamma interferon is an antitumor agent that activates the antineoplastic function of macrophages. Recombinant bovine gamma interferon has been shown to inhibit in vitro replication of cattle leukemia virus (Sentsui H. et al., Cytokine, 2001, v.16, p.227-231). Given that this disease is widespread, the need for such a medicine for veterinary medicine and agriculture is not in doubt.

However, systematic studies of the spectrum of the biological action of gamma-interferon, as well as its use as a medicine, are difficult, since currently there are no bull gamma-interferon preparations in Russia.

A promising approach for producing gamma interferon in bovine in significant quantities is the use of microorganisms as producers of this drug. There are strains of Escherichia coli that synthesize bovine gamma interferon (T. Kashima et al., J. Vet.Med.Sci. 1999, v. 61, p. 171-173). But Escherichia coli is a conditionally pathogenic microorganism, and gamma-interferon preparations obtained from bacterial cells may contain endotoxins.

All of the above indicates the promise of creating strains producing bovine gamma interferon based on other microorganisms, in particular yeast. The use of non-pathogenic microorganisms (yeast) that do not contain toxic and pyrogenic factors as producers of animal proteins allows the use of recombinant proteins in veterinary practice. The yeast strain Saccharomyces cerevisiae VKPM Y-1223, an intracellular producer of bovine immune interferon, synthesized about 10 mg of recombinant interferon per liter of culture previously created in the laboratory of biochemical genetics at the Biological Research Institute of St. Petersburg State University (Myasnikov A.N. et al. RF Patent, SU 1660388, 1991).

The use of methylotrophic yeast Pichia pastoris for heterologous expression is of particular interest. The advantages of this yeast are the accumulation of significant biomass during cultivation on inexpensive mineral nutrient media and a higher level of synthesis of recombinant proteins (K. Sreekrishna et al., J. Basic. Microbiol, 1988, v. 28, p. 265-278). Currently, the bull’s immune interferon gene has been cloned into vectors that ensure the production of recombinant protein by Pichia pastoris yeast cells and the secretion of the synthesized protein into the culture fluid (Nikolaev Y.V. et al. Cytokines and inflammation, 2002, v. 1, p. 28- 29). However, the use of strains accumulating the recombinant protein inside the cell as a probiotic feed additive seems equally promising.

The use of an immunoprobiotic feed supplement has certain advantages over medicinal products. Yeast is a valuable protein-vitamin product and is widely used in animal husbandry and poultry farming as feed additives to the main diet. Proteins synthesized by yeast are more easily absorbed than proteins of animal origin, in addition, increase the biological value of other feeds. Phosphorus and calcium, which are part of the yeast, contribute to the normal development of bone tissue; B vitamins contained in yeast cells are regulators of fat metabolism. In addition, it was shown that the lipopolysaccharides that make up the cell wall of microorganisms activate the local immunity system and, interacting with interferon regulatory factors, induce the production of their own cytokines (Navarro L., David M., J. Biol. Chem, 1999, v 274, p. 35535-35538; Coates NJ, McColl SR, J. Immunol, 2001, v. 166, p. 5176-5182).

It is known that strains of Bacillus subtilis synthesizing human α2-interferon, when administered orally, remain in the gastrointestinal tract of experimental animals for at least 96 hours and provide the synthesis of recombinant protein (Bakulina L.F. et al. Biotechnology, 2001, No. 2, p. .48-56). This allows us to confidently expect that the Pichia pastoris yeast strain, the producer of bovine gamma-interferon, under similar conditions will ensure the production of recombinant protein in animals, which will be accompanied by a specific increase in the bactericidal, fungicidal and antineoplastic activity of animal macrophages. Moreover, the features of creating producer strains exclude the possibility of bacterial DNA sequences entering the yeast cells. The use of such an immunoprobiotic feed additive is also cost-effective, as it eliminates the time-consuming procedures for the isolation and purification of recombinant interferon. Experiments on the use of strains of Pichia pastoris yeast that synthesize bird growth hormone as a feed additive in poultry farming have been successful (Chen C. M. et al., 2000, Life Sci., V. 67, p. 2103-2115).

The task to which the present invention is directed is to create a strain of the yeast Pichia pastoris PS105 (pBIG) -, which synthesizes the intracellular immune immune interferon of the bovine, to obtain recombinant plasmid DNA pBIG, which provides biosynthesis of the intracellular immune interferon of the bovine, and to develop a method for constructing the recombinant plasmid pB DNA.

To solve this problem, a method was developed for constructing recombinant plasmid DNA pBIG, which ensures biosynthesis of intracellular immune immune interferon of a bull, as a result, a strain of yeast Pichia pastoris PS105 (pBIG), a producer of intracellular immune immune interferon of a bull, was created.

The recombinant plasmid DNA pBIG, which ensures the biosynthesis of the intracellular immune immune interferon of a bull by the yeast cells transformed by it, consists of the following elements:

- BamHI-EcoRI - a fragment of plasmid DNA of a bifunctional bacterial yeast vector pIC9 of 7.75 kbp. (from which the preproregion of the Saccharomyces cerevisiae yeast MFα gene with a size of 0.25 kb, which secures the secretion of the recombinant protein into the culture medium, has been removed), including the bacterial gene for ampicillin resistance, the bacterial replication initiation region, yeast HIS4 gene; a fragment of the 5'-non-coding region of the yeast gene AOX1 with a size of 0.95 kbp containing a region that activates the transcription of this gene in the presence of methanol as a carbon source in the culture medium; a 0.33 kbp AOX1 gene fragment containing a transcription termination region of this gene; a fragment of the 3'-untranslated region of the AOX1 gene of 0.76 kbp .;

- XhoI-EcoRI — 0.42 kbp fragment containing the coding portion of the bovine gamma-interferon gene except for the region encoding the signal peptide;

- Unique recognition sites for the following restriction enzymes: EcoRI - 1.39 tp; XbaI - 2.20 kbp .; SalI - 3.34 kb; SphI - 4.96 kb; PstI - 7.35 kbp

The scheme of plasmid DNA pBIG with a restriction map is depicted in figure 1. The total size of the plasmid pBIG - 8170 p. (5.39 MDa).

A method of constructing a plasmid that provides for the biosynthesis of intracellular immune interferon in yeast cells is illustrated by the construction scheme of the plasmid pBIG shown in Fig.2.

From the plasmid pPIC9 (IFN-γ) (Nikolaev Y.V. et al. Cytokines and inflammation, 2002, v. 1, p. 28-29), the preproregion of the Saccharomyces cerevisiae yeast MFα gene is secreted using restriction enzymes BamHI and XhoI. gamma-interferon into the culture medium, then Escherichia coli cells are transformed with a ligase mixture and clones containing recombinant plasmid pBIG DNA are selected.

The choice of plasmid design for the production of immune interferon (gamma-interferon) of a bull is due to the following reasons. Plasmid pBIG was obtained on the basis of the shuttle bacterial-yeast integrative vector pIC9. As a result of transformation by a linearized plasmid and subsequent homologous recombination, the expression cassette is inserted into the chromosome of the Pichia pastoris yeast, which ensures stable maintenance of the cloned gamma-interferon gene. The plasmid contains the yeast HIS4 gene, which makes it possible to selectively select transformants when yeast strains with mutations in this gene are used as recipients.

The expression of the bovine gamma-interferon gene in the plasmid pBIG is controlled by the promoter of the AOX1 gene, which contains regions that activate transcription in the presence of methanol in the culture medium, as well as the region of transcription initiation. The AOX1 gene promoter is one of the most powerful yeast promoters. The level of expression of genes under the control of the AOX1 promoter is effectively regulated by carbon sources. Transcription of the AOX1 gene is completely blocked when yeast is grown on a medium with glucose; only a basal level of gene expression is observed on a medium with glycerin. The use of methanol as the sole carbon source significantly enhances the expression of the AOX1 gene and, therefore, genes controlled by the AOX1 promoter. This allows you to regulate the synthesis of gamma interferon bovine in yeast cells. Regulated expression of the cloned gene significantly reduces the metabolic load on the yeast cell.

As a producer of gamma interferon, strain PS105 (pBIG) is used. Strain PS105 (pBIG) was obtained by transforming the yeast strain PS99 (his4 pp4 :: PHO85) with plasmid DNA pBIG. Strain PS99 carries a mutation in the HIS4 gene, which makes it possible to selectively select transformants carrying plasmid DNA pBIG. A mutation in the PEP4 gene leads to a lack of activity of proteases A and B, as well as carboxypeptidase Y in yeast cells, which is accompanied by increased stability of heterologous recombinant proteins (Hisch HH et al. In: Walton EF, Yarranton GT, Eds., Molecular and Cell Biology of Yeast 1989, p.134-200).

The strain of the yeast Pichia pastoris PS105 (pBIG) is characterized by the following features.

Morphological signs.

The cells are round, slightly oval in shape, 5-10 microns in size, some of the cells on the surface of the kidney or connected to daughter cells.

Cultural signs.

Cells grow well on a complete organic YEPD medium - 2% peptone, 1% yeast extract, 2% glucose or 1% glycerol.

In addition, cells grow well on SC mineral medium: 1.34% Yeast Nitrogen Base (Difco, USA), 2% glucose (1% glycerol, 0.5% methanol), as well as other synthetic yeast media.

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

When grown in liquid media, they form an intense, even suspension. The culture has a characteristic smell of methylotrophic yeast.

Physiological and biochemical characteristics.

Cells grow in the range from 4 to 37 ° C. The optimum temperature for growing is 30 ° C. With growth under aerobic conditions, the cells slightly alkalize the medium. The optimum pH for growth is 4.5-6.5.

Cells can use many simple compounds as a carbon source, such as glucose, glycerin, methanol.

As a source of nitrogen, cells can use mineral salts in the ammonium form, amino acids, urea.

Cells are capable of aerobic and anaerobic growth.

An essential sign of the strain is the lack of need for histidine.

A method for constructing pBIG plasmid DNA is illustrated by the following example.

EXAMPLE.

Escherichia coli bacteria cells containing the plasmid pPIC9 (IFN-γ) were grown at 37 ° C overnight in 1 L of LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride) containing ampicillin at a concentration of 50 mg / l Cells are harvested by centrifugation at 5000 rpm for 10 minutes at 4 ° C, suspended in 20 ml of 25 mM Tris-chloride buffer (pH 8.0) containing 10 mM EDTA and 50 mM glucose, 30 mg lysozyme are added and incubated 10 minutes at room temperature. Then add 40 ml of 0.2 M sodium hydroxide containing 1% sodium dodecyl sulfate, mix gently and incubate for 10 minutes at 4 ° C. The solution is neutralized by adding 30 ml of 3 M sodium acetate (pH 5.0) and incubated for 10 minutes at 4 ° C. Then centrifuged at 14000 rpm for 40 minutes at 4 ° C. 0.6 volumes of isopropyl alcohol are added to the supernatant, incubated for 20 minutes at room temperature and centrifuged at 14000 rpm for 20 minutes at 20 ° C. The resulting precipitate was washed with 70% ethyl alcohol, dried in vacuo and dissolved in 4 ml of distilled water. Then 4.2 g of cesium chloride and 0.36 ml of ethidium bromide solution (10 mg / ml) are added. The resulting solution was incubated for 1 hour at 4 ° C, then centrifuged at 15,000 rpm for 15 minutes. The supernatant is centrifuged at 70,000 rpm for 16 hours in a TL100 centrifuge (“Beckman”). After centrifugation, a strip of plasmid DNA is selected (the lower of the two bands fluorescent in ultraviolet light), ethidium bromide is extracted twice with an equal volume of isoamyl alcohol, diluted twice with distilled water, and plasmid DNA is precipitated with two volumes of ethyl alcohol and 1/15 volume of 3 M sodium acetate ( pH 5.0). The precipitate was collected by centrifugation at 10,000 rpm for 10 minutes, washed with 70% ethanol and dissolved in 0.5-1 ml of TE buffer (10 mM Tris-chloride buffer, pH 8.0, containing 1 mM EDTA). The concentration of plasmid DNA is determined by the absorption of the solution at a wavelength of 260 nm. The purity of the drug is controlled by electrophoresis in a 0.7% agarose gel in TBE buffer (0.1 M Tris-borate buffer, pH 8.3, containing 1 mM EDTA).

Hydrolysis of plasmid pPIC9 (IFN-γ) with restriction enzymes BamHI and XhoI is carried out in 10 mM Tris-chloride buffer (pH 8.0) containing 100 mM potassium chloride, 5 mM magnesium chloride, 0.02% X-100 triton and 0.1 mg / ml bovine serum albumin.

To 5 μg of plasmid DNA in a volume of 20 μl add 5 units. each restrictase, after which the sample is incubated for 2 hours at 37 ° C.

Isolation of linearized plasmid DNA from agarose gel is carried out according to the method developed by QIAGEN. A strip of gel with a DNA fragment is placed in a tube and QX1 solution (300 μl per 100 mg of gel) is added. The sample is heated to 50 ° C, QIAEX reagent (10 μl per 5 μg DNA) is added and incubated at 50 ° C for 10 minutes, stirring occasionally. Then it is centrifuged for 30 seconds at 15000 rpm, the supernatant is discarded, the precipitate is extracted twice with QX2 and QX3 solutions, and the supernatant is removed by centrifugation at 15000 rpm for 30 seconds. The precipitate was dried in air, dissolved in 20 μl of TE buffer, centrifuged for 30 seconds at 15,000 rpm, the supernatant was transferred to a new tube.

The isolated linear DNA is treated with a Klenov fragment of DNA polymerase I, which ensures the completion of truncated 3'-ends. The reaction is carried out in 50 mM Tris-chloride buffer containing 10 mM magnesium chloride, 1 mM dithiothreetol, 50 μg / ml bovine serum albumin, 1 mmol of deoxyribonucleotides and 1 unit. enzyme per 1 μg of DNA. The reaction mixture was incubated for 15 min at 37 ° C, then the enzyme was inactivated by heating at 65 ° C for 10 min.

To obtain the pBIG plasmid, 1 μg of the pPIC9 plasmid (IFN-γ) is ligated, digested with BamHI and XhoI restriction enzymes and treated with the Maple DNA polymerase I fragment in 10 μl of 70 mM Tris-chloride buffer (pH 7.6) containing 5 mM dithiothreetol, 5 mM magnesium chloride, 1 mM ATP, adding 10 units. Phage T4 DNA ligases and incubating at 14 ° C overnight.

The obtained ligase mixture transform cells of the strain DH5α Escherichia coli (F '/ endA1 hsdR17 (r $\genfrac{}{}{0ex}{}{\text{-}}{\text{k}}$ m $\genfrac{}{}{0ex}{}{\text{+}}{\text{k}}$ ) supE44 thi-1 recA1 gyrA (Nal ^r ) relA1 Δ (lacZYA-argF) U169 deoR (φ80dlacΔ (lacZ) M15) Escherichia coli. For this, Escherichia coli cells are grown in 100 ml of LB medium at 37 ° C until the culture reaches a cell suspension density corresponding to 0.4-0.6 units. optical density at a wavelength of 550 nm. The cell suspension is cooled in an ice bath, centrifuged at 5000 rpm for 10 minutes at 4 ° C. Cells are suspended in 100 ml of 10 mm sodium chloride, collected by centrifugation under the same conditions. Then the cells are suspended in 50 ml of 75 mm calcium chloride, kept in an ice bath for 40 minutes, precipitated by centrifugation under the same conditions and suspended in 1 ml of 75 mm calcium chloride. Glycerin is added to a suspension of competent cells to a final concentration of 15%, aliquoted and stored at -70 ° C. Before transformation, a suspension of competent cells is thawed in an ice bath, a ligase mixture is added and incubated in an ice bath for 40 minutes. Next, the cells are subjected to heat shock at 42 ° C for 2 minutes, after which they are incubated in 1.5 ml of LB medium at 37 ° C for 1 hour. Cells were harvested by centrifugation at 5000 rpm for 10 minutes and plated on Petri dishes with LB medium containing 2% agar and 50 mg / L ampicillin. Cups are incubated at 37 ° C for 12-16 hours.

Plasmid DNA was isolated from the grown individual clones of transformants using the technique used to obtain plasmid pPIC9 (IFN-γ), except that Escherichia coli cells were grown in 10 ml LB and, accordingly, the volume of all solutions was reduced by 100 times. In addition, instead of a centrifugation step in the density gradient of cesium chloride, DNA is treated with pancreatic RNase. For this, nucleic acids precipitated with isopropyl alcohol are dissolved in 100 μl of TE buffer, 10 μl of RNase solution (1 mg / ml) is added and incubated for 30 minutes at 37 ° C.

Next, the obtained plasmid DNA is hydrolyzed by restriction enzymes PstI and SalI in 50 mM Tris-chloride buffer containing 10 mM magnesium chloride, 100 mM sodium chloride and 0.1 mg / ml bovine serum albumin. With restriction of the desired plasmid pBIG and subsequent electrophoresis in 0.7% agarose gel, fragments of 4.17 and 4.0 kb are detected. From the clone thus identified, the pBIG plasmid was preliminarily isolated as described for the plasmid pPIC9 (IFN-γ) and digested with BglII restriction enzyme in 50 mM Tris-chloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride . The BglII fragment of the pBIG plasmid, size 5.77 kb, was isolated according to the QIAGEN method described above, excluding the heating step, and used to transform yeast cells, as described in Example 2.

EXAMPLE 2

To obtain the yeast strain Pichia pastoris, a producer of bovine gamma interferon, the yeast cells of strain PS99 are transformed with pBIG plasmid DNA.

Yeast cells are grown in 100 ml of YEPD medium at 30 ° C until the culture reaches an optical density corresponding to 2-4 units. absorption at a wavelength of 600 nm. Cells are washed twice with sterile water, after which they are suspended in 0.3 ml of a 100 mM lithium acetate solution and incubated at 30 ° C for 30 minutes. To 50 μl of the obtained cell suspension, 0.1-1 μg of plasmid DNA, 50 μg of salmon sperm DNA pre-denatured by heating (10 minutes at 100 ° C) and 0.3 ml of a solution of 100 mM lithium acetate containing 40% polyethylene glycol 4000 are added. The sample is then incubated for 30 minutes at 30 ° C and 20 minutes at 42 ° C, placed for 15 seconds in an ice bath and centrifuged for 10 seconds at 10,000 rpm. Cells are suspended in 1 ml of sterile water and plated on SC solid medium. Clones of transformants grow in 2-3 days. The grown clones are subcultured on plates with SC medium containing 2% glucose in separate colonies, then reprinted on MM medium (1.34% Yeast Nitrogen Base (Difco, USA), 0.5% methanol, 2% agar (Difco ”, USA) for the selection of transformants characterized by a decrease in the growth rate on a medium with methanol, which indicates the integration of a foreign gene into the AOXI locus (phenotype Met ^S ).

To analyze the production of bovine gamma-interferon by cells of Met ^S phenotype transformants, they are grown at 30 ° C in 100 ml of MGY liquid medium (1.34% Yeast Nitrogen Base (Difco, USA), 1% glycerol, 0.04 mg biotin up to stationary growth phase for 2 days. Cells are harvested by centrifugation at 5000 rpm for 10 minutes, the supernatant is drained and the whole biomass is transferred into 100 ml of liquid MM (1.34% Yeast Nitrogen Base (Difco, USA), 0 , 5% methanol, 0.04 mg biotin) to induce the expression of gamma-interferon Induction is carried out at 30 ° C for 4 days. by 5,000 rpm for 10 minutes, resuspended in TE buffer, add glass beads (⌀ = 0.5 mm) and destroy on a disintegrator (Braun Metsunger). The resulting suspension is separated from the beads and centrifuged 10,000 rpm 20 minutes in the supernatant determine the content of gamma interferon by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate and subsequent hybridization with specific antibodies to gamma interferon. The high degree of homology of human and bovine gamma-interferons allows in this test to replace monoclonal antibodies that are not on sale for bovine gamma-interferon with antibodies to a similar human protein (Myasnikov A.N. et al. RF Patent, SU 1660388 A1, 1991). Protein separation is carried out in a 15% polyacrylamide gel in a standard buffer system (electrode buffer: 25 mm Tris, 192 mm glycine, 0.1% sodium dodecyl sulfate, pH 8.3; gel buffer: 375 mm Tris-chloride buffer, pH 8, 8). In parallel, the separation of proteins of the control strain grown under identical conditions is carried out. Carbonic anhydrase, trypsin inhibitor, myoglobin, lysozyme are used as molecular weight standards. After electrophoresis, the proteins were renatured, keeping the gels for 15 minutes in 10 mM Tris-chloride buffer (pH 7.5) containing 4 M urea, 20 mM EDTA, and transferred onto a nitrocellulose membrane in 25 mM Tris-192 mM glycine buffer (pH 8, 3) containing 20% methyl alcohol, at 30-40 V, for 1.5 hours. Next, the membrane was kept in TBST buffer (10 mM Tris-chloride buffer (pH 8.0), 150 mM sodium chloride, 0.05% tween-20, 1% bovine serum albumin) for 2 hours at 37 ° C. The membrane is then placed in the same buffer containing 1000-fold diluted murine monoclonal antibodies to human gamma interferon (Protein circuit, St. Petersburg), and incubated for 2 hours at 37 ° C. Then, the membrane is washed three times with TBST buffer and incubated for 1 hour at 37 ° C with a conjugate of species-specific antibodies to mouse immunoglobulins and horseradish peroxidase diluted 7000 times (Protein circuit, St. Petersburg). After washing the membrane with PBST buffer (58 mM disubstituted sodium phosphate, 17 mM monosubstituted sodium phosphate, 68 mM sodium chloride, 0.1% tween-20), a solution of peroxidase substrates was added: 0.02% DAB (3, 3-diaminobenzidine tetrahydrochloride ), 0.006% hydrogen peroxide in 10 mM Tris-chloride buffer, pH 7.5. In parallel, the gels are stained with a 0.15% solution of Coomassie G250 in 25% isopropanol and 10% acetic acid and washed in 10% acetic acid. The level of synthesis of gamma interferon of a bovine is determined by comparing the intensity of staining of the band of the recombinant protein with the band of marker protein of the corresponding molecular weight.

According to the data obtained, yeast cells of strain PS104 (pBIG) synthesize at least 15-20 mg of gamma-interferon per liter of yeast culture, which exceeds the productivity of the prototype strain VKPM Y1223.

Summarizing the above, it can be concluded that the obtained strain of yeast Pichia pastoris PS105 (pBIG) synthesizes gamma interferon of bovine and can be used in veterinary medicine and agriculture as an immunoprobiotic feed additive that increases the resistance of animals to various infectious and oncological diseases. The level of synthesis of the recombinant protein allows the use of this strain also for the isolation of bovine gamma-interferon.

The yeast strain Pichia pastoris PS105 (pBIG), the producer of the intracellular immune immune interferon of the bull, was deposited in the All-Russian collection of industrial microorganisms under the number VKPM-U2990.

Claims (3)

1. The yeast strain Pichia pastoris PS105 (pBIG), deposited in the All-Union collection of industrial microorganisms under the number BKPM-Y2990 - producer of bovine gamma-interferon. 2. Recombinant plasmid DNA pBIG, providing biosynthesis of intracellular gamma-interferon bovine, having a size of 8.17 kb molecular weight 5.39 MDa and consisting of the following elements: BamHI-EcoRI - a fragment of the plasmid DNA of the bifunctional bacterial-yeast vector pIC9 obtained by removing the prepreg region of the Saccharomyces cerevisiae yeast MFα gene, which secures the secretion of the recombinant protein into the culture medium, size 0.25 t bp having a size of 7.75 bp and comprising a bacterial gene for ampicillin resistance; bacterial replication initiation region; yeast HIS4 gene; a fragment of the 5'-non-coding region of the yeast gene AOX1 with a size of 0.95 kb containing a sequence that activates the transcription of this gene in the presence of methanol as a carbon source in the culture medium; a 0.33 kbp AOX1 gene fragment containing a transcription termination region of this gene; a fragment of the 3'-untranslated region of the AOX1 gene, 0.76 kbp; XhoI-EcoRI — 0.42 kbp fragment containing the coding portion of the bovine gamma interferon gene except for the region encoding the signal peptide; the following unique restriction enzyme recognition sites: EcoRI 1.39 kbp; XbaI - 2.20 kbp .; SalI - 3.34 kb; SphI - 4.96 kb; PstI - 7.35 kbp 3. A method of constructing a recombinant plasmid DNA pBIG, providing synthesis of intracellular gamma-interferon bovine in yeast cells Pichia pastoris, which consists in the fact that from the plasmid pPIC9 (IFN-γ) using the restriction enzymes BamHI and XhoI cut out the prepreg region of the Saccharis cerevis MFarces gene the protruding ends of the plasmid DNA are completed and ligated, then Escherichia coli cells are transformed with the ligase mixture and clones containing the recombinant plasmid pBIG DNA are selected.

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