KR900007867B1 - Method for producing human γ-interferon from yeast cells by synthetic genes - Google Patents

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Description

Method for producing human γ-interferon from yeast cells by synthetic genes

FIG. 1 shows the nucleotide sequence of 30 kinds of oligonucleotides corresponding to total γ-interferon (including cysteine (Tys), tyrosine (Tyr), cislane (Cys), etc.) and the conjugation strategy of oligonucleotides. Will be

Figure 2 illustrates the entire γ-interferon gene recombination process to plasmid pAB112 for the conjugation of synthetic oligonucleotides,

3 shows the conjugation of spontaneous γ-interferon (cysteine, tyrosine, cysteine removed) to promoters and terminators for yeast production and recombination into yeast expression carriers.

Figure 4 shows the γ-interferon band in which the whole protein of γ-interferon-expressed yeast cells was subjected to Coomassie staining after electrophoresis of SDS-Poly acrylamide gel. 1 is a protein standard molecule, lanes 2, 3 and 4 are spontaneously induced samples cultured in 2% glucose medium and lanes 5 and 6 are samples induced γ-interferon in 3% ethanol culture.

The present invention relates to a method for mass production of chemically synthesized human natural γ-interferon gene in yeast cells through genetic engineering process.

Since the discovery of a substance with antiviral activity called interferon in 1957, its usefulness as a therapeutic agent for antiviral diseases and anticancer agents has led to a great deal of research in the industrial and academic fields about its effect, mechanism of action and pure separation, and mass production methods. come.

Currently known interferons include α-interferon, which is induced by viral infection in leukocytes of leukocytes, and β-interferon and lymphocytes, which are induced by viral or double-stranded RNA in Fibroblast cells. There are three types of γ-interferon produced by lymphocyte).

Of these, γ-interferon is known as immune interferon because it has an immunomodulatory function, and is reported to be superior to α or β-interferon.

(Reference Stewart et al. (1979) The Interferon System, Springenverlag, New York., Rubin et al. (1980), Proc. Natl. Acad. Sci. USA 78, 5928-5932). Such γ-interferon is conventionally known as T-type lymphocytes (Lymphocytes) in human peripheral blood lymphocytes (Peripheral Blood Lymphocytes) or various types of myogens [12-0-tetra decanoyl phorbo1-13-Acetate (TPA). ), Phytohemagglutimin (PHA). desacetylthymosine-d1)], but the amount is so small that it has been limited to study the mechanism of action and research as antiviral, anticancer drugs.

Recently, however, research has been conducted on a method for mass production of γ-interferon using animal cells, E. coli and yeast cells through genetic factor recombination technology.

The method of producing γ-interferon using animal cells (Marcucci et al., The Biology of The Interferon System p91, Elsevier, 1983) has the advantage of producing glycosylated γ-interferon, but is produced in comparison with the use of E. coli or yeast cells. Yield is extremely low and also because the production cost is expensive because the expensive bovine serum to be supplied to the culture medium. In addition, methods using Escherichia coli cells [Tessier et al. (1984) Nucleic Biol Research 12, 7663-7675., Simon et al. (1984) Gene 28, 55-64. Jay et al. (1984) Proc. Nat1. Acad. Sci 81. 2290-2294] has a high production yield, but the difficulty of refining has become a problem due to the possibility of endotoxin and pyrogen contamination due to E. coli protein in the separation and purification process. On the other hand, the method using yeast cells (Derynck et al. (1983 in experimental manipulation of gene Expression Inouye, M. (ed) pp. 297-258. Academic press; Derynck et al. (1983) Nucleic Acids Research 11, 1819-l837) Not only is this high but there is no concern about contamination of resistant toxins.

Therefore, the present inventors have attempted a method for mass production of γ-interferon using yeast cells, and a gene codon (Bennetzen et al. (1982), J. Biol. Chem. 257, 3026-3031) was used to selectively synthesize genes of natural mature γ-interferon to be mass-produced in yeast cells, and by using inducible yeast promoters (Inducible promotor). Γ-interferon is produced independently of the growth pattern, and the invention is characterized in that a large amount of γ-interferon is induced to be produced after high concentration of yeast cell growth.

That is, this invention is an invention characterized by the following.

The present invention changes and synthesizes the human γ-interferon gene sequence into an amino acid codon selectively used in yeast cells, and clones the bacterial carrier according to the oligonucleotide conjugation strategy, and then the three amino acids of the amino terminus cysteine, tyrosine, The cysteine was removed and cloned into the carrier (pBS 100) together with the synthetic linker to prepare a cassette of the effector- [gamma] -interferon-terminating gene, which was then cloned into the yeast expression carrier (pYLBCIO5) to produce the final yeast expression carrier (pYLBC-). As a method for producing human γ-interferon, characterized in that after the production of γIFN-4) induced induction, the conjugation strategy of the oligonucleotide is expressed as follows,

here,

gIFN-1 (24) CTAGATAAAAGATGTTCTGTCAG

gIFN-2 (30) GATCCATACGTTAAGGAAGCTGAAAACCTA

gIFN-3 (30) AAGAAATACTTCAACGCTGGTCACTCTGAC

gIFN-4 (30) GTTGCTGACAACGGTACCTTGTTCTTGGGT

gIFN-5 (30) ATCTTGAAAAACTGGAAGGAAGAATCTGAC

gIFN-6 (32) AGAAAGATCATGCAATCCCAAATCGTTTCTTT

gIFN-7 (32) CTACTTCAAGTTGTTCAAGAACTTCAAGGACG

gIFN-8 (32) ACCAATCTATCCAAAAGTCTGTTGAAACCATC

gIFN-9 (33) AAGGAAGACATGAACGTTAAGTTCTTCAACTCT

gIFN-10 (32) AACAAGAAGAAGAGAGACGACTTCGAAAAGCT

gIFN-11 (32) TACCAACTACTCTGTTAACGACTTGAACGTTC

gIFN-12 (32) AAAGAAAGGCTATCCACGAATTGATCCAAGTT

gIFN-13 (31) ATGGCTGAATTGTCTCCAGCTGCTAAGACCG

gIFN-14 (31) GTAAGAGAAAGAGATCTCAAATCTTGTTCAG

gIFN-15 (30) AGGTAGAAGAGCTTCTCAATAATAGCGTCG

gIFN-16 * (34) TTACGTATGGATCCTGACAGTAACATCTTTTAT

gIFN-17 * (31) GTTGAAGTATTTCTTTAGGTTTTCAGCTTCC

gIFN-18 * (32) GTACCGTTGTCAGCAACGTCAGASTGACCAGC

gIFN-19 * (32) CCTTCCAGTTTTTCAAGATACCCAAGAACAAG

glFN-20 * (30) GGGATTGCATGATCTTTCTGTCASATTCTT

gIFN-21 * (30) TGAACAACTTGAAGTAGAAAGAAACGATTT

gIFN-22 * (33) ACTTTTGGATAGATTGGTCGTCCTTGAAGTTCT

gIFN-23 * (32) AACGTTCATGTCTTCCTTGATGGTTTCAACAG

gIFN-24 * (31) CTCTCTTCTTCTTGTTAGAGTTGAAAGACTT

gIFN-25 * (32) AACAGAGTAGTTGGTAAGCTTTTCGAAGTCGT

gIFN-26 * (31) GGATAGCCTTTCTTTGAACGTTCAAGTCGGT

gIFN-27 * (32) AGACAATTCAGCCATAACTTGGATCAATTCGT

gIFN-28 * (31) ATCTCTTTCTCTTACCGGTCTTAGCAGCTGG

gIFN-29 * (32) AGAAGCTCTTCTACCTCTGAACAACATTTGAG

gIFN-30 * (18) TCGACGACGCTATTATTG

The bacterial carrier cloning was performed by cloning the fragments of the restriction enzymes Hind III and Sal I among the oligonucleotides bound with the binding enzyme according to the conjugation strategy to the bacterial carriers, followed by fragments of the restriction enzymes Xba-1 and Hind III. And cloned each fragment into one, and the removal of the three amino acids, cysteine, tyrosine, and cysteine at the amino terminus is performed by treating the carrier (γ-IFN) with restriction enzymes BamHl and Sal I. Cloning in the carrier (BS-100) was performed by treating the carrier (BS-l00) with restriction enzymes Sal I and NCOI, and then cloning the synthetic linkage to the γ-interferon gene from which cysteine, tyrosine, and cysteine were removed. Also, the synthetic linker was synthesized to be linked to NcoI and BamH1, and the cassette of the promoter-γ-indiferon-terminated gene restricts the carrier (pBS-γIFN). The final yeast expression carrier (pYLBC-γIFN4) was prepared by treating the carrier (pY LBC105) with restriction enzymes BamH1 and phosphatase followed by binding of a cassette of promoter-y-interferon-terminated gene. It is a method for producing a human γ-interferon characterized in that.

Carrier pBS100 in the present invention is purchased from Reference (Yeast 2, 72, 1986 or Chiron Corp., Emeryville, CA., USA), or the vector (ATCC No. 37179 or No. 33875) from ATCC, a strain depositing institution in the United States Vector, pJDB (Catalog No. N338) and Amersham, USA (Beggs, JD Molecular Genetics in Yeast, Alfred Benzon Symposium 383, 1981: ICN-UCLA Symposium 15,325, 1979: Gene 20,347,1982: Gene 26,243,1983) Promoter and terminator can be purchased from Clontech's Yeast Nucleic Acid Library (Catalog No. YL100lb) and synthesized with a DNA synthesizer (Ref. Young, DC). , et al., J. Molecular Biology 148, 355, 1981: Russel, DW, et al., J. Biological Chemistry 258, 2674, 1983: Ho1land, JP and Holland, MJJ Biological Chemistry 255,2596, 1980), genes Cloning method (Maniatis, T., et al., Molecular Cloning-A Laboratory Manual, Co ld Spring Harbor Lab., Cold Spring Harbor, N. Y., U. S. A, 1982).

Yeast cloning vector pYLBC105 is available from ATCC in USA (Catalog 37115) or Amersham in USA (Catalog No. N338) Yeast Genetic Stock Center, University of California, Berkeley, CA., USA, or Dr. . Purchased from Jeremy Thorner (Department of Microbiology and Immunology, University of California, Berkeley, CA., USA), the final expression vector pYLBC-γ-IFN4 was prepared from pYLBC105 and pBS-γ-IFN as described above, Documents: Mainiatis, T., et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor, Lab, CSH, NY, US A, 1982), and the γ-IFN gene is known amino acid sequence. Refer to (Rinderknecht, E., J. Biological Chemistry 259,6790, 1984) by artificially selecting genes commonly used in yeast (Bennetzon, JL and Hall, BD, J. Biological Chemistry, 257,3026, 1982). Gamma interferon gene was synthesized before using a nucleic acid synthesizer (mode1 380B) of App1ied Biosystems, USA, and cloned according to the method of Maniatis, T., et al., (1982).

The content of the present invention will be described in detail as follows.

Human γ-interferon gene sequencing (Gray et al. (1982) 295,503, Devos et al. (Nucleic Acid Research (1982), 10, 2487)) to the entire genetic factor by changing the amino acid codons selectively used in yeast cells Was synthesized by a gene factor synthesizer (Applied Biosystems, Mode 1 38OB, USA) through phosphoramidite method and the oligonucleotide and conjugation strategy of these synthetic genes are shown in FIG. The synthesis of these oligonucleotides was designed as a system that separates γ-interferon into extracellular culture in yeast cell culture. For this purpose, the α-factor secretion system of yeast was attempted but changed later. Therefore, the synthesis of oligonucleotides is carried out by the gene codon and termination codon (TAA) corresponding to 146 amino acids of γ-interferon, restriction enzyme Sal I at 3 'end and α-factor precursor at 5' end. Lys. For the recognition of proteolytic enzymes when converting from material to mature. A total of 461 base pairs were synthesized by adding codons corresponding to Arg, starting codon (ATG), and restriction enzyme Xba-1 recognition site.

Each of the oligonucleotides synthesized above was phosphorylated at the 5 ′ end with T4 polynucleotide kinase, and then the entire γ-interferon gene was converted to pAB112 [(Brake et al. 91984) Proc. Natl. Acad. Sci. 81,4642) to prepare pγ-IFN.

Isolates BamHI-SalI restriction enzyme fragments corresponding to partial γ-interferon from pγIFN and initiates codons (ATG), amino acids to allow promoter and partial mature γ-interferon genes to be conjugated as shown in FIG. Inducible promoter that is depleted in yeast cells by depletion of reduced carbon source (e.g. glucose) in the yeast cells by using the gene-synthesizing linkage including the codon corresponding to glutamine and the recognition sites of restriction enzymes NcoI and BamHI. Carrier pBS-γIFN having a promoter and terminator was prepared.

The BamHI fragment (approximately 2560 base pairs) containing the promoter γ-interferon and the terminating gene site was isolated from the pBS-γIFN to spontaneously replicate in E. coli and yeast cells and lack leucine deficiency in yeast cells. PYLBC-γIFN was prepared by inserting at the BmHI position of the expression carrier pYLBC105 for cultivation in a leucine deficient selection medium (FIG. 3), and the DNA was prepared by Hinn et al. (Proc. Natl. Acsd. Sci. USA (1978). 75. Porotpolast transformation to yeast cells (Saccharomyces cerevisiae AB110) by the method of 1929-1933), and then transformed yeast cells were aged in γPD medium containing 2% glucose voluntary interferon induction (Auto induction) sikimeuroseo γ- were to be produced by more than 20% of the total protein in yeast cells, the biological activity of interferon (NIH Standard asay, the interferons, NIH, USA 1983) is 2.4 × 10 ⁹ per liter culture It was cut.

Therefore, the present invention is characterized in that the gene was synthesized using the amino acid codon selectively used by yeast cells for mass production in yeast cells, and second, fermentation conditions of normal yeast cells by using an inducible promoter. Cells do not produce γ-interferon but are induced when the available carbon source is depleted in the culture medium, and induced by ethanol in the culture medium, so there is no growth inhibitory factor by γ-interferon produced in the yeast cells. Cell culture is possible with a high yield and is characterized in that the production method to increase the production of γ-interferon per unit culture in response to the high cell yield.

Third, the γ-interferon produced in these yeast cells is a natural mature γ-interferon without the 3 amino acids cysteine (Cys), tyrosine (Tyr), and cysteine (Cys) residues at the amino-terminal site, so 3 amino acids It is characterized by an increase in solubility than γ-interferon.

(Example 1)

Ligation of Synthetic γ-Interferon Oligonucleotides and Cloning to Carrier pABl12.

Using the amino acid codons selectively used by the yeast cells, the total mature γ-interferon gene was synthesized as shown in FIG. 1, and then, for their ligation, as shown in FIG. Each oligonucleotide corresponding to the carboxyl Hind III-SalI fragment of the base pairs was taken at 260 nm to have an absorbance of 0.05, followed by drying and 50 mM Tris-HCI (pH 7.5), 1 mM ATP in 30 μl total volume. T1 polynucleotide kinase 4 unlt was added under 1mM DTT, 10mM MgCl ₂ buffer, and reacted at 37 ° C for 30 minutes to phosphorylate and phosphorylate the 5'-terminal residues of the nucleotides. After treating with the same volume of phenol and chloroform, they were precipitated with ethanol, and then they were dissolved in 53 μl of buffer (60 mM Tris-HCl, pH 7.5), 1 mM DTT, 10 mM MgCl ₂ ) and then immersed in a 95 ° C. water tank. Allow 6 hours at room temperature to allow each oligonucleotide to form base pairs with complementary strands, then add 20 units of T4 DNA ligase and 5 μl of 10 mM ATP to the oligomer for 10 minutes at room temperature. The 5'-terminus and 3'-terminus of (Oligomer) were connected, and ethanol was precipitated after treating phenol and chloroform.

Precipitated DNA was reacted for 1 hour at 37 ° C by adding 10 units of Hind III and SalI restriction enzymes under 60 mM Tris (pH 7.6) 10 mM MgCl ₂ and 100 mM NaCl buffer, and these were subjected to 7% polyacrylamide gel electrophoresis.

After electrophoresis, portions of 120 to 200 base pairs in the gel were cut out to allow these DNAs to flow out of the gel under the same conditions as electrophoresis, followed by ethanol precipitation and dissolving again in 20 μl of distilled water.

3 μl of this DNA and 1 μl of other carriers pAB112 (5 ng) and 10 μl conjugation buffer (600 mM Tris-HCI (pH 7.5), 1 μl of 10 mM DTT, 100 mM MgCl ₂ ), 1 μl of 10 mM ATP and 10 units of ligase The total reaction volume was added to 10 μl and reacted at 14 ° C. for 16 hours.

Thereafter, E. Coli HB101 competent cells were added to the conjugate reaction solution, and the cells were transformed according to Hanahan's method (J. Mol. Biol. 116,557 (1983)) and aged at 37 ° C overnight. Clones were screened and clones with the recombinant carrier p3'-γIFN were selected by the method of Dolly and Dolly (Nucleic acid res. 7, 1513 (1979)). (Figure 2)

The p3'-γIFN was cleaved with XbaI and Hind III and then the 160 base pair Hind III-SalI described above to insert the remaining xbaI-Hind III gene fragment of the amino-terminal region of γ-interferon corresponding to 201 base pairs in this carrier. The fragment was carried out in the same manner as the insertion into the pAB 112 carrier cleaved with Hind III and SalI, and the clone thus obtained, ie, a clone into which 461 base pairs of the total γ-interferon genes were inserted, was called pγIFN. (See Figure 2)

(Example 2)

Cloning experiments into pBS100 for yeast intracellular expression of synthetic γ-interferon gene.

During the whole γ-interferon gene factor synthesis and cloning process, the inventors attempted to make some modifications to the design for expression in yeast cells. According to a recent publication (Rinderknecht et al. J. Biol. Chem. 259,6790 (1984)), the amino acid cysteine, tyrosine and the amino terminal portion of the amino-terminal portion of the base sequence synthesized by the inventors corresponding to the natural γ-interferon 146 amino acid Cysteine-free and its biological activity was maintained, and the production of γ-interferon into the extracellular culture using the alpha-factor production system of yeast, which was originally designed, resulted in extremely low production efficiency (10 ⁵ -10 per liter culture). ⁶ units). First, the amino acid glutamine was used to remove cysteine, tyrosine, and cysteine seamino acid.

In other words, the BamHI SalI restriction enzyme was treated from pγIFN to isolate the 437 cheap pair of gene fragments by agarose electrophoresis, and the Nco I recognition site and the initiation site were cloned for cloning to the Nco I site of the carrier pBS 100 having an inducible promoter. Complementary adapters 5'-CATGCAA-3 'and 5'-GATCTTG-3' with codons corresponding to cotamine at the codon and amino terminus were synthesized using a DNA synthesizer and then 0.05A 260 units. And dried respectively and phosphorylated in the same manner as in the synthesis of γ-interferon γ-interferon in Example 1, 1 μl of each reaction solution and 3 μl of the isolated BamHI SalI fragment gene and Nco I, After dissolving 1 μl of the pBS100 transporter cut into SalI, it was left to stand at 65 ° C. for 15 minutes and slowly cooled at room temperature. After forming the spiral structure, 2μl of 10mM ATP, 2μl of 10-fold concentrated conjugation buffer, 1μl of ligase and 8μl of distilled water were added and reacted at 14 ° C for 16 hours. Then, as described in Example 1, E. coli HB 101 was transformed in the same manner to prepare a recombinant carrier pBS-γIFN into which a synthetic linker and a mature γ-interferon were inserted. (Figure 3)

pBS100 has a hybrid promoter (ADH2: GAPDH Hybrid Promotor) of the promoter of the alcohol dehydrogenase II (ADH2) and the promoter of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used by the inventors In the promoter portion of the alcohol dehydrogenase induced when ethanol is depleted due to the depletion of the carbonated (glucose) available as a carrier, and the 3'-terminal portion with the TATA box and the CAP position to which RNA polymerase binds. More RNA synthesis was produced by substituting with that of the promoter of glyceraldehyde 3 ′ phosphate dihydrogenase. Therefore, this hybrid promoter has the inducible properties of the ADH2 promoter and the stronger RNA synthesis properties of the GAPDH promoter, thus having a stronger promoter than the promoter of ADH2 itself.

This carrier was obtained by the courtesy of CHIRON, USA (4560 Horton St. Emeryville, CA 94608).

(Example 3)

Cloning experiments into enzyme-expressing carrier (pYLBC105) for yeast production of γ-interferon.

10 μg of the carrier pBS-γIFN was added to 10 units of restriction enzyme BamHI for 1 hour at 37 ° C. to terminate the hybrid promoter, γ-interferon gene, and glyceraldehyde 3′-phosphate dehydrogenase by 1% agarose electrophoresis. BamHI fragment of 2560 base pair BamHI fragment containing the gene site was isolated and dissolved in 20 μl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) and the BamHI site of pYLBC105, a derivative of the carrier pJDB (Amershom Co.USA) The recombinant gene carrier selected after inserting the BamHI fragment under the conjugation conditions described above and transforming the bacterium HB101 was named pYLBC-γIFN-4.

This pYLBC-γIFN-4 was applied to the method of yeast strain Saccharomyces cerevisiae AB110 (Yeast Genetic Stock Center, USA) in the method of Hinn (Proc. Natl. Acad. Sci USA 75 1929, (1978)). Protoplasts were transformed and plated on leucine deficient agaplate (6.7 g Yeast nitrogen base wlthout amino acids, 182 g Sorbitol, 2% glucose, 0.25% Leu-supplements, 20% Bacto agar). After incubation at 30 ° C. for 3-5 days, the grown corona was selected to identify γ-interferon, and the production yield of γ-interferon was estimated by electrophoresis of all proteins.

(Example 4)

Culture of Yeast Cells for the Production of γ-Interferon and Quantification of γ-Interferon Expression.

Yeast strains transformed with the carrier pYLBC-γIFN4 were incubated at 30 ° C for 24 hours in 3μl leucine deficient culture (6.7g Yeast nitrogen base without amino acids, 182g Sorbitol, 2% glucose, 0.2g Leu-supplements) After centrifugation, the supernatant was discarded and the precipitated cells were divided into two, dissolved in 5 mL 2% YEPD (1% Yeast Extract, 2% Peptone, and 3% ethanol), respectively, aged at 30 ° C. for 8 hours, followed by redimensionalization of the yeast cells. And then dissolved in 400 μl of buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM Phenylme-than sulfonyl fluoride, 4M UREA) and glass beads (Glass bead, 6N HCl washed, Thomas) of the same volume of diameter. scientific, USA) and then shaken strongly to break the cell wall, γ-interferon is eluted with buffer, 10μl of the eluate is taken and γ-interferon quantitatively measured by NIH standard biological activity assay and forced induction (Autoinduction) Forc In the case of ed induction, the similar expression efficiency was 2-4 × 10 ⁹ units per 1μl culture.

On the other hand, the result of electrophoresis of the whole protein of the yeast eluate destroyed by glass beads by SD polyacrylamide gel electrophoresis is shown in FIG. 4, where γ-interferon is a dark band corresponding to the estimated molecular weight of l7.7Kdalton. Densitometric scanning results in about 20% of the total protein production.

Claims (8)

The human γ-interferon gene sequence is converted into amino acid codons selectively used in yeast cells, and then chemically synthesized and cloned into bacterial carriers according to the oligonucleotide conjugation strategy. Three amino acids, cysteine, tyrosine, and cysteine, respectively, are then The final yeast expression carrier (pYLBC-γIFN-4) was removed by cloning it in a carrier (pBS100) with a synthetic linker and producing a cassette of the promoter γ-interferon-terminating gene and then cloning it into the yeast expression carrier (pYLBC105). Method for producing human γ-interferon characterized in that after the induction of the production. The method of producing a human γ-interferon according to claim 1, wherein the conjugation strategy of the oligonucleotide is expressed as follows.

here, gIFN-1 (24) CTAGATAAAAGATGTTCTGTCAG gIFN-2 (30) GATCCATACGTTAAGGAAGCTGAAAACCTA gIFN-3 (30) AAGAAATACTTCAACGCTGGTCACTCTGAC glFN-4 (30) GTTGCTGACAACGGTACCTTGTTCTTGGGT gIFN-5 (30) ATCTTGAAAAACTGGAAGGAAGAATCTGAC gIFN-6 (32) AGAAAGATCATGCAATCCCAAATCGTTTCTTT gIFN-7 (32) CTACTTCAAGTTGTTCAAGAACTTCAAGGACG gIFN-8 (32) ACCAATCTATCCAAAAGTCTGTTGAAACCATC gIFN-g (33) AAGGAAGACATGAACGTTAAGTTCTTCAACTCT gIFN-10 (32) AACAAGAAGAAGAGAGACGACTTCGAAAAGCT gIFN-11 (32) TACCAACTACTCTGTTAACGACTTGAACGTTC gIFN-12 (32) AAAGAAAGGCTATCCACGAATTGATCCAAGTT gIFN-13 (31) ATGGCTGAATTGTCTCCAGCTGCTAAGACCG gIFN-14 (31) GTAAGAGAAAGAGATCTCAAATGTTGTTCAG gIFN-15 (30) AGGTAGAAGAGCTTCTCAATAATAGCGTCG gIFN-16 * (34) TTACGTATGGATCCTGACAGTAACATCTTTTAT gIFN-17 * (31) GTTGAAGTATTTCTTTAGGTTTTCAGCTTCC gIFN-18 * (32) GTACCGTTGTCAGCAACGTCAGASTGACCAGC gIFN-19 * (32) CCTTCCAGTTTTTCAAGATACCCAAGAACAAG gIFN-20 * (30) GGGATTGCATGATCTTTCTGTCASATTCTT gIFN-21 * (30) TGAACAACTTGAAGTAGAAAGAAACGATTT glFN-22 * (33) ACTTTTGGATAGATTGGTCGTCCTTGAAGTTCT gIFN-23 * (32) AACGTTCATGTCTTCCTTGATGGTTTCAACAG gIFN-24 * (31) CTCTCTTCTTCTTGTTAGAGTTGAAAGACTT gIFN-25 * (32) AACAGAGTAGTTGGTAAGCTTTTCGAAGTCGT gIFN-26 * (31) GGATAGCCTTTCTTTGAACGTTCAAGTCGGT gIFN-27 * (32) AGACAATTCAGCCATAACTTGGATCAATTCGT gIFN-28 * (31) ATCTCTTTCTCTTACCGGTCTTAGCAGCTGG gIFN-29 * (32) AGAAGCTCTTCTACCTCTGAACAACATTTGAG gIFN-30 * (18) is TCGACGACGCTATTATTG The method of claim 1, wherein the cloning of the bacterial carrier is performed by first cloning the fragment of the restriction enzymes Hind III and Sal I among the oligonucleotides bound by the binding element according to the conjugation strategy, followed by the restriction enzymes Xba-1 and Hind. A method for producing human γ-interferon, characterized by cloning a fragment of III. The method for preparing human γ-interferon according to claim 1, wherein the three amino acids of the amino terminus, cysteine, tyrosine, and cysteine are removed by treating the carrier (pγ-IFN) with restriction enzymes BamHl and Sal I. The method of claim 1, wherein the cloning to the carrier (pBS-100) is treated with the restriction enzymes Sal I and Nco I to the carrier (pBS-100) after binding to the γ-interferon gene is removed cysteine, tyrosine, cysteine A method for producing human γ-interferon, characterized by cloning the synthetic linker. The method according to claim 1 or 5, wherein the synthetic linkage is

Method for producing human γ-interferon characterized in that the recognition site. The method of claim 1, wherein the cassette of the promoter- [gamma] -interferon-end gene is prepared by treating a carrier (pBS- [gamma] IFN) with a restriction enzyme BamHl. The method of claim 1, wherein the final yeast expression carrier (pYLBC-γIFN4) is prepared by combining the cassette of the promoter-γ-interferon-end gene after treatment of the carrier (pYLBC105) with restriction enzymes BamHl and phosphatase Method for preparing γ-interferon.

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