RU2764787C1 - RECOMBINANT PLASMID DNA ENCODING CHIMERIC INTERFERON ALPHA2b, RECOMBINANT STRAIN OF YEAST P. PASTORIS X33 - PRODUCER OF CHIMERIC INTERFERON ALPHA2b, AND METHOD FOR PRODUCING THIS PROTEIN - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and the pharmaceutical industry and can be used in creating antiviral medicinal products of prolonged action. Proposed is a chimeric human interferon alpha2b exhibiting antiviral activity, with an amino acid residue of 434, a molecular weight of 49.360 kDa and SEQ ID NO 1, including, from the N-terminus, the amino acid sequence of a mature human interferon alpha2b with an amino acid residue of 165, a molecular weight of 19.268 kDa and SEQ ID NO 2, fused, from the C-terminus, with the amino acid sequence of a linker with an amino acid residue of 20, a molecular weight of 1.469 kDa and SEQ ID NO 3, fused, in turn, from the C-terminus, with the amino acid sequence of mature human apolipoprotein A-I with an amino acid residue of 243, a molecular weight of 28.076 kDa and SEQ ID NO 4. Also proposed are a recombinant plasmid DNA pPICZ αA:IFNα2b-linker-ApoA-I, ensuring synthesis of said protein in cells of yeast Pichia pastoris X33, a recombinant strain of yeast Pichia pastorisX33 transformed by said recombinant plasmid DNA, and also proposed is a method for producing said protein.

EFFECT: inventions ensure production of a chimeric recombinant human interferon alpha2b of prolonged action exhibiting high biological activity.

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Description

The present invention relates to biotechnology and the pharmaceutical industry and can be used to create long-acting antiviral drugs.

Interferons are important components of the defense of vertebrates against infectious agents. Currently, more than twenty genetic varieties of human alpha interferons (IFN-α1a, -α1b, -α2b, -α4a, -α4b, -α5, -α6, etc.) have been isolated and characterized, differing in their primary structure, but possessing similar functional properties [Rubinstein M. The purification and characterization of alpha interferons and related cytokine receptors-a personal account // Cytokine Growth Factor Rev. 2007. V. 18. No. 5-6. P. 519-524; Emanuel S.L., Pestka, S.J. Human interferon-alpha A, -alpha 2, and -alpha 2(Arg) genes in genomic DNA // Biol. Chem. 1993. V. 268. No. 17. P. 12565-12569]. Varieties of alpha-interferons provide antiviral protection of the body, have antimicrobial, anti-inflammatory, immunomodulatory, antitumor and radioprotective effects [Baron S., Tyring S.K., Fleischmann W.R. Jr., Coppenhaver D.H., Niesel D.W., Klimpel G.R., Stanton G.J., Hughes T.K. The interferons. Mechanisms of action and clinical applications // JAMA. 1991. V. 266. No. 10. P. 1375-1383; Pestka S., Langer J.A., Zoon K.C., Samuel C.E. Interferons and their actions // Annu. Rev. Biochem. 1987. V. 56. P. 727-777; Chelbi-Alix M.K., Wietzerbin J. Interferon, a growing cytokine family: 50 years of interferon research // Biochimie. 2007. V. 89. No. 6-7. P. 713-771.]. In the human genome, the variant of the interferon alpha 2b locus is dominant [Gewert D., Salom C., Barber K., Macbride S., Cooper H., Lewis A., Wood J., Crowe S. Analysis of interferon-alpha 2 sequences in humangenomic DNA // J. Interferon Res. 1993. V. 13. No. 3. P. 227-231].

Mature human leukocyte interferon alpha 2b (IFNα2b) is a protein with a molecular weight of 19300 Da, consisting of 165 amino acid residues. The protein has two disulfide bonds formed by four cysteine residues at positions Cys1-Cys98 (first disulfide bond) and Cys29-Cys138 (second disulfide bond), the latter being important for the antiviral activity of IFNα2b.

The ability of alpha-interferons to have antiviral, immunomodulatory, antitumor effects have led to their widespread use in medicine as active ingredients of drugs. Most of these drugs contain IFNα2b and are used to treat hepatitis C [JP 2011173898 (A) Combination therapy comprising ribavirin and interferon alpha in antiviral treatment naive patient having chronic hepatitis C infection. 2011. A61K 38/21], oncological diseases: basal carcinomas [Application for invention US 5028422(A) Treatment of basal cell carcinoma intralesionally with recombinant human alpha interferon. 1991-07-02. A61K 38/21, A61K 37/66], Kaposi's sarcoma [Patent for the invention of the Russian Federation No. 2140270 Method for the treatment of idiopathic type of Kaposi's sarcoma. Published 10/27/1999. IPC A61K 31/495, A61K 38/21], bladder cancer [Patent for the invention of the Russian Federation No. 2257912 (C1) Method for the treatment of bladder cancer. Published 08/10/2005. IPC A61K 38/21, A61K 31/345, A61M 25/01, A61R 35/00].

In recent years, there has been an increase in the consumption of pharmaceutical preparations containing recombinant interferon as an active ingredient all over the world. This, in turn, dictates the need to increase the production of leukocyte interferon IFNα2b. However, interferons, like most other proteins with a small molecular weight, are unstable and easily degraded in the blood serum by proteases and, therefore, have a short half-life in the human body (1.5-2 hours).

There are several ways to increase the half-life of injectable recombinant interferons:

1) encapsulation of IFNα2b in polymer or lipid nanoparticles that provide slow and gradual release of the cytokine [Patent for the invention of the Russian Federation No. 2552851 Liposomal antiviral agent based on human interferon alpha-2b in encapsulated form for vaginal use. Published 06/10/2015. IPC A61K 9/48, A61K 9/12, A61K 38/21, A61K 47/48, A61P 31/12];

2) chemical modification of IFNα2b molecules with polyethylene glycol molecules (PEGylation), resulting in the formation of a PEG-modified protein molecule with fundamentally new physicochemical properties, having a higher molecular weight, high hydrophilicity, and resistance to proteolysis; pegylated interferon α-2b preparations have a half-life of 35 to 65 hours [Glue P., Fang JW, Rouzier-Panis R., Raffanel C., Sabo R., Gupta SK, Salfi M., Jacobs S. Pegylated interferon-alpha2b : pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data. Hepatitis C Intervention Therapy Group // Clin. Pharmacol. Ther. 2000. V. 68. No. 5. P. 556-567];

3) site-directed mutagenesis - replacement of protease-sensitive amino acids in the IFNα2b molecule, which reduces cytokine degradation in blood serum and increases its half-life [CN 107556376 (A) - 2018-01-09 Interferon alpha-2b mutant as well as preparation method and application thereof - A61K 38/21; A61P 31/12; C07K 14/56; C12N 15/70];

4) technology of genetic engineering creation of hybrid molecules containing chimeric interferon α2 (IFNα2), fused with long-term circulating blood proteins [Application for invention CN 101768601 (A) Method for producing recombinant human serum albumin-interferon alpha 2b. 2010. IPC A61P 31/14, C07K 1/16, C07K 1/18, C07K 1/20, C07K 19/00, C12N 15/81, C12P21/02, C12R1/84; RF Patent No. 2262510 Fusion protein with biological activity of interferon-alpha, dimeric fusion protein, pharmaceutical composition containing them, DNA molecule (variants) and method for addressing interferon-alpha in liver tissue. 2005. IPC C07K 14/715, C07K 14/52, C12N15/21, C12N 15/63, C12N 15/62, C12N 15/86, C12N15/863, A61K 38/21].

Disadvantages of encapsulation methods for recombinant interferons: low encapsulation rate, possible protein denaturation during encapsulation and, accordingly, loss of interferon biological activity [van de Weert M., Hennink WE, Jiskoot W. Protein instability in poly(lactic-co-glycolic acid) microparticles // Pharm. Res. 2000. V. 17. No. 10. P. 1159-1167. Review].

The disadvantages of pegylation technology are limited diffusion rates and low biological activity of pegylated interferons. In addition, the preparations are a mixture of many individual modifications, as well as a small amount of unmodified interferon, which leads to difficulties in its production. The technology of site-directed mutagenesis is associated with difficulties in designing the mutation site and predicting the nature of the mutant protein.

The production of chimeric proteins, including interferons, by genetic engineering methods is a relatively new and promising approach to increasing the half-life of these proteins, however, the biological activity and stability of chimeric interferons depends on the nature of the selected fusion protein with the interferon molecule and the choice of cell producing the chimeric cytokine.

When choosing a fusion protein with IFNα2b known use of serum albumin [Application for invention CN 101768601 (A) Method for producing recombinant human serum albumin-interferon alpha 2b. 2010. IPC A61P 31/14, C07K 1/16, C07K 1/18, C07K 1/20, C07K 19/00, C12N 15/81, C12P 21/02, C12R 1/84], immunoglobulin [Patent for the invention of the Russian Federation No. 2262510 Fusion protein with biological activity of interferon-alpha, dimeric fusion protein, pharmaceutical composition containing them, DNA molecule (options) and method for addressing interferon-alpha in liver tissue. Published October 20, 2005. IPC C07K 14/715, C07K 14/52, C12N 15/21, C12N 15/63, C12N 15/62, C12N 15/86, C12N 15/863, A61K 38/21].

The most common solution for obtaining chimeric interferon alpha of prolonged action is the use of human serum albumin (HSA) fused with it, produced by the yeast Kluyveromyces lactis [Osborn BL, Olsen HS, Nardelli B., Murray JH, Zhou JX, Garcia A., Moody G., Zaritskaya LS, Sung C. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys // J. Pharmacol. Exp.Ther. 2002. V. 303. No. 2. P. 540-548]. Also known recombinant interferons-alpha, fused with albumin, produced by the yeast Saccharomyces cerevisiae [Application for invention CN 101665798 (A) - 2010-03-10. Method for preparing recombinant human serum albumin and interferon alpha fusion protein. IPC C12N 15/62] or E. coli strain [Application for invention WO 2012048653 (A1) - 2012-04-19 Long-life interferon fusion protein and use thereof. 2012. IPC A61K 38/21; A61K 47/48; A61P 31/12; A61P 35/00; C07K 19/00; C12N 1/12; C12N 1/15; C12N 15/62; C12N 15/63; C12N 5/10 (24, 25)].

As mentioned above, the biological activity and stability of chimeric interferons depends not only on the nature of the selected fusion protein with the interferon molecule, but also on the choice of the producer cell.

The basis of modern biotechnological production of therapeutic proteins, both similar to natural ones and their chimeric forms, is the use of appropriate cultures of human and other mammalian cells, recombinant microorganisms as their producers.

The use of mammalian cells as producers of recombinant cytokines makes it possible to obtain glycosylated proteins that are closest in structure to human proteins, however, the process of culturing mammalian cells is lengthy (8-10 days), requiring special aeration conditions and maintenance of CO ₂ concentration, the use of expensive media, strict observance of sterile cultivation conditions, as well as the involvement of highly qualified personnel in the production.

Yeast & cerevisiae and K. lactis are characterized by excessive glycosylation of proteins, which leads to a significant decrease in their biological activity [Loren D. Schultz, Jerry Tanner, Kathryn J. Hofmann, Emilio A. Emini, Jon H. Condra, Raymond E. Jones, Elliott Kieff, Ronald W. Ellis. Expression and secretion in yeast of a 400-kda envelope glycoprotein derived from epstein-barr virus // Gene. 1987. V.54. no. 1. P. 13-123; Yeh P., Fleer R., Maury I., Mayaux J.-F. Secretion of naturally N-glycosylated human proteins in Kluyveromyces lactis. Abstract D14, 6th International Symposium on Genetics of Industrial Organisms, Strasbourg. 1990].

E. coli and P. pastor is expression systems are currently the most widely used for large scale production of various recombinant pharmaceutically important proteins. However, E. coli bacteria, as producers of therapeutic proteins, have a number of disadvantages, such as the inability to carry out post-translational modifications of proteins, in particular, human interferon IFNα2b, which are characteristic of eukaryotic organisms; accumulation of the bulk of this synthesized IFNα2b in inclusion bodies in the form of high molecular weight polymers [Babu KR, Swaminathan S., Marten S., Khanna N., Rinas U. Production of interferon-alpha in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins // Appl. microbiol. Biotechnol. 2000. V.53. no. 3. P. 655-660; Patent of the Russian Federation No. 2165455 Recombinant plasmid DNA pss5 encoding the synthesis of recombinant human alpha-2b interferon, Escherichia coli ss5 strain is a producer of recombinant human alpha-2b interferon and a method for producing interferon alpha-2b. 2001. IPC C12N 15/21, C12N 1/21, C12R 21/02, C12R 1/19; Valente S.A., Monteiro G.A., Cabral J.M., Fevereiro M., Prazeres D.M. Optimization of the primary recovery of human interferon alpha2b from Escherichia coli inclusion bodies // Protein Expr. Purif. 2005. V. 45. No. 1. P. 226-234], which significantly complicates the procedure for obtaining a recombinant protein [Gavrikov A.V., Ryazanov I.A., Kaluga V.E., Mashko S.V. Dependence of the procedure for isolation and purification of recombinant human interferon α-2b on the conditions of its accumulation in the cells of the producer strain during controlled cultivation // Biotechnology. 2006. No. 5. S. 23-31]; the presence of endotoxins in the synthesized protein, the complete release from impurities of which significantly complicates and increases the cost of the purification procedure for recombinant IFNα2b [Vu T.T., Jeong B., Krupa M., Kwon U., Song JA, Do BH, Nguyen MT, Seo T. , Nguyen AN, Joo CH, Choe H. Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag // J. Mol. microbiol. Biotechnol. 2016. V. 26. P. 359-368].

One of the most promising approaches to the production of biologically active recombinant eukaryotic interferons is the use of methylotrophic yeast Pichia pastoris cells for expression of the genes encoding them. The yeast P. pastoris is non-pathogenic for humans and animals and does not contain toxic and pyrogenic compounds, which makes it possible to use these microorganisms as producers of recombinant interferons used in clinical practice. Previously, the following advantages of P. pastoris as producers of other recombinant proteins have been shown, due to:

a) the use of integration vectors for transformation of yeast cells containing a strong regulated promoter of the methanol-induced alcohol oxidase (AOX1) gene [Cregg J.M., Madden K.R., Barringer K.J., Thill G.P., Stillman C.A. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris // Mol. Cell biol. 1989. V. 9. No. 3. P. 1316-1323];

b) high genetic stability of recombinant producer strains due to the insertion of the target genes cloned in them into the genome of yeast cells [Cregg J.M., Barringer K.J., Hessler A.Y., Madden K.R. Pichia pastoris as a host system for transformations // Mol. Cell biol. 1985. V. 5. No. 12. P. 3376-3385];

c) the possibility of obtaining multiple insertion of the target gene into the yeast cell genome and, as a result, a significant increase in the synthesis of the protein it encodes [Shen W., Shu M., Ma L., Ni H., Yan H. High level expression of organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly // Protein Expr. Purif. 2016. V. 119. P. 110-116; Shu M., Shen W., Yang S., Wang X., Wang F., Wang Y., Ma L. High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration // Enzyme Microb . Technol. 2016. V. 92. P. 56-66];

d) cultivation of yeast cells using inexpensive media and low energy consumption (cells grow at low temperatures of 25-28°C), achieving high cell densities (up to 100 g/l or 500 OE ₆₀₀ /ml) [Sreekrishna K., Potenz RH, Cruze JA, McCombie WR, Parker KA, Nelles L., Mazzaferro PK, Holden KA, Harrison RG, Wood PJ, et al. High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris // J. Basic. microbiol. 1988. V. 28. No. 4. P. 265-278; Stratton J., Chiruvolu V., Meagher M. High cell-density fermentation // Methods Mol. Biol. 1998. V. 103. P. 107-120].

e) the ability of P. pastoris to carry out such post-translational modifications of proteins as: proteolytic processing, folding, formation of disulfide bonds and glycosylation similar to that in humans [Pichia protocol. Higgins D.R., Cregg J.M. editors. // Methods in Molecular Biology. 1998. V.103. P. 1-270];

f) the use of special types of expression vectors that provide the target recombinant protein in a form secreted into the culture medium, which greatly simplifies the processes of isolation and purification of the target protein, which accounts for up to 80% of the total amount of proteins secreted by P. pastoris yeast [Tschopp JF, Sverlow G., Kosson R., Craig W. Grinna L. High-Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast, Pichia Pastoris // Bio/Technology. 1987. V. 5. P. 1305-1308; Chen X., Li J., Sun H., Li S., Chen T., Liu G., Dyson P. High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin / / Sc. Rep. 2017.7(1):14543. doi: 10.1038/s41598-017-15149-3];

g) a high level of synthesis and secretion of target recombinant proteins [Clare J.J., Rayment F.B., Ballantine S.P., Sreekrishna K., Romanos M.A. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene // Biotechnology (N Y). 1991. V. 9. No. 5. P. 455-460; Paifer E., Margolles E., Cremata J., Montesino R., Herrera L., Delgado J.M. Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences // Yeast. 1994. 10. no. 11. P. 1415-1419].

In the world patent literature, the following patents are known for the creation of recombinant strains of Pichia pastoris - producers of chimeric human IFNα2b, which are analogues of the claimed invention.

A known method of obtaining a strain of P. pastoris GS115 - a producer of chimeric interferon alpha, including IFNα2b, with human serum albumin (IFNα2b-HSA), with a prolonged action. The proteins in the chimera are connected by a (GlySer)n-linker [Application for invention CN 101200503 (A) - 2008-06-18 Fusion protein for seralbumin and interferon. IPC C07K 14/76, C07K 19/00, C12N 1/19, C12N 15/62, C12N 15/63]. The disadvantage of this invention is the production of synthetic genes for interferon and albumin using cDNA technology, i.e. by synthesizing the coding regions of these genes on natural mRNA templates by reverse transcription. Genes thus obtained contain a codon composition that is not optimal for expression in P. pastoris yeast cells. cDNA technology reduces the yield of the target protein, which, as a result, leads to an increase in the percentage of the part of the target protein degraded by proteases, and, accordingly, to a decrease in its biological activity.

A known method of obtaining a strain of P. pastoris KM71 - producer of chimeric interferon alpha 2 with human serum albumin (IFNα2-HSA) with prolonged action [Application for invention CN 1831124 (A) - 2006-09-13 Method for preparing fusion protein contg. human interferon-alpha 2 and human seralbumin and its products. 2006. IPC C07K 14/56, C07K 14/765, C07K 19/00, C12N 1/19, C12N 15/09, C12N 15/14, C12N 15/21, C12N 15/62]. The invention does not provide data on the quantitative yield of the protein and its specific biological activity, however, the direct connection of two synthetic genes IFNα2 and HSA without the use of binding linkers may adversely affect the biological activity of the chimeric protein.

A known method of obtaining a strain of P. pastoris GS115 producer of chimeric interferon alpha 2b with human serum albumin (IFNα2b-4CA). The genes encoding chimeric interferon are linked by a nucleotide sequence encoding a GlySer linker [Application for invention CN 1807646 (A) - 2006-07-26 Production method of recombinant fusion protein of human serum albumin-interferon alpha 2b. IPC C12N 1/19, C12N 15/14, C12N 15/21, C12N 15/62]. The disadvantage of analogue is the low specific biological activity of the chimeric protein, amounting to 3.0×10 ⁶ IU/mg.

The closest analogue, adopted as a prototype, is a method for obtaining a strain of P. pastoris X33, transformed by recombinant plasmid DNA encoding a chimeric human interferon alpha 2b of prolonged action, including the nucleotide sequence of the human interferon alpha 2b gene (IFNα2b) and the human serum albumin gene (HSA ). The IFNα2b and HSA genes are linked in plasmid DNA by a nucleotide sequence encoding a (GlySer)n-linker [Application for invention CN 101768601 (A) Method for producing recombinant human serum albumin-interferon alpha 2b. 2010. IPC A61P 31/14, C07K 1/16, C07K 1/18, C07K 1/20, C07K 19/00, C12N 15/81, C12P 21/02, C12R 1/84]. The disadvantage of the prototype is the low antiviral activity of the chimeric protein, amounting to 1.5×10 ⁶ IU/mg. This, apparently, is due to the fact that albumin, which has a large molecular weight, can affect the conformation of IFNα2b fused with it and, as a consequence, lead to a significant decrease in the specific biological activity of the latter.

The objective of the present invention is to create, by genetic engineering methods, a producer strain containing a plasmid encoding a chimeric interferon a2b of prolonged action, having a higher antiviral activity, and a method for obtaining the specified protein.

The technical result is the construction of a recombinant plasmid DNA that provides the expression of a gene of chimeric recombinant human interferon alpha2b of prolonged action, having a higher antiviral activity, the creation of a yeast strain Pichia past oris X33, transformed with the specified plasmid DNA, a producer of chimeric human interferon alpha2b, including amino acid sequences of interferon alpha2b human and human apolipoprotein AI, and a method for producing said protein.

Disclosure of the essence of the invention

A group of inventions is claimed, including a chimeric human alpha2b interferon with antiviral activity; recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I, providing synthesis of chimeric interferon alpha2b human in Pichia pastoris X33 yeast cells; recombinant yeast strain Pichia pastoris X33, transformed with recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I, producing chimeric human interferon alpha2b; a method for producing chimeric human interferon alpha2b.

Chimeric human interferon alpha2b (hereinafter IFNα2b-linker-ApoA-I), 434 a.a., molecular weight 49.360 kDa (SEQ ID NO 1), which has antiviral activity, includes the amino acid sequence of mature human interferon alpha2b from the N-terminus (hereinafter - IFNα2b), 165 a.a., molecular weight 19.268 kDa (SEQ ID NO 2), fused at the C-terminus to the amino acid sequence of the linker, 20 a.a., molecular weight 1.469 kDa (SEQ ID NO 3), which, in in turn, fused at the C-terminus with the amino acid sequence of mature human apolipoprotein AI (hereinafter referred to as apoA-1), 243 aa, molecular weight 28.076 kDa (SEQ ID NO 4).

Recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I, 4792 bp in size, which ensures the synthesis of chimeric human interferon alpha2b (IFNα2b-linker-ApoA-I) in yeast Pichia pastoris X33 cells (IFNα2b-linker-ApoA-I), contains the AOX-1 promoter in the direction from left to right size 940 b.p. (numbers of base pairs in the nucleotide sequence of the plasmid 1-940 bp); a fragment of the MFα gene of the yeast Saccharomyces cerevisiae, 249 bp in size. (bp numbers 941-1189) encoding the a-factor secretion signal and allowing the secretion of IFNα2b-linker-ApoA-I into the culture medium; a synthetic gene for chimeric human interferon alpha2b (SEQ ID NO 1) 1296 bp in size, including a gene for mature human interferon alpha2b (SEQ ID NO 2) 495 bp in size. (bp numbers 1208-1702), linker gene (SEQ ID NO 3) 60 bp in size. (bp numbers 1709-1768 bp) and the mature ApoA-I gene (SEQ ID NO 4) 729 bp in size. (bp numbers 1781-2509 bp); transcription terminator of the AOX-1 gene, 342 bp in size. (base pair numbers 2540-2881); 412 bp TEF1 gene promoter (base pair numbers 2882-3293); EM7 prokaryotic promoter, 68 bp in size. (bp numbers 3294-3361 bp); the 375 bp neomycin transferase Zeo(R) gene conferring resistance to zeocin (bp numbers 3362-3736); transcription terminator of the cytochrome C1 (CYC1) gene, 318 bp in size. (base pair numbers 3737-4054); pUC plasmid origin of replication 674 bp (base pair numbers 4065-4738)

Recombinant yeast strain Pichia pastoris X33/pPICZαA:IFNα2b-linker-ApoA-I - producer of chimeric interferon IFNα2b-linker-ApoA-I (SEQ ID NO 1) was obtained by transformation of yeast Pichia pastoris X33 with linearized plasmid DNA pPICZαA:IFNα2b-linker-ApoA -I, inserted as a result of recombination into the genome of transformed yeast.

Method for obtaining chimeric interferon IFNα2b-linker-ApoA-I (SEQ ID NO 1) includes microbiological synthesis of this protein during cultivation of the yeast strain Pichia pastoris X33/pPICZαA:IFNα2b-linker-ApoA-I; sedimentation of cells of the specified strain with separation of the supernatant by centrifugation; precipitation from the supernatant of chimeric interferon IFNα2b-linker-ApoA-I with ammonium sulfate; suspending and dissolving the precipitate of this protein; purification of IFNα2b-linker-ApoA-I by reverse-phase chromatography in an acetonitrile gradient and subsequent dialysis of this purified protein.

List of figures, drawings and other materials

Fig. 1. Comparative analysis of the nucleotide sequences of the native mature human IFNα2b gene from nucleotides 1 to 495 and the corresponding synthetic mature human IFNα2b gene optimized for expression in P. pastoris yeast cells.

Designations: IFN-W - native mature human IFNα2b gene from nucleotides 1 to 495, IFN-Pp - synthetic mature human IFNα2b gene from nucleotides 1 to 495.

Underlined are synonymous nucleotide substitutions in the synthetic gene compared to the native human IFNα2b gene (120 substitutions in total).

Fig. 2. Physical map of the recombinant plasmid pJ201-mature-ApoA-I

pUC ori - pUC plasmid origin of replication (bp 41-844); rpn txn terminator - transcription terminator of the rpn txn gene (1016-1129 bp); bla txn terminator of the bla gene (bp 1136-1436); pTF3 - TF3 gene promoter (bp 1320-1345); mature apoA-I - chemically synthesized gene of mature human apoA-I (bp 1479-2207); pTR - promoter (bp 2346-2362); rrnB1 B2 txn terminator - transcription terminator of the B1 and B2 genes (bp 2263-2437); Kanamycin-R - neomycin phosphotransferase II gene (bp 2622-3416).

Fig. 3. Physical map of recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I

AOX1 promoter - alcohol oxidase gene promoter region (bp 1-940); Alpha factor signal - a fragment of the MFα gene of the yeast Saccharomyces cerevisiae, encoding a signal for the secretion of the alpha factor (941-1189 bp); IFNα2b-linker-ApoA-I is a 1296 bp synthetic chimeric interferon gene containing the nucleotide sequence of the mature human alpha2-b interferon gene (1208-1702 bp) joined by an oligonucleotide linker (Linker) (1709-1768 bp) with the mature human apoA-I gene (1208-2509 bp); AOX1 TT - transcription terminator of the AOX1 gene (2540-2881 bp); TEF1 - promoter of the translation elongation factor gene from Saccharomyces cerevisiae (2882-293); EM7 - prokaryotic promoter (bp 3294-3361); the Zeo(R) gene, the product of which provides resistance to the antibiotic Zeocin (bp 3362-3736); CYC1 transcription terminator - cytochrome C1 gene transcription terminator region (bp 3737-4054); pUC origin - origin of pUC replication (4065-4738 bp).

Fig. Fig. 4. Electropherogram of proteins present in culture liquids of Pichia pastoris clones analyzed for the production of chimeric interferon IFNα2b-linker-ApoA-I after 6 days of cultivation in BMGY medium on an orbital shaker in a 96-well plate.

Clones were induced with 1% methanol (v/v) for 96 h at 28°C. Electrophoresis was carried out in 12% SDS-PAGE plates. Lanes: 1 - Fermentas protein molecular weight marker (12-116 kDa); 2-10 - proteins in the culture fluids of the analyzed methanol-induced clones.

Fig. 5. Western blot analysis of IFNα2b-linker-ApoA-I chimeric interferon produced by one of the clones of Pichia pastoris.

Electrophoresis was carried out in 12% SDS-PAGE plates. Lanes: 1 - protein molecular weight marker (Bio-Rad) (10-250 kDa); 2 - chimeric interferon IFNα2b-linker-ApoA-I.

Fig. 6. Electropherogram of purified chimeric interferon IFNα2b-linker-ApoA-I.

Electrophoresis was carried out in 12% SDS-PAGE plates. Lanes: M - protein molecular weight marker (Sib Enzyme) (10-200 kDa); 1 - chimeric interferon IFNα2b-linker-ApoA-I.

Fig. 7. Pharmacokinetic curves of IFNα2b and IFNα2b-linker-ApoA-I concentrations in blood sera after their single subcutaneous administration to CD-I male mice at doses of 10 µg/kg and 25 µg/kg, respectively.

Animals were injected with equimolar amounts of IFNα2b and IFNα2b-linker-ApoA-I. Numerical data are presented as mean ± standard deviation (n=5).

Implementation of the invention

Below are examples of a specific implementation of a group of inventions.

Example 1 Design and synthesis of IFNα2b-linker and ApoA-I protein genes

Example 1a. Design and synthesis of the IFNα2b-linker chimeric protein gene

At the first stage, a theoretical calculation of the nucleotide sequence of the projected gene encoding human IFNα2b fused with the nucleotide sequence encoding the oligopeptide linker is carried out in order to develop an optimal composition of degenerate codons for expression in P. pastoris cells and exclude other factors that affect the efficiency of gene expression at the level transcription and translation. The work is carried out using three computer programs: using the Gene designer computer program (ATOM, USA), the VisualGeneDeveloper software package (http://www.visualgenedeveloper.net/Download.html) and the GeneOptimizer™ program from Invitrogen. As a result of computer calculations, the primary structure of the artificial mature human IFNα2b gene fused to the nucleotide sequence of the linker (IFNα2b-linker) optimized for expression in P. pastoris cells is developed. In addition, the restriction sites necessary for cloning are included in the composition of the artificial gene. In FIG. Figure 1 shows the nucleotide sequence of a projected mature human IFNα2b gene optimized for expression in the yeast P. pastoris compared to the nucleotide sequence of the natural gene for this protein. As can be seen from FIG. 1, the artificial IFNα2b gene has 120 nucleotide substitutions in the coding region of the gene relative to the natural IFNα2b gene. The marked substitutions are synonymous, i.e. do not lead to the replacement of amino acid residues encoded by the corresponding codons. Next, chemical-enzymatic synthesis of the artificial IFNα2b-linker gene is carried out, including the mature IFNα2b gene fused at the 3'-end with the nucleotide sequence of the linker, followed by the EcoRI restriction site, and the 5'-terminal region of the gene contains a sequence encoding sites of hydrolysis by proteases Ste13 and Kex2 and XhoI restriction site.

Example 16 Amplification of the apoA-I gene by PCR

The nucleotide sequence of the human apoA-I gene (SEQ ID NO 4) is 729 bp in size. synthesized by PCR using forward 373-F: 5'-GA GAATTC GATGAGCCACCACAGTCCC-3' and reverse 431-R: 5'-ACGG CTCGAG TCATTGCGTGTTTAACTTCTTAGTGTACTC-3' primers on the DNA of plasmid pJ201-mature-ApoA-I used as a template (Fig. 2). Plasmid pJ201-mature-ApoA-I contains the mature human apoA-I gene previously optimized for expression in Pichia pastoris cells [Mamaev A.L., Beklemishev A.B. Cloning and analysis of the expression of synthetic human apolipoprotein A1 genes in Escherichia coli and methylotrophic yeast Pichia pastoris // Bul. SO RAMN. 2014. V. 34. No. 5. S. 37-49] (Example 1). The primers used to amplify the mature apoA-I gene contain the EcoRI and XhoI restriction sites at the 5' ends, which are necessary for subsequent cloning of the amplicon in the pPICZαA vector at the EcoRI and SaiI sites (Example 1).

Example 2 Construction of the claimed recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I

First, the pPICZαA:ApoA-I plasmid is constructed. The mature human apoA-I gene amplicon is digested with XhoI and EcoRI and ligated with the plasmid vector pPICZαA digested at the EcoRI and SaiI sites. The ligation mixture transforms the electrocompetent cells of E. coli pcs. TOP10 and then grown and selected transformants on LB selective low-salt medium containing 25 μg/ml zeocin. Grown clones are analyzed for the presence of the target recombinant plasmid pPICZαA:ApoA-I by colony PCR. PCR is carried out in the presence of a pair of primers: forward 423F (5'-TACTATTGCCAGCATTGCTGC-3') and reverse 424R (5'-GCAAATGGCATTCTGACATCC-3'), specific for flanking the mature human apoA-I gene regions of the pPICZαA:ApoA-I recombinant plasmid. The size of the amplicons is determined by electrophoresis in 0.8% agarose gel. One of the PCR-positive clones is used for the preparative production of the pPICZαA:ApoA-I plasmid.

At the next stage, the synthesized chimeric IFNα2b-linker gene is hydrolyzed at the EcoRI and XhoI sites and ligated with the pPICZαA:ApoA-I plasmid, previously digested with restriction enzymes at the same sites. Received ligase mixture transform cells E. coli piece. TOP10. Then, transformants are grown and selected on selective low-salt LB medium containing 25 μg/ml zeocin. Grown clones are analyzed for the presence of the recombinant plasmid by colony PCR in the presence of forward 423F and reverse 424R primers. The size of the amplicons is determined by electrophoresis in 0.8% agarose gel. One of the PCR-positive clones was used for the preparative production of recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I (Fig. 3) by alkaline lysis.

Example 3 Transformation of the yeast P. pastoris X33 with recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I

The isolated recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I is treated with BstXI restriction enzyme and its linearized form is used to transform P. pastoris X33 electrocompetent yeast cells. Transformed yeast cells are seeded and selected on YPD selective agar medium containing 500 and 2000 μg/ml Zeocin. The selected colonies are grown in flasks with baffles for 2 days on an orbital shaker at 300 rpm in BMGY medium, after which methanol is added to cultures daily for 2 to 3 days up to 1%. At the end of the induction, the cells are pelleted by centrifugation (3500g, 20 min, +4°C). Proteins from supernatants with a volume of 1-0.5 ml are precipitated with 10% trichloroacetic acid. Protein precipitates were washed with acetone and analyzed by denaturing electrophoresis on a 12% polyacrylamide gel (SDS-PAGE) (FIG. 4). The clone producing the largest amount of the target recombinant IFNα2b-linker-ApoA-I chimeric interferon is selected for the implementation of the method for obtaining the specified protein.

Example 4. Method for obtaining chimeric interferon IFNα2b-linker-ApoA-I

P. pastoris X33 clone transformed with linearized plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I and producing the highest amounts of recombinant chimeric interferon IFNα2b-linker-ApoA-I is used to implement the claimed method for producing chimeric interferon IFNα2b-linker-ApoA-I . Clone cultivation is carried out in conical flasks with a volume of 250-300 ml with baffles, containing 50 ml of BMGY medium on an orbital shaker at 28°C and 250 rpm for 2 days. Before induction, 0.2% (w/v) of Tween 20 detergent is added to the flasks. Microbiological synthesis of chimeric interferon IFNα2b-linker-ApoA-I is induced daily by adding methanol to flasks with recombinant yeast cultures to a final concentration of 1%. On the 4th day after induction, the cells are pelleted by centrifugation at +5°C, 3750xg for 25 min, with the separation of the supernatant.

Recombinant chimeric interferon IFNα2b-linker-ApoA-I is precipitated from the supernatant with ammonium sulfate. To do this, ammonium sulfate powder is added to the main part of the supernatant to 50% saturation and incubated for 12-16 hours at +4°C. The culture liquid with the formed precipitates is centrifuged at 39000xg for 30 min at +4°C. Precipitates containing chimeric interferon IFNα2b-linker-ApoA-I, after ammonium sulfate precipitation, are suspended and dissolved in 0.02% Tween 20, pH 4.5. The resulting suspension is centrifuged for 5 min at 12000 rpm to obtain a homogeneous clarified supernatant, which is used for further chromatographic purification of chimeric interferon IFNα2b-linker-ApoA-I (Example 5).

The presence of the apoA-I amino acid sequence in the IFNα2b-linker-ApoA-I chimeric interferon was confirmed by immunoblot analysis. For this, proteins secreted by the selected yeast clone and standard biotinylated marker proteins were separated by electrophoresis in 12% PAAG followed by transfer to a nitrocellulose membrane (Millipore, USA). The membrane was incubated for 1 h at room temperature in buffer A (50 mM sodium phosphate, pH 7.0, 150 mM sodium chloride, 5% (w/v) BSA) to block non-specific binding of the membrane to antibodies and conjugate. The membrane was then incubated for 1 h at room temperature in buffer A containing primary rabbit antibodies against human apoA-I, and after washing with buffer A, incubated for 1 h with horseradish peroxidase-labeled goat anti-rabbit immunoglobulin antibodies (dilution 1:500). Proteins on the membrane were visualized with a mixture of 4-chloro-1-naphthol chromogenic dye and H ₂ O ₂ solution. In FIG. 5 shows the results of immunoblotting.

Example 5 Chromatographic Purification of Chimeric Interferon IFNα2b-linker-ApoA-I

Proteins from the clarified supernatant (Example 4) are purified by reverse phase chromatography with an acetonitrile gradient. TFA is added to the supernatant to a final concentration of 0.3% and applied to a chromatographic column. The column was washed with a buffer containing 20% acetonitrile, 0.1% TFA. The target protein is eluted from the column with a solution containing 0.1% TFA and increasing amounts (gradient) of acetonitrile (20%-80%). Fractions containing maximally purified chimeric interferon are pooled, dialyzed against a buffer containing 10 mM sodium phosphate, 0.02% Tween 20, 1 mM EDTA, pH 7.4, and analyzed by denaturing electrophoresis in 12% SDS-PAGE. The purity of the final preparation of chimeric interferon IFNα2b-linker-ApoA-I with the described purification method is ~90-93% according to polyacrylamide gel densitometry (Fig. 6). The concentration of the purified protein is measured spectrophotometrically in UV absorption at a wavelength of 280 nm, taking into account the molar extinction coefficient, as well as polyacrylamide gel densitometry using Gel Pro programs, etc.

Example 6. Determination of antiviral activity of chimeric interferon IFNα2b-linker-ApoA-I

The antiviral activity of the chimeric interferon IFNα2b-linker-ApoA-I is determined on a continuous bovine kidney cell line (MDBK) infected with the equine vesicular stomatitis virus, in accordance with the instructions described in the General Pharmacopoeia Monograph (OFS.1.7.2.0002L 5) "Biological methods testing of interferon preparations using cell cultures”. The magnitude of antiviral activity is assessed by the ability of chimeric interferon IFNα2b-linker-ApoA-I to suppress the cytopathic effect of the vesicular stomatitis virus of horses (VSV, GKV No. 600, Indiana strain, deposited in the State Collection of Viruses of the D.I. Ivanovsky Research Institute of Virology) on MDBK cells. The virus is introduced at a dose corresponding to 100 TCID 50/0.1 ml (50% of tissue cytopathic infectious doses). In the above example, it was found that the chimeric interferon IFNα2b-linker-ApoA-I increased antiviral activity in a dose-dependent manner. According to the results of the analysis, a sample of chimeric interferon IFNα2b-linker-ApoA-I has an antiviral activity of 1.6*10 IU/mg, which corresponds to that of the European standard interferon preparation.

Example 7. Analysis of the pharmacokinetics of chimeric interferon IFNα2b-Iinker-ApoA-1

The pharmacokinetics of chimeric interferon IFNα2b-linker-ApoA-I was studied in comparison with that of authentic recombinant human IFNα2b. The study is carried out on groups of laboratory mice of the CD1 line. Animals are weighed and randomly assigned to 2 experimental groups. Mice of the first experimental group are injected subcutaneously with 200 μl of phosphate-buffered saline (PBS) containing 1 μg / ml of IFNα2b (10 μg / kg of mouse), mice of the second experimental group - 200 μl of PBS containing 2.5 μg / ml of IFNα2b- linker-ApoA-I (25 µg/kg mouse), 5 mice per group. Mice of the control group are injected with 200 μl of FSB. Blood is taken from animals anesthetized with ether immediately after decapitation, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after the introduction of the solutions into tubes with heparin. To obtain blood plasma, the samples are centrifuged for 15 minutes at 3000 rpm. Samples are frozen at -70°C until analysis. The content of the studied proteins in plasma is determined by the method of enzyme-linked immunosorbent assay.

taking into account the molar extinction coefficient, as well as polyacrylamide gel densitometry using Gel Pro programs, etc.

Example 6. Determination of antiviral activity of chimeric interferon IFNα2b-linker-ApoA-I

The antiviral activity of the chimeric interferon IFNα2b-linker-ApoA-I is determined on a continuous bovine kidney cell line (MDBK) infected with the equine vesicular stomatitis virus, in accordance with the instructions described in the General Pharmacopoeia Monograph (OFS.1.7.2.0002L 5) "Biological methods testing of interferon preparations using cell cultures”. The magnitude of antiviral activity is assessed by the ability of chimeric interferon IFNα2b-linker-ApoA-I to suppress the cytopathic effect of the vesicular stomatitis virus of horses (VSV, GKV No. 600, Indiana strain, deposited in the State Collection of Viruses of the D.I. Ivanovsky Research Institute of Virology) on MDBK cells. The virus is introduced at a dose corresponding to 100 TCID 50/0.1 ml (50% of tissue cytopathic infectious doses). In the above example, it was found that the chimeric interferon IFNα2b-linker-ApoA-I increased antiviral activity in a dose-dependent manner. According to the results of the analysis, a sample of chimeric interferon IFNα2b-linker-ApoA-I has an antiviral activity of 1.6*10 IU/mg, which corresponds to that of the European standard interferon preparation.

Example 7. Analysis of the pharmacokinetics of chimeric interferon IFNα2b-Iinker-ApoA-1

The pharmacokinetics of chimeric interferon IFNα2b-linker-ApoA-I was studied in comparison with that of authentic recombinant human IFNα2b. The study is carried out on groups of laboratory mice of the CD1 line. Animals are weighed and randomly assigned to 2 experimental groups. Mice of the first experimental group are injected subcutaneously with 200 μl of phosphate-buffered saline (PBS) containing 1 μg / ml of IFNα2b (10 μg / kg of mouse), mice of the second experimental group - 200 μl of PBS containing 2.5 μg / ml of IFNα2b- linker-ApoA-I (25 µg/kg mouse), 5 mice per group. Mice of the control group are injected with 200 μl of FSB. Blood is taken from animals anesthetized with ether immediately after decapitation, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after the introduction of the solutions into tubes with heparin. To obtain blood plasma, the samples are centrifuged for 15 minutes at 3000 rpm. Samples are frozen at -70°C until analysis. The content of the studied proteins in plasma is determined by the method of enzyme-linked immunosorbent assay.

In addition, apoA-I is the main HDL protein and has many properties in the body, such as anti-atherogenic [Rubin E.M., Krauss R.M., Spangler E.A., Verstuyft J.G., Clift S.M. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI // Nature. 1991. V. 353. No. 6341. P. 265-267], anti-inflammatory [Beck W.H., Adams C.P., Biglang-Awa I.M., Patel A.B., Vincent H., Haas-Stapleton E.J., Paul M.M. weers. Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: role for lysine residues in antimicrobial properties // Biochim. Biophys. acta. 2013. V. 1828. No. 6. P. 1503-1510], antioxidant [Garner B., Waldeck A.R., Witting P.K., Rye K.A., Stacker R. Oxidation of highdensity lipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residues of apolipoproteins AI and All // J. Biol. Chem. 1998. V. 273. No. 11. P. 6088-6095]. It has been shown that a decrease in the level of apoA-I in the body directly correlates with the development of a number of oncological diseases [Mao, M., Wang, X., Sheng, N., Liu, Y., Zhang, L., Dai, S., Chi, PD A novel score based on serum apolipoprotein A-l and C-reactive protein is a prognostic biomarker in hepatocellular carcinoma patients // BMC Cancer. 2018. V. 18. P. 1178; Lin X., Hong S., Huang J., Chen Y., Chen Y., Wu Z. Plasma apolipoprotein AI levels at diagnosis are independent prognostic factors in invasive ductal breast cancer // Discov. Med. 2017. V. 23. No. 127. P. 247-258; Shi H., Huang H., Pu J., Shi D., Ning Y., Dong Y., Han Y., Zarogoulidis P., Bai C. Decreased pretherapy serum apolipoprotein AI is associated with extent of metastasis and poor prognosis of non-small-cell lung cancer // Onco Targets Ther. 2018. V. 11. P. 6995-7003]. In addition, apoA-I is able to interact with phospholipids and spontaneously form lipoprotein particles, which can further protect transported therapeutic agents. It is assumed that the use of apoA-I as a protector in the construction of the claimed chimeric interferon will allow combining the immunological activity of IFNα2b with the pharmacokinetic advantages of apoA-I.

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List of nucleotide and amino acid sequences

<110> Beklemishev Anatoly Borisovich, Pykhtina Maria Borisovna

<120> Recombinant plasmid DNA encoding chimeric interferon alpha2b,

recombinant yeast strain P. pastoris X33 - producer of chimeric

interferon alpha2b and a method for obtaining said protein

<160> 4

<210> 1

<211> 1302

<212> DNA

<213> Pichia Pastoris

<400> 1

tgc gat cta ccc caa act cac agt ttg ggt agt cgt aga aca cta atg 48

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

ctt ttg gct cag atg agg cgt atc tct ctc ttc agc tgt ctg aaa gac 96

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

aga cat gat ttc ggt ttt cct caa gag gaa ttt ggt aat caa ttc caa 144

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

aag gca gag act att cct gtg ctg cat gaa atg att caa caa atc ttt 192

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

aac ttg ttc tcg acc aag gat tcc tcc gcc gct tgg gac gaa aca tta 240

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

ctt gac aaa ttc tac act gaa ctg tat cag caa ctt aat gac ttg gag 288

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

gct tgt gta att caa gga gtg gga gtt acg gaa act cca tta atg aaa 336

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

100 105 110

gag gat tct ata ttg gca gtt aga aag tat ttt cag cgt atc aca tta 384

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

tac ttg aag gag aaa aag tac tca cca tgt gca tgg gag gtc gtt agg 432

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

gct gaa atc atg aga tcc ttt tca ctg tct acc aac ttg cag gaa agc 480

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

ttg aga tcc aaa gag ggt acc gga tcc tcc ggc agc ggt gga tcc tca 528

Leu Arg Ser Lys Glu Gly Thr Gly Ser Ser Gly Ser Gly Gly Ser Ser

165 170 175

gga tcg ggt tcc ggt agt agt ggt gga tct ggt gaa ttc gct agc gat 576

Gly Ser Gly Ser Gly Ser Ser Gly Gly Ser Gly Glu Phe Ala Ser Asp

180 185 190

gag cca cca cag tcc cct tgg gac cgt gtt aag gac ctg gca act gta 624

Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val

195 200 205

tat gtt gac gtg ttg aag gat tca ggc aga gac tac gtt tct caa ttc 672

Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe

210 215 220

gaa ggt tcc gca cta gga aaa cag ctg aac ttg aaa ctt ttg gac aat 720

Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn

225 230 235 240

tgg gat tct gtc act tct aca ttt tct aaa cta aga gaa cag tta ggt 768

Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly

245 250 255

cca gtt acc caa gag ttc tgg gat aat ctt gaa aaa gaa acc gaa ggt 816

Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly

260 265 270

ttg aga caa gag atg tct aag gat ttg gag gag gtt aaa gct aag gtg 864

Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val

275 280 285

caa ccc tac tta gat gac ttt caa aag aaa tgg cag gaa gaa atg gag 912

Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu

290 295 300

cta tat cga caa aag gta gaa cca ctt cgt gca gag ctg caa gaa ggg 960

Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly

305 310 315 320

gcc aga caa aag cta cat gaa ttg caa gag aaa ctg tca cct ttg gga 1008

Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly

325 330 335

gaa gaa atg agg gac cgt gct aga gcc cat gtc gat gca tta cga acg 1056

Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr

340 345 350

cac ctg gcc ccc tac agt gat gag ctc aga caa agg tta gcc gct agg 1104

His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg

355 360 365

ttg gag gct ctt aag gag aat gga ggt gcc aga ctt gct gag tat cat 1152

Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His

370 375 380

gct aag gct aca gaa cac cta tct aca tta agt gaa aaa gct aag cca 1200

Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro

385 390 395 400

gct ctg gag gat ttg aga cag ggc ctt tta cct gtc ttg gaa tcc ttt 1248

Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe

405 410 415

aag gtg tcc ttc ttg tca gct ttg gaa gag tac act aag aag tta aac 1296

Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn

420 425 430

acg caa 1302

Thr Gln

<210> 2

<211> 495

<212> DNA

<213> Pichia Pastoris

<400> 2

tgc gat cta ccc caa act cac agt ttg ggt agt cgt aga aca cta atg 48

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

ctt ttg gct cag atg agg cgt atc tct ctc ttc agc tgt ctg aaa gac 96

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

aga cat gat ttc ggt ttt cct caa gag gaa ttt ggt aat caa ttc caa 144

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

aag gca gag act att cct gtg ctg cat gaa atg att caa caa atc ttt 192

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

aac ttg ttc tcg acc aag gat tcc tcc gcc gct tgg gac gaa aca tta 240

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

ctt gac aaa ttc tac act gaa ctg tat cag caa ctt aat gac ttg gag 288

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

gct tgt gta att caa gga gtg gga gtt acg gaa act cca tta atg aaa 336

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

100 105 110

gag gat tct ata ttg gca gtt aga aag tat ttt cag cgt atc aca tta 384

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

tac ttg aag gag aaa aag tac tca cca tgt gca tgg gag gtc gtt agg 432

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

gct gaa atc atg aga tcc ttt tca ctg tct acc aac ttg cag gaa agc 480

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

ttg aga tcc aaa gag 495

Leu Arg Ser Lys Glu

165

<210> 3

<211> 60

<212> DNA

<213> Pichia Pastoris

<400> 3

gga tcc tcc ggc agc ggt gga tcc tca gga tcg ggt tcc ggt agt agt 48

Gly Ser Ser Gly Ser Gly Gly Ser Ser Gly Ser Gly Ser Gly Ser Ser

1 5 10 15

ggt gga tct ggt 60

Gly Gly Ser Gly

twenty

<210> 4

<211> 729

<212> DNA

<213> Pichia Pastoris

<400> 4

gat gag cca cca cag tcc cct tgg gac cgt gtt aag gac ctg gca act 48

Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr

1 5 10 15

gta tat gtt gac gtg ttg aag gat tca ggc aga gac tac gtt tct caa 96

Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln

20 25 30

ttc gaa ggt tcc gca cta gga aaa cag ctg aac ttg aaa ctt ttg gac 144

Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp

35 40 45

aat tgg gat tct gtc act tct aca ttt tct aaa cta aga gaa cag tta 192

Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu

50 55 60

ggt cca gtt acc caa gag ttc tgg gat aat ctt gaa aaa gaa acc gaa 240

Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu

65 70 75 80

ggt ttg aga caa gag atg tct aag gat ttg gag gag gtt aaa gct aag 288

Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys

85 90 95

gtg caa ccc tac tta gat gac ttt caa aag aaa tgg cag gaa gaa atg 336

Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met

100 105 110

gag cta tat cga caa aag gta gaa cca ctt cgt gca gag ctg caa gaa 384

Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu

115 120 125

ggg gcc aga caa aag cta cat gaa ttg caa gag aaa ctg tca cct ttg 432

Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu

130 135 140

gga gaa gaa atg agg gac cgt gct aga gcc cat gtc gat gca tta cga 480

Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg

145 150 155 160

acg cac ctg gcc ccc tac agt gat gag ctc aga caa agg tta gcc gct 528

Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala

165 170 175

agg ttg gag gct ctt aag gag aat gga ggt gcc aga ctt gct gag tat 576

Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr

180 185 190

cat gct aag gct aca gaa cac cta tct aca tta agt gaa aaa gct aag 624

His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys

195 200 205

cca gct ctg gag gat ttg aga cag ggc ctt tta cct gtc ttg gaa tcc 672

Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser

210 215 220

ttt aag gtg tcc ttc ttg tca gct ttg gaa gag tac act aag aag tta 720

Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu

225 230 235 240

aac acg caa 729

Asn Thr Gln

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Claims (4)

1. Chimeric human interferon alpha2b, 434 a.a., molecular weight 49.360 kDa (SEQ ID NO 1), having antiviral activity, including from the N-terminus the amino acid sequence of mature human interferon alpha2b, 165 a.a., molecular weight 19.268 kDa (SEQ ID NO 2) fused at the C-terminus to the amino acid sequence of the linker, 20 a.a., molecular weight 1.469 kDa (SEQ ID NO 3), which in turn is fused at the C-terminus to the amino acid sequence of the mature apolipoprotein Human AI, 243 aa, molecular weight 28.076 kDa (SEQ ID NO 4). 2. Recombinant plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I, 4792 bp in size, providing the synthesis in Pichia pastoris X33 yeast cells of the chimeric human interferon alpha2b according to claim 1, containing the AOX-1 gene promoter, 940 bp in size. ; a fragment of the MFα gene of the yeast Saccharomyces cerevisiae, 249 bp in size, encoding a signal for secretion of the α-factor; a synthetic gene for chimeric human interferon alpha2b (SEQ ID NO 1) of 1296 bp, including a mature human interferon alpha2b gene (SEQ ID NO 2) of 495 bp, a linker gene of 60 bp. (SEQ ID NO 3) and the 729 bp mature human apolipoprotein A-I gene. (SEQ ID NO 4); transcription terminator of the AOX-1 gene, 342 bp in size; TEF1 gene promoter 412 bp in size; EM7 promoter, 68 bp; a 375 bp neomycin transferase gene conferring resistance to zeocin; transcription terminator of the CYC1 gene, 318 bp in size; 674 bp pUC plasmid origin of replication 3. Recombinant yeast strain Pichia pastoris X33/pPICZαA:IFNα2b-linker-ApoA-I, obtained by transformation of yeast P. pastoris X 33 with linearized plasmid DNA pPICZαA:IFNα2b-linker-ApoA-I according to item 2, is a producer of chimeric interferon alpha2b person according to item 1. 4. The method of obtaining chimeric human interferon alpha2b according to claim 1, including the microbiological synthesis of this protein during the cultivation of the yeast strain Pichia pastoris X33/pPICZαA:IFNα2b-linker-ApoA-I according to claim 2; sedimentation of cells of the specified strain with separation of the supernatant by centrifugation; precipitation from the supernatant of chimeric human interferon alpha2b with ammonium sulfate; suspending and dissolving the precipitated protein, purifying it by reverse phase chromatography in an acetonitrile gradient, and then dialysing the purified protein.

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