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Fusion protein comprising pig albumin and pig interferon gamma, preparation method of fusion protein, and recombination pig long-acting interferon gamma

A technology of fusion protein and porcine interferon, applied in the field of biological genetic engineering, can solve the problems of high cost, short half-life and unfavorable clinical application of interferon, and achieve the effect of reducing cost and improving half-life

Inactive Publication Date: 2018-11-20
WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The common long-acting interferon on the market is represented by polyethylene glycol fusion interferon, which only partially solves the problem of short half-life due to the small molecular weight of interferon at the molecular weight level. At the same time, the cost of polyethylene glycol fusion interferon is very high. High, not conducive to clinical application

Method used

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  • Fusion protein comprising pig albumin and pig interferon gamma, preparation method of fusion protein, and recombination pig long-acting interferon gamma
  • Fusion protein comprising pig albumin and pig interferon gamma, preparation method of fusion protein, and recombination pig long-acting interferon gamma
  • Fusion protein comprising pig albumin and pig interferon gamma, preparation method of fusion protein, and recombination pig long-acting interferon gamma

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] A fusion protein composed of porcine albumin and porcine interferon gamma, the preparation method of which is as follows:

[0070] 1. Acquisition and amplification of target genes of porcine albumin (Alb) and porcine interferon-γ (IFN-γ)

[0071] Primer design:

[0072] Design synthetic primers according to the target gene sequence reported in Genebank, see Table 1, introduce EcoRI restriction site and Linker sequence into the upstream primer and downstream primer of porcine albumin, respectively, and introduce the upstream primer and downstream primer of porcine interferon gamma Linker sequence and HindⅢ restriction site were introduced respectively.

[0073] Table 1 PCR amplification primers

[0074]

[0075] RT-PCR to obtain the target gene:

[0076] RNA was extracted from pig liver tissue, and the target genes of Alb and IFN-γ were obtained by reverse transcription, and the gene sequences of the two were shown in SEQUENCE LISTING 400 and SEQUENCE LISTING 400, ...

Embodiment 2

[0109] A fusion protein composed of porcine albumin and porcine interferon gamma, the others are the same as in Example 1, except that the Escherichia coli BL21(DE3) competent cells are replaced with BL21(DE3) competent cells carrying pGro7 plasmid. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 102KD in the supernatant is thicker, indicating that after the introduction of molecular chaperone pGro7, the expression of the target protein in the supernatant is better, and the obtained The amount of fusion protein is higher. Most of the proteins expressed in E. coli exist in inclusion bodies; by introducing molecular chaperones into the expression strains, the co-expressed proteins can be correctly folded to achieve protein soluble expression.

[0110] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano Biotech, C...

Embodiment 3

[0112] A fusion protein composed of porcine albumin and porcine interferon gamma, the preparation method of which is as follows:

[0113] 1. Acquisition and amplification of target genes of porcine albumin (Alb) and porcine interferon-γ (IFN-γ)

[0114] Alb and IFN-γ in Example 1 are optimized, and Alb and IFN-γ target genes are artificially synthesized. After optimization, the nucleotide sequences of the two are as shown in SEQUENCE LISTING 400 and SEQUENCE LISTING 400 respectively Show.

[0115] 1.1 Codon optimization

[0116] There are 64 genetic codes, but most organisms tend to use a subset of these. Those that are most frequently used are called optimal codons, and those that are not frequently used are called rare or low-usage codons. Virtually every organism commonly used for protein expression or production (including E. coli, yeast, mammalian cells, plant cells, and insect cells) exhibits some degree of difference or bias in codon usage. The expression efficiency...

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Abstract

The invention discloses a fusion protein comprising a pig albumin and a pig interferon gamma, a preparation method of the fusion protein, and a recombination pig long-acting interferon gamma. The fusion protein is formed by connecting the pig albumin and the pig interferon gamma through a flexible linker, and the recombination pig long-acting interferon gamma is obtained by blending the fusion protein and a freezing and drying protecting agent, and freeze-drying. The recombination pig long-acting interferon gamma obviously improves the half-life period of the pig interferon, the half-life period is improved by 15 times or more in comparison with the common pig interferon gamma, the recombination pig long-acting interferon gamma has a broad-spectrum antiviral effect, and the immune responseof the pig is improved.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a fusion protein composed of porcine albumin and porcine interferon gamma, a preparation method thereof and a recombinant porcine peginterferon gamma. Background technique [0002] In recent years, large-scale and intensive pig farms have emerged rapidly in my country. The number of pigs on hand and the total amount of pork production in my country rank first in the world. Traditional methods of prevention and control of pig diseases are far from being able to adapt to the control of infectious diseases in large-scale intensive pig production. Nearly 20 kinds of livestock and poultry infectious diseases have emerged in my country in the past 20 years. In addition to the original animal diseases, they have caused huge economic losses to my country's breeding industry. According to incomplete statistics, the mortality rate of livestock and poultry caused b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/04C12R1/19
CPCA61P31/12A61P37/04C07K14/57C07K14/76C07K2319/00C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 徐慕珍徐文俊高耀辉周炜刘家炉王红朵凡玉芳
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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