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Fusion protein consisting of porcine albumin and porcine interferon gamma, preparation method of fusion protein and recombinant porcine pegylated interferon gamma

A technology of fusion protein and porcine interferon, applied in the field of biological genetic engineering, can solve the problems of unfavorable clinical application, low molecular weight of interferon, high cost of interferon, avoid denaturation and renaturation, shorten expression time, improve immunity effect of response

Inactive Publication Date: 2017-11-17
WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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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 consisting of porcine albumin and porcine interferon gamma, preparation method of fusion protein and recombinant porcine pegylated interferon gamma
  • Fusion protein consisting of porcine albumin and porcine interferon gamma, preparation method of fusion protein and recombinant porcine pegylated interferon gamma
  • Fusion protein consisting of porcine albumin and porcine interferon gamma, preparation method of fusion protein and recombinant porcine pegylated interferon gamma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] A fusion protein composed of porcine albumin and porcine interferon gamma, the preparation method of which is as follows:

[0074] 1. Acquisition and amplification of target genes of porcine albumin (Alb) and porcine interferon-γ (IFN-γ)

[0075] Primer design:

[0076] According to the target gene sequence reported in Genebank, the synthetic primers were designed and synthesized as shown in Table 1. The EcoRI restriction site and Linker sequence were introduced into the upstream primer and downstream primer of porcine albumin respectively, and the upstream primer and downstream primer of porcine interferon gamma Linker sequence and HindⅢ restriction site were introduced respectively.

[0077] Table 1 PCR amplification primers

[0078]

[0079] RT-PCR to obtain the target gene:

[0080] 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 LIST...

Embodiment 2

[0114] 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.

[0115] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano Biotech, C...

Embodiment 3

[0117] A fusion protein composed of porcine albumin and porcine interferon gamma, the preparation method of which is as follows:

[0118] 1. Acquisition and amplification of target genes of porcine albumin (Alb) and porcine interferon-γ (IFN-γ)

[0119] 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.

[0120] 1.1 Codon optimization

[0121] 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 composed of porcine albumin and porcine interferon gamma, a preparation method thereof and a recombinant porcine peginterferon gamma. The fusion protein is composed of porcine albumin and porcine interferon gamma through a flexible linker The recombinant porcine long-acting interferon gamma can be obtained by lyophilizing after the fusion protein is mixed with the freeze-drying protective agent. The recombinant porcine long-acting interferon gamma can significantly increase the half-life of porcine interferon, which is more than 18 times higher than that of ordinary porcine interferon gamma, has broad-spectrum antiviral effect and can improve the immune response of pigs themselves.

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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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/04C12R1/19
CPCC07K14/57C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽韩国祥汪良青高耀辉戚仕梅马腾飞王利利周炜
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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