Fusion protein consisting of pig interleukin 2, pig interferon gamma and pig interferon alpha and preparation method thereof

A technology of interleukin and fusion protein, applied in the field of fusion protein and its preparation, can solve the problems of unfavorable clinical application, small molecular weight of interferon, short half-life, etc.

Inactive Publication Date: 2018-11-20
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

[0008] 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 pig interleukin 2, pig interferon gamma and pig interferon alpha and preparation method thereof
  • Fusion protein consisting of pig interleukin 2, pig interferon gamma and pig interferon alpha and preparation method thereof
  • Fusion protein consisting of pig interleukin 2, pig interferon gamma and pig interferon alpha and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] A fusion protein composed of porcine interleukin 2, porcine interferon gamma and porcine interferon alpha, the preparation method of which is as follows:

[0081] 1. Acquisition and amplification of porcine interleukin 2 (IL-2), porcine interferon γ (IFN-γ) and porcine interferon α (IFN-α) target genes

[0082] Primer design:

[0083] Design and synthesize primers according to the target gene sequence reported in Genebank, see Table 1. NcoI restriction site and Linker sequence were introduced into the upstream primer and downstream primer of porcine interleukin 2, respectively, and the upstream primer and downstream primer of porcine interferon gamma Linker sequences were introduced into the primers, and Linker sequences and XhoI restriction sites were introduced into the upstream primers and downstream primers of porcine interferon α, respectively.

[0084] Table 1 PCR amplification primers

[0085]

[0086] RT-PCR to obtain the target gene:

[0087] RNA was extr...

Embodiment 2

[0124] A fusion protein composed of porcine interleukin 2, porcine interferon γ and porcine interferon α, the others are the same as in Example 1, except that the competent cells of Escherichia coli BL21 (DE3) are replaced by BL21 with pGro7 plasmid (DE3) Competent cells. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 80.6KD in the supernatant is relatively thick, indicating that after the introduction of molecular chaperone pGro7, the expression of the target protein in the supernatant is better, and it is 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.

[0125] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Tec...

Embodiment 3

[0127] A fusion protein composed of porcine interleukin 2, porcine interferon gamma and porcine interferon alpha, the preparation method of which is as follows:

[0128] 1. Acquisition and amplification of porcine interleukin 2 (IL-2), porcine interferon γ (IFN-γ) and porcine interferon α (IFN-α) target genes

[0129] The porcine IL-2, porcine IFN-γ and porcine IFN-α in Example 1 were optimized, and the target genes of porcine IL-2, porcine IFN-γ and porcine IFN-α were artificially synthesized. After optimization, the nucleosides of the three The acid sequences are shown in SEQUENCE LISTING 400, SEQUENCE LISTING 400 and SEQUENCE LISTING 400, respectively.

[0130] 1.1 Codon optimization

[0131]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 pr...

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Abstract

The invention discloses a fusion protein consisting of pig interleukin 2, pig interferon gamma and pig interferon alpha and a preparation method thereof. The fusion protein is formed by connecting thepig interleukin 2, pig interferon gamma and pig interferon alpha through a flexible linker, and a recombinant pig long-acting interferon can be obtained through freeze drying after the fusion proteinis compounded with a freeze-drying protective additive. The recombinant pig long-acting interferon can obviously improve the half-life period of the pig interferon, the half-life period of the pig interferon is prolonged by more than 24 times by compared with that of common pig interferon, the fusion protein has a broad-spectrum antiviral role, and the immune response of pigs can be 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 interleukin 2, porcine interferon gamma and porcine interferon alpha and a preparation method thereof. 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 by various v...

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
CPCC07K14/57A61P31/12A61P37/04C07K14/55C07K14/56C07K2319/00C12N15/70C12N2800/22
Inventor 高耀辉周炜郭志燕何志远单雪芹鲍可兵付超
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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