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Fusion protein composed of bovine interleukin 2, bovine interferon gamma and bovine 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, short half-life, and high cost of interferon

Inactive Publication Date: 2018-11-13
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 composed of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha and preparation method thereof
  • Fusion protein composed of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha and preparation method thereof
  • Fusion protein composed of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] A fusion protein consisting of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha, the preparation method of which is as follows:

[0090] 1. Acquisition and amplification of bovine interleukin 2 (IL-2), bovine interferon γ (IFN-γ) and bovine interferon α (IFN-α) target genes

[0091] Primer design:

[0092] 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 bovine interleukin 2, respectively, and introduce the upstream primer and downstream primer of bovine interferon gamma The Linker sequence was introduced into the primers, and the Linker sequence and the XhoI restriction site were respectively introduced into the upstream primer and downstream primer of bovine interferon α.

[0093] Table 1 PCR amplification primers

[0094]

[0095] RT-PCR to obtain the target gene:

[0096] RNA was extrac...

Embodiment 2

[0131] A fusion protein composed of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha, the others are the same as in Example 1, except that the Escherichia coli BL21 (DE3) competent cells 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 74.1KD in the supernatant is relatively thick, indicating that after the introduction of the 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.

[0132] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science...

Embodiment 3

[0134] A fusion protein consisting of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha, the preparation method of which is as follows:

[0135] 1. Acquisition and amplification of bovine interleukin 2 (IL-2), bovine interferon γ (IFN-γ) and bovine interferon α (IFN-α) target genes

[0136]Bovine IL-2, bovine IFN-γ and bovine IFN-α in Example 1 were optimized, and bovine IL-2, bovine IFN-γ and bovine IFN-α target genes were artificially synthesized. After optimization, the nucleotide sequences of the three They are respectively shown in SEQUENCE LISTING 400, SEQUENCE LISTING 400 and SEQUENCE LISTING 400.

[0137] 1.1 Codon optimization

[0138] 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...

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Abstract

The invention discloses fusion protein composed of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha and a preparation method thereof. The fusion protein is formed by conducting flexible linker connection on the bovine interleukin 2, the bovine interferon gamma and the bovine interferon alpha, and after the fusion protein and a freeze-drying protective additive are mixed, through freeze drying, recombinant bovine pegylated interferon can be obtained. A half-life period of the recombinant bovine pegylated interferon is significantly improved, compared with the half-lifeperiod of ordinary bovine interferon, the half-life period of the recombinant bovine pegylated interferon is improved by 16 times or more, and the recombinant bovine pegylated interferon has a broad-spectrum antiviral effect and can improve immune response of bovine.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a fusion protein composed of bovine interleukin 2, bovine interferon gamma and bovine interferon alpha and a preparation method thereof. Background technique [0002] Cattle are one of the important livestock species in my country. With the continuous development of intensive and large-scale breeding, the incidence of bovine viral infectious diseases is increasing year by year. Many infectious diseases of cattle have been prevalent in domestic large-scale pastures for a long time. Because of the rapid spread of diseases, the high morbidity and mortality seriously restrict the healthy development of my country's cattle breeding industry; what is more serious is that some zoonotic infectious diseases such as cattle Brucellosis, tuberculosis, etc. directly threaten human life and health. [0003] The current approach to the prevention and treatment of infec...

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 单雪芹凡玉芳王亚男许高涛高耀辉周炜徐慕珍
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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