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Fused protein composed of sheep interleukin 2, sheep interferon gamma and sheep interferon tau and preparation method of fused protein

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

Inactive Publication Date: 2017-11-24
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

[0010] 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, and 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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  • Fused protein composed of sheep interleukin 2, sheep interferon gamma and sheep interferon tau and preparation method of fused protein
  • Fused protein composed of sheep interleukin 2, sheep interferon gamma and sheep interferon tau and preparation method of fused protein
  • Fused protein composed of sheep interleukin 2, sheep interferon gamma and sheep interferon tau and preparation method of fused protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] A fusion protein composed of goat interleukin 2, goat interferon gamma and goat interferon tau, the preparation method of which is as follows:

[0092] 1. Acquisition and amplification of sheep interleukin 2 (IL-2), sheep interferon gamma (IFN-gamma) and sheep interferon tau (IFN-τ) target genes

[0093] Primer design:

[0094] 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 sheep interleukin 2, respectively, and introduce the upstream primer and downstream primer of sheep interferon gamma The Linker sequence was introduced into the primers, and the Linker sequence and XhoI restriction site were respectively introduced into the upstream primer and downstream primer of sheep interferon τ.

[0095] Table 1 PCR amplification primers

[0096]

[0097]

[0098] RT-PCR to obtain the target gene:

[0099] RNA was extr...

Embodiment 2

[0134] A fusion protein composed of goat interleukin 2, goat interferon gamma and goat interferon tau, 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 77.3KD 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 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.

[0135] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Lt...

Embodiment 3

[0137] A fusion protein composed of goat interleukin 2, goat interferon gamma and goat interferon tau, the preparation method of which is as follows:

[0138] 1. Acquisition and amplification of sheep interleukin 2 (IL-2), sheep interferon gamma (IFN-gamma) and sheep interferon tau (IFN-τ) target genes

[0139] Goat IL-2, goat IFN-γ and goat IFN-τ in Example 1 were optimized, and the target genes of goat IL-2, goat IFN-γ and goat IFN-τ 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.

[0140] 1.1 Codon optimization

[0141] 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, y...

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Abstract

The invention discloses a fusion protein composed of goat interleukin 2, goat interferon gamma and goat interferon tau and a preparation method thereof. The fusion protein is composed of goat interleukin 2, goat interferon gamma and goat interferon τ is formed by connecting with a flexible linker, the fusion protein is mixed with a freeze-drying protective agent, and then freeze-dried to obtain recombinant sheep peginterferon. The recombinant sheep long-acting interferon can significantly increase the half-life of sheep interferon, which is more than 15 times higher than that of ordinary sheep interferon, has broad-spectrum antiviral effect and can improve the immune response of sheep itself.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a fusion protein composed of sheep interleukin 2, sheep interferon gamma and sheep interferon tau and a preparation method thereof. Background technique [0002] Animal infectious diseases caused by viruses have seriously restricted the healthy development of the breeding industry in various countries and regions. China is the country with the largest sheep stock, slaughter and mutton production in the world. The mutton sheep industry is also one of the important pillar industries of my country's animal husbandry. . With the continuous development of the sheep breeding industry, it is inevitable to face the problem of diseases caused by viruses. Livestock diseases not only cause huge economic losses to sheep farmers, but more seriously, some zoonotic infectious diseases also cause serious problems. potential threat to human health. [0003] At present, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/04C12R1/19
CPCC07K14/555C07K14/55C07K14/57C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽沈咏舟李树启高耀辉戚仕梅王利利周炜梅志强
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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