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Fused protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2 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

[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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  • Fused protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2 and preparation method of fused protein
  • Fused protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2 and preparation method of fused protein
  • Fused protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2 and preparation method of fused protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] A fusion protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2, the preparation method of which is as follows:

[0090] 1. Acquisition and amplification of chicken albumin (Alb), chicken interferon gamma (IFN-gamma) and chicken interleukin 2 (IL-2) 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 chicken albumin, respectively, and introduce the upstream primer and downstream primer of chicken interferon gamma The Linker sequence was introduced respectively, and the Linker sequence and the XhoI restriction site were respectively introduced into the upstream primer and the downstream primer of chicken interleukin 2.

[0093] Table 1 PCR amplification primers

[0094]

[0095]

[0096] RT-PCR to obtain the target gene:

[0097] RNA was ...

Embodiment 2

[0132] A fusion protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2, others are the same as in Example 1, except that 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 121.9KD 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 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.

[0133] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co.,...

Embodiment 3

[0135] A fusion protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2, the preparation method of which is as follows:

[0136] 1. Acquisition and amplification of chicken albumin (Alb), chicken interferon gamma (IFN-gamma) and chicken interleukin 2 (IL-2) target genes

[0137]Chicken Alb, chicken IFN-γ and chicken IL-2 in Example 1 are optimized, and chicken Alb, chicken IFN-γ and chicken IL-2 target genes are artificially synthesized. After optimization, the nucleotide sequences of the three are respectively as follows SEQUENCE LISTING 400, SEQUENCELISTING 400 and SEQUENCE LISTING 400.

[0138] 1.1 Codon optimization

[0139] 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,...

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Abstract

The invention discloses a fusion protein composed of chicken albumin, chicken interferon gamma and chicken interleukin 2 and a preparation method thereof. The fusion protein is composed of chicken albumin, chicken interferon gamma and chicken interleukin 2. Formed by flexible linker connection, the fusion protein is mixed with a lyoprotectant and freeze-dried to obtain the recombinant chicken peginterferon. The recombinant chicken long-acting interferon can significantly increase the half-life of chicken interferon, which is more than 21 times higher than that of common chicken interferon, has broad-spectrum antiviral effect and can improve the immune response of chicken itself.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a fusion protein composed of chicken albumin, chicken interferon gamma and chicken interleukin-2 and a preparation method thereof. Background technique [0002] In recent years, with the scale of the breeding industry and the rapid development of the circulation of livestock and poultry and their products, China's poultry industry has made great progress, forming a huge industry with an annual output value of more than 100 billion yuan. However, due to our country's weak animal epidemic prevention system, poultry disease is still a serious problem facing poultry production in our country, which has become an important factor restricting the development of poultry breeding industry in our country. There are many poultry diseases and large losses, the mortality rate is higher than 15%, and the death rate is 20% to 25% (less than 5% in developed countries)...

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
CPCC07K14/57C07K14/55C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽符修乐王利利夏兵兵徐慕珍何志远徐文俊杨建伟
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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