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Fusion protein composed of chicken albumin, chicken interferon gamma and chicken interferon alpha and preparation method thereof

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

Inactive Publication Date: 2017-10-24
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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  • Fusion protein composed of chicken albumin, chicken interferon gamma and chicken interferon alpha and preparation method thereof
  • Fusion protein composed of chicken albumin, chicken interferon gamma and chicken interferon alpha and preparation method thereof
  • Fusion protein composed of chicken albumin, chicken interferon gamma and chicken interferon alpha and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0090] 1. Acquisition and amplification of chicken albumin (Alb), chicken interferon gamma (IFN-gamma) and chicken interferon alpha (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 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 interferon alpha.

[0093] Table 1 PCR amplification primers

[0094]

[0095]

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

[00...

Embodiment 2

[0133] A fusion protein composed of chicken albumin, chicken interferon gamma and chicken interferon alpha, the others are the same as in Example 1, except that the competent cells of Escherichia coli BL21(DE3) are replaced by BL21(DE3) with pGro7 plasmid ) 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 127.6KD 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.

[0134] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Scienc...

Embodiment 3

[0136] A fusion protein consisting of chicken albumin, chicken interferon gamma and chicken interferon alpha, the preparation method of which is as follows:

[0137] 1. Acquisition and amplification of chicken albumin (Alb), chicken interferon gamma (IFN-gamma) and chicken interferon alpha (IFN-α) target genes

[0138]Chicken Alb, chicken IFN-γ and chicken IFN-α in Example 1 are optimized, and chicken Alb, chicken IFN-γ and chicken IFN-α 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.

[0139] 1.1 Codon optimization

[0140] 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. c...

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Abstract

The invention discloses a fusion protein composed of chicken albumin, chicken interferon gamma and chicken interferon alpha and a preparation method thereof. The fusion protein is composed of chicken albumin, chicken interferon gamma and chicken interferon alpha through a flexible linker The recombinant chicken peginterferon can be obtained after the fusion protein is mixed with the freeze-drying protective agent and freeze-dried. The recombinant chicken long-acting interferon can significantly increase the half-life of chicken interferon, which is more than 22 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 specifically relates to a fusion protein composed of chicken albumin, chicken interferon gamma and chicken interferon alpha 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 countrie...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/04C12R1/19
CPCC07K14/57C07K14/56C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽汪良青王利利夏兵兵徐慕珍朱亚召付超杨建伟
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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