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Recombinant chicken long-acting interferon gamma, fusion protein for preparing recombinant chicken long-acting interferon gamma, and preparation method of fusion protein

A technology of fusion protein and chicken interferon, applied in the field of biological genetic engineering, can solve the problems of short half-life, unfavorable clinical application, high cost of interferon, and achieve the effect of improving half-life

Inactive Publication Date: 2017-10-24
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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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  • Recombinant chicken long-acting interferon gamma, fusion protein for preparing recombinant chicken long-acting interferon gamma, and preparation method of fusion protein
  • Recombinant chicken long-acting interferon gamma, fusion protein for preparing recombinant chicken long-acting interferon gamma, and preparation method of fusion protein
  • Recombinant chicken long-acting interferon gamma, fusion protein for preparing recombinant chicken long-acting interferon gamma, and preparation method of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] A fusion protein composed of chicken interleukin 2 and chicken interferon gamma, the preparation method of which is as follows:

[0074] 1. Acquisition and amplification of chicken interleukin 2 (IL-2) and chicken interferon gamma (IFN-gamma) target genes

[0075] Primer design:

[0076] 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 interleukin 2, respectively, and introduce the upstream primer and downstream primer of chicken interferon gamma Linker sequence and SalI restriction site were introduced respectively.

[0077] Table 1PCR amplification primers

[0078]

[0079]

[0080] RT-PCR to obtain the target gene:

[0081] RNA was extracted from chicken liver tissue, and the target genes of IL-2 and IFN-γ were obtained by reverse transcription, and the gene sequences of the two were shown in SEQUENCE...

Embodiment 2

[0114] A fusion protein composed of chicken interleukin 2 and chicken interferon gamma, the others are the same as in Example 1, except that Escherichia coli BL21 (DE3) competent cells are replaced by BL21 (DE3) competent cells with pGro7 plasmid cell. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 53.2KD 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.

[0115] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano Bio...

Embodiment 3

[0117] A fusion protein composed of chicken interleukin 2 and chicken interferon gamma, the preparation method of which is as follows:

[0118] 1. Acquisition and amplification of chicken interleukin 2 (IL-2) and chicken interferon gamma (IFN-gamma) target genes

[0119] IL-2 and IFN-γ in Example 1 are optimized, and IL-2 and IFN-γ target genes are artificially synthesized. After optimization, the nucleotide sequences of the two are as SEQUENCE LISTING 400 and SEQUENCE LISTING 400 respectively As shown in .

[0120] 1.1 Codon optimization

[0121] 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, mammalian cells, plant cells, and insect cells) exhibits some degree of difference or bias in codon usage. The ex...

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Abstract

The invention discloses a recombinant chicken long-acting interferon gamma, a fusion protein for preparing the recombinant chicken long-acting interferon gamma, and a preparation method of the fusion protein; the fusion protein is formed by linking chicken interleukin 2 and chicken interferon gamma via a flexible linker; the fusion protein is mixed with a lyoprotectant, and the mixture is lyophilized to obtain the recombinant chicken long-acting interferon gamma. The recombinant chicken long-acting interferon gamma can provide significantly extended half-life period for chicken interferons which is more than 13 times longer than that of common chicken interferons, has broad-spectrum antiviral action, and can improve autologous immune response of chicken.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a recombinant chicken peginterferon gamma, a fusion protein for preparing the peg interferon gamma 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
CPCC07K14/57C07K14/55C07K2319/00C12N15/62C12N15/70C12N2800/22
Inventor 赵俊赵雨高耀辉戚仕梅马腾飞周炜陈毅王亚男
Owner ANHUI JIUCHUAN BIOTECH
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