Recombinant bovine long-term interferon alpha and fusion protein for preparing long-term interferon and its preparation method

A technology of fusion protein and bovine interferon, which is applied in the field of biogenetic engineering, can solve the problems of small molecular weight of interferon, high cost of interferon, unfavorable application, etc., and achieve the effects of avoiding denaturation and renaturation, improving immune response, and increasing half-life

Inactive Publication Date: 2017-10-20
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

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 in livestock

Method used

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  • Recombinant bovine long-term interferon alpha and fusion protein for preparing long-term interferon and its preparation method
  • Recombinant bovine long-term interferon alpha and fusion protein for preparing long-term interferon and its preparation method
  • Recombinant bovine long-term interferon alpha and fusion protein for preparing long-term interferon and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] A fusion protein consisting of bovine interferon gamma and bovine interferon alpha, the preparation method of which is as follows:

[0072] 1. Acquisition and amplification of bovine interferon gamma (IFN-γ) and bovine interferon alpha (IFN-α) target genes

[0073] Primer design:

[0074] According to the target gene sequence reported in Genebank, the synthetic primers were designed and synthesized as shown in Table 1. The EcoRI restriction site and Linker sequence were respectively introduced into the upstream primer and downstream primer of bovine interferon gamma, and the upstream primer and downstream primer of bovine interferon alpha The Linker sequence and the XhoI restriction site were respectively introduced in.

[0075] Table 1 PCR amplification primers

[0076]

[0077] RT-PCR to obtain the target gene:

[0078] RNA was extracted from bovine liver tissue, and the target genes of IFN-γ and IFN-α were obtained by reverse transcription, and the gene sequenc...

Embodiment 2

[0111] A fusion protein composed of 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 (DE3) competent cells with pGro7 plasmid . The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 56KD 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 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.

[0112] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano Biotec...

Embodiment 3

[0114] A fusion protein consisting of bovine interferon gamma and bovine interferon alpha, the preparation method of which is as follows:

[0115] 1. Acquisition and amplification of bovine interferon gamma (IFN-γ) and bovine interferon alpha (IFN-α) target genes

[0116] The IFN-γ and IFN-α in Example 1 are optimized, and the IFN-γ and IFN-α 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 .

[0117] 1.1 Codon optimization

[0118] 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 usa...

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Abstract

The invention discloses a recombinant bovine peginterferon α, a fusion protein for preparing the peginterferon and a preparation method thereof. The fusion protein is formed by connecting bovine interferon γ and bovine interferon α through a flexible linker, and the fusion protein is connected with bovine interferon α. After the lyoprotectant is mixed, the recombinant bovine peginterferon α can be obtained by lyophilization. The recombinant bovine long-acting interferon alpha can significantly increase the half-life of bovine interferon, which is more than 11 times higher than that of common bovine interferon, has broad-spectrum antiviral effect and can improve the immune response of cattle itself.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a recombinant bovine peginterferon alpha, a fusion protein for preparing the peginterferon 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 infectious bovine...

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
CPCC07K14/57C07K14/56C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 赵俊符修乐夏兵兵徐慕珍程正阳徐文俊单雪芹郭志燕
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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