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Recombinant canine long-acting interferon alpha, fusion protein for preparing long-acting interferon, and preparation method of fusion protein

A technology of fusion protein and canine 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 effect of avoiding denaturation and renaturation, improving immune response, and increasing half-life

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

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 canine long-acting interferon alpha, fusion protein for preparing long-acting interferon, and preparation method of fusion protein
  • Recombinant canine long-acting interferon alpha, fusion protein for preparing long-acting interferon, and preparation method of fusion protein
  • Recombinant canine long-acting interferon alpha, fusion protein for preparing long-acting interferon, and preparation method of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] A fusion protein composed of canine interferon gamma and canine interferon alpha, the preparation method of which is as follows:

[0073] 1. Acquisition and amplification of canine interferon gamma (IFN-gamma) and canine interferon alpha (IFN-α) target genes

[0074] Primer design:

[0075] Synthetic primers were designed and synthesized according to the target gene sequence reported in Genebank, as shown in Table 1. The EcoRI restriction site and Linker sequence were respectively introduced into the upstream primer and downstream primer of canine interferon gamma, and the upstream primer and downstream primer of canine interferon alpha The Linker sequence and the XhoI restriction site were respectively introduced in.

[0076] Table 1 PCR amplification primers

[0077]

[0078] RT-PCR to obtain the target gene:

[0079] RNA was extracted from canine liver tissue, and the target genes of IFN-γ and IFN-α were obtained by reverse transcription. The gene sequences of ...

Embodiment 2

[0111] A fusion protein composed of canine interferon gamma and canine 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 58KD 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 composed of canine interferon gamma and canine interferon alpha, the preparation method of which is as follows:

[0115] 1. Acquisition and amplification of canine interferon gamma (IFN-gamma) and canine 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 u...

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Abstract

The invention discloses recombinant canine long-acting interferon alpha, fusion protein for preparing the long-acting interferon, and a preparation method of the fusion protein. The fusion protein is formed by connecting canine interferon gamma and canine interferon alpha through flexible linker, and the fusion protein is blended with a lyoprotectant and then is freeze-dried so as to obtain the recombinant canine long-acting interferon alpha. The recombinant canine long-acting interferon alpha can remarkably prolong the half-life period of the canine interferon and prolongs the half-life period by 11 times or more compared with common canine interferon, has a broad-spectrum anti-virus effect, and improves the immune response of the canine.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a recombinant canine peginterferon alpha, a fusion protein for preparing the peginterferon and a preparation method thereof. Background technique [0002] According to the analysis of the current situation of China's pet industry, the market size of my country's pet industry has reached 105.8 billion yuan by the end of 2014, while China's pet medical industry currently only accounts for about 20%. Compared with the relatively mature US market, the output value of its pet medical The ratio reaches 50%, and there is huge room for development in China's pet medical industry. [0003] Dogs are loyal friends of human beings. With the substantial increase in domestic pet dogs and other dogs, the morbidity and mortality of canine diseases continue to increase, causing both spiritual and economic losses to human beings. The close contact between dogs and humans ...

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
CPCC07K14/56C07K14/57C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 赵俊沈咏舟符修乐徐慕珍程正阳徐文俊单雪芹夏兵兵
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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