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Fusion protein prepared from canine albumin and canine interferon gamma as well as preparation method thereof and recombinant canine pegylated interferon gamma

A fusion protein, canine interferon technology, applied in interferon, biochemical equipment and methods, cytokine/lymphokine/interferon and other directions, can solve the problems of short half-life, high cost of interferon, unfavorable clinical application, etc. Achieve the effect of increasing half-life and reducing cost

Inactive Publication Date: 2018-11-23
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 prepared from canine albumin and canine interferon gamma as well as preparation method thereof and recombinant canine pegylated interferon gamma
  • Fusion protein prepared from canine albumin and canine interferon gamma as well as preparation method thereof and recombinant canine pegylated interferon gamma
  • Fusion protein prepared from canine albumin and canine interferon gamma as well as preparation method thereof and recombinant canine pegylated interferon gamma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0074] 1. Acquisition and amplification of canine albumin (Alb) and canine 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 BamHI restriction site and Linker sequence into the upstream primer and downstream primer of canine albumin, respectively, and introduce the upstream primer and downstream primer of canine interferon gamma Linker sequence and XhoI restriction site were introduced respectively.

[0077] Table 1 PCR amplification primers

[0078]

[0079] RT-PCR to obtain the target gene:

[0080] RNA was extracted from canine liver tissue, and the target genes of Alb and IFN-γ were obtained by reverse transcription. The gene sequences of the two are shown in SEQUENCE LISTING 400 and SEQUENCE LISTING 400, resp...

Embodiment 2

[0113] A fusion protein composed of canine albumin and canine interferon gamma, the others are the same as in Example 1, except that the Escherichia coli BL21(DE3) competent cells are replaced with BL21(DE3) competent cells carrying 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 104.5KD 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.

[0114] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano B...

Embodiment 3

[0116] A fusion protein composed of canine albumin and canine interferon gamma, the preparation method of which is as follows:

[0117] 1. Acquisition and amplification of canine albumin (Alb) and canine interferon gamma (IFN-gamma) target genes

[0118] Alb and IFN-γ in Example 1 are optimized, and the target genes of Alb and IFN-γ are artificially synthesized. After optimization, the nucleotide sequences of the two are as shown in SEQUENCE LISTING 400 and SEQUENCE LISTING 400 respectively Show.

[0119] 1.1 Codon optimization

[0120] 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 expression ef...

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Abstract

The invention discloses fusion protein prepared from canine albumin and canine interferon gamma as well as a preparation method thereof and a recombinant canine pegylated interferon gamma. The fusionprotein is prepared from the canine albumin and the canine interferon gamma through flexible linker connection, and the fusion protein and a freeze-drying protective additive are mixed and freeze dried to obtain the recombinant canine pegylated interferon gamma. The recombinant canine pegylated interferon gamma can obviously improve a half-life period of the canine interferon; compared with ordinary canine interferon gamma, the half-life period of the canine interferon is improved by 13 times or more; furthermore, the recombinant canine pegylated interferon gamma has a broad-spectrum antiviraleffect and can improve canine self immune response.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to a fusion protein composed of canine albumin and canine interferon gamma, a preparation method thereof and a recombinant canine peginterferon gamma. 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 c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/04
CPCA61P31/12A61P37/04C07K14/57C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 周炜鲍可兵高耀辉杨建伟蒋敏之付超许高涛王红朵
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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