Canine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and canine long-acting interferon

A fusion protein, interferon alpha technology, applied in interferon, biochemical equipment and methods, cytokine/lymphokine/interferon, etc., can solve the problems of short half-life, unfavorable clinical application, and high cost of interferon

Inactive Publication Date: 2018-11-20
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

[0009] 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, and 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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  • Canine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and canine long-acting interferon
  • Canine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and canine long-acting interferon
  • Canine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and canine long-acting interferon

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] The preparation method of canine albumin-interferon alpha-interleukin 2 fusion protein comprises the following steps:

[0093] 1. Acquisition and amplification of canine albumin (Alb), canine interferon-α (IFN-α) and canine interleukin-2 (IL-2) target genes

[0094] Primer design:

[0095] Design synthetic primers according to the target gene sequence reported in Genebank, see Table 1, introduce the EcoRI restriction site into the upstream primer of canine albumin, introduce the Linker sequence into the downstream primer, and introduce the upstream primer and downstream primer of canine interferon α The Linker sequence was introduced into the canine interleukin 2 upstream primer and the SalI restriction site was introduced into the downstream primer.

[0096] Table 1 PCR amplification primers

[0097]

[0098] RT-PCR to obtain the target gene:

[0099] RNA was extracted from canine liver tissue, and the target genes of canine Alb, canine IFN-α and canine IL-2 were...

Embodiment 2

[0135] A preparation method of canine albumin-interferon α-interleukin 2 fusion protein, the preparation method is 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 state cells. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1. The dominant expression band at about 125KD 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.

[0136] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., ...

Embodiment 3

[0139] A preparation method of canine albumin-interferon alpha-interleukin 2 fusion protein, the preparation method is as follows:

[0140]1. Acquisition and amplification of canine albumin (Alb), canine interferon-α (IFN-α) and canine interleukin-2 (IL-2) target genes

[0141] Dog Alb, dog IFN-α and dog IL-2 in Example 1 were optimized, and the target genes of dog Alb, dog IFN-α and dog IL-2 were artificially synthesized. After optimization, the nucleotide sequences of the three were respectively as follows SEQUENCE LISTING 400, SEQUENCELISTING 400 and SEQUENCE LISTING 400.

[0142] 1.1 Codon optimization

[0143] 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 cell...

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Abstract

The invention discloses a canine albumin-interferon alpha-interleukin 2 fusion protein, a preparation method thereof and a coding gene thereof, and a canine long-acting interferon. Canine albumin, canine interferon alpha and canine interleukin 2 are connected by a flexible linker to obtain the canine albumin-interferon alpha-interleukin 2 fusion protein. The coding gene of the fusion protein is designed and optimized to finally obtain a recombinant canine long-acting interferon, which can significantly prolong the half-life of the canine interferon by more than 18 times in comparison with a common canine interferon, has a broad-spectrum antiviral effect and can improve the immune response of dogs.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to canine albumin-interferon alpha-interleukin 2 fusion protein, a preparation method and its coding gene, and a canine peginterferon. Canine albumin, canine interferon α and canine interleukin 2 were connected through a flexible linker to obtain canine albumin-interferon α-interleukin 2 fusion protein, and its coding gene was designed and optimized to finally prepare recombinant canine peginterferon. 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] D...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C07K14/56C12R1/19
CPCC07K14/56C07K14/55C07K2319/31C12N15/70C12N2800/22
Inventor 高耀辉鲍可兵徐慕珍杨建伟王红朵周炜蒋敏之刘家炉
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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