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Recombinant canine long-acting interferon as well as fusion protein preparing long-acting interferon as well as 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, unfavorable application, and short half-life of interferon, and achieve the effects of avoiding denaturation and renaturation, increasing half-life, and increasing expression

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

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 poultry

Method used

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  • Recombinant canine long-acting interferon as well as fusion protein preparing long-acting interferon as well as preparation method of fusion protein
  • Recombinant canine long-acting interferon as well as fusion protein preparing long-acting interferon as well as preparation method of fusion protein
  • Recombinant canine long-acting interferon as well as fusion protein preparing long-acting interferon as well as preparation method of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] A fusion protein composed of canine interleukin 2 and canine interferon α, the preparation method of which is as follows:

[0076] 1. Acquisition and amplification of canine interleukin-2 (IL-2) and canine interferon-α (IFN-α) target genes

[0077] Primer design:

[0078] 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 interleukin 2, respectively, and introduce the upstream primer and downstream primer of canine interferon α Linker sequence and XhoI restriction site were introduced respectively.

[0079] Table 1 PCR amplification primers

[0080]

[0081] RT-PCR to obtain the target gene:

[0082] RNA was extracted from canine liver tissue, and the target genes of IL-2 and IFN-α were obtained by reverse transcription. The gene sequences of the two are shown in SEQUENCE LISTING 400 and SEQUENCE LISTING 400, ...

Embodiment 2

[0116] A fusion protein composed of canine interleukin 2 and canine interferon α, 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 cell. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 56.8KD 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.

[0117] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano Biotech...

Embodiment 3

[0119] A fusion protein composed of canine interleukin 2 and canine interferon α, the preparation method of which is as follows:

[0120] 1. Acquisition and amplification of canine interleukin-2 (IL-2) and canine interferon-α (IFN-α) target genes

[0121] IL-2 and IFN-α in Example 1 are optimized, and IL-2 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 .

[0122] 1.1 Codon optimization

[0123] 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 efficie...

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Abstract

The invention discloses a recombinant canine long-acting interferon as well as a fusion protein preparing the long-acting interferon as well as a preparation method of the protein. The fusion proteinis formed by connecting canine interleukin 2 and canine interferon alpha by virtue of flexible linker, after the fusion protein is mixed with a freeze drying protection agent, the mixture is freeze dried, thus the recombinant canine long-acting interferon is obtained. By adopting the recombinant canine long-acting interferon, the half-life period of the canine interferon can be significantly prolonged and can be increased by 10 times or more compared with the half-life period of the ordinary canine interferon, a broad-spectrum antivirus effect can be achieved, and the immune response of the dog can be improved.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a recombinant canine peginterferon, 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 the number of 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...

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
CPCC07K14/56A61P31/12A61P37/04C07K14/55C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 周炜赵雨夏兵兵徐慕珍付超杨建伟徐文俊单雪芹
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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