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Recombinant bovine long-term interferon and fusion protein for preparing same and preparation method of recombinant bovine long-term interferon

A technology of fusion protein and bovine interferon, applied in the field of biological genetic engineering, can solve the problems of small molecular weight of interferon, unfavorable application, short half-life, etc., and achieve the effects of avoiding denaturation and renaturation, improving half-life and shortening expression time.

Inactive Publication Date: 2017-10-17
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 bovine long-term interferon and fusion protein for preparing same and preparation method of recombinant bovine long-term interferon
  • Recombinant bovine long-term interferon and fusion protein for preparing same and preparation method of recombinant bovine long-term interferon
  • Recombinant bovine long-term interferon and fusion protein for preparing same and preparation method of recombinant bovine long-term interferon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] A fusion protein consisting of bovine interleukin 2 and bovine interferon α, the preparation method of which is as follows:

[0073] 1. Acquisition and amplification of bovine interleukin 2 (IL-2) and bovine interferon α (IFN-α) target genes

[0074] Primer design:

[0075] 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 interleukin 2, and the upstream primer and downstream primer of bovine interferon alpha Linker sequence and XhoI restriction site were introduced respectively.

[0076] Table 1 PCR amplification primers

[0077]

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

[0079] RNA was extracted from bovine liver tissue, and the target genes of IL-2 and IFN-α were obtained by reverse transcription, and the gene sequences of the two were shown in SE...

Embodiment 2

[0112] A fusion protein composed of bovine interleukin 2 and bovine 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 inspection result of its fusion protein is compared with embodiment 1, and the dominant expression band at about 55.9KD in the supernatant is thicker, indicating that after introducing molecular chaperone pGro7, the expression of the target protein in the supernatant is more Well, higher amounts of fusion protein are obtained. 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.

[0113] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano Biotech, Cat. No...

Embodiment 3

[0115] A fusion protein consisting of bovine interleukin 2 and bovine interferon α, the preparation method of which is as follows:

[0116] 1. Acquisition and amplification of bovine interleukin 2 (IL-2) and bovine interferon α (IFN-α) target genes

[0117] 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 .

[0118] 1.1 Codon optimization

[0119] 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 effic...

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Abstract

The invention discloses a recombinant bovine peginterferon, a fusion protein for preparing the peginterferon and a preparation method thereof. The fusion protein is formed by connecting bovine interleukin 2 and bovine interferon α through a flexible linker. The fusion protein and After the lyoprotectant is mixed, Hu Jing freeze-dries to obtain the recombinant bovine peginterferon. The recombinant bovine long-acting interferon can significantly increase the half-life of bovine interferon, which is more than 10 times higher than that of common bovine interferon, has broad-spectrum antiviral effect and can improve the immune response of bovine itself.

Description

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

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
CPCC07K14/56C07K14/55C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽赵雨徐文俊夏兵兵徐慕珍陈毅单雪芹凡玉芳
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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