Fusion protein formed by sheep albumin and sheep interferon gamma and preparation method of fusion protein and recombinant sheep long-term interferon gamma

A fusion protein and interferon technology, applied in the field of biogenetic engineering, can solve the problems of unfavorable clinical application, small molecular weight of interferon, high cost of interferon, etc., to avoid denaturation and renaturation, shorten expression time, and improve immune response Effect

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

[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

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  • Fusion protein formed by sheep albumin and sheep interferon gamma and preparation method of fusion protein and recombinant sheep long-term interferon gamma
  • Fusion protein formed by sheep albumin and sheep interferon gamma and preparation method of fusion protein and recombinant sheep long-term interferon gamma
  • Fusion protein formed by sheep albumin and sheep interferon gamma and preparation method of fusion protein and recombinant sheep long-term interferon gamma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] A fusion protein composed of ovine albumin and ovine interferon gamma, the preparation method of which is as follows:

[0076] 1. Acquisition of sheep albumin (Alb) and sheep interferon gamma (IFN-gamma) target genes and design of amplification primers:

[0077] Design synthetic primers according to the target gene sequence reported in Genebank, as shown in Table 1. Introduce the EcoRI restriction site and Linker sequence into the upstream primer and downstream primer of ovine albumin, respectively, and introduce the upstream and downstream primers of ovine interferon gamma Linker sequence and XhoI restriction site were introduced respectively.

[0078] Table 1PCR amplification primers

[0079]

[0080] RT-PCR to obtain the target gene:

[0081] RNA was extracted from sheep liver tissue, and the target genes of Alb and IFN-γ were obtained by reverse transcription, and the gene sequences of the two were shown in SEQUENCE LISTING 400 and SEQUENCE LISTING 400, respect...

Embodiment 2

[0115] A fusion protein composed of ovine albumin and ovine 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 104.4KD 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 it is 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.

[0116] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simbano Biotech, C...

Embodiment 3

[0118] A fusion protein composed of ovine albumin and ovine interferon gamma, the preparation method of which is as follows:

[0119] 1. Acquisition and amplification of sheep albumin (Alb) and sheep interferon gamma (IFN-gamma) target genes

[0120] 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.

[0121] 1.1 Codon optimization

[0122] 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 effici...

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Abstract

The invention discloses a fusion protein composed of ovine albumin and ovine interferon gamma, a preparation method thereof and a recombinant ovine long-acting interferon gamma. The fusion protein is composed of ovine albumin and ovine interferon gamma through a flexible linker The recombinant sheep peginterferon gamma can be obtained after mixing the fusion protein with the freeze-drying protective agent and freeze-drying. The recombinant sheep long-acting interferon gamma can significantly increase the half-life of sheep interferon, which is more than 12 times higher than that of ordinary sheep interferon gamma, has broad-spectrum antiviral effect and can improve the immune response of sheep itself.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a fusion protein composed of ovine albumin and ovine interferon gamma, a preparation method thereof and a recombinant ovine long-acting interferon gamma. Background technique [0002] Animal infectious diseases caused by viruses have seriously restricted the healthy development of the breeding industry in various countries and regions. China is the country with the largest sheep stock, slaughter and mutton production in the world. The mutton sheep industry is also one of the important pillar industries of my country's animal husbandry. . With the continuous development of the sheep breeding industry, it is inevitable to face the problem of diseases caused by viruses. Animal diseases not only cause huge economic losses to sheep farmers, but more seriously, some zoonotic infectious diseases are also Potential threat to human health. [0003] At present, t...

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
CPCC07K14/57C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽沈咏舟汪良青高耀辉戚仕梅马腾飞周炜朱亚召
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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