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Recombinant goat long-acting interferon gamma, fusion protein for preparing long-acting interferon gamma and preparation method thereof

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

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

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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  • Recombinant goat long-acting interferon gamma, fusion protein for preparing long-acting interferon gamma and preparation method thereof
  • Recombinant goat long-acting interferon gamma, fusion protein for preparing long-acting interferon gamma and preparation method thereof
  • Recombinant goat long-acting interferon gamma, fusion protein for preparing long-acting interferon gamma and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] A fusion protein composed of sheep interleukin 2 and sheep interferon gamma, the preparation method is as follows:

[0074] 1. Obtain and amplify the target genes of sheep interleukin 2 (IL-2) and sheep interferon gamma (IFN-γ)

[0075] Primer design:

[0076] The primers were designed and synthesized according to the target gene sequence reported in Genebank, see Table 1. The EcoRI restriction site and Linker sequence were introduced into the upstream primer and downstream primer of sheep interleukin 2, respectively, and the upstream primer and downstream primer of sheep interferon gamma The Linker sequence and XhoI restriction site were introduced respectively.

[0077] Table 1 PCR primers

[0078]

[0079] Obtain the target gene by RT-PCR:

[0080] RNA was extracted from sheep liver tissue, and the target genes of IL-2 and IFN-γ were obtained by reverse transcription. The gene sequences of the two are shown in SEQUENCE LISTING 400 and SEQUENCE LISTING 400 respectively;

[0081]...

Embodiment 2

[0114] A fusion protein composed of sheep interleukin 2 and sheep interferon gamma. Others are the same as in Example 1, except that the competent cells of E. coli BL21 (DE3) are replaced with BL21 (DE3) competent cells with pGro7 plasmid cell. The SDS-PAGE electrophoresis results of the fusion protein were compared with those in Example 1. The dominant expression band at about 54.6KD in the supernatant was thicker, indicating that after the molecular chaperone pGro7 was introduced, the expression of the target protein in the supernatant was better. The amount of fusion protein is higher. Most of the proteins expressed by Escherichia coli are present in inclusion bodies; by introducing molecular chaperones into the expression strains, the co-expressed proteins can be folded correctly to achieve protein soluble expression.

[0115] The BL21(DE3) competent cell with pGro7 plasmid was purchased from Shanghai Nearshore Technology Co., Ltd. / Xinbainuo Biotechnology, catalog number V20...

Embodiment 3

[0117] A fusion protein composed of sheep interleukin 2 and sheep interferon gamma, the preparation method is as follows:

[0118] 1. Obtain and amplify the target genes of sheep interleukin 2 (IL-2) and sheep interferon gamma (IFN-γ)

[0119] The IL-2 and IFN-γ in Example 1 were optimized, and the IL-2 and IFN-γ target genes were artificially synthesized. After optimization, the nucleotide sequences of the two were as SEQUENCE LISTING 400 and SEQUENCE LISTING 400, respectively As shown in .

[0120] 1.1 Codon optimization

[0121] There are 64 genetic codes, but most organisms tend to use some of these codons. Those that are used most frequently are called optimal codons, and those that are not frequently used are called rare or low-usage codons. In fact, every organism (including Escherichia coli, yeast, mammalian cells, plant cells and insect cells) commonly used for protein expression or production shows a certain degree of difference or preference in codon usage. The expressio...

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Abstract

The invention discloses a recombinant goat long-acting interferon gamma, a fusion protein for preparing the long-acting interferon gamma and a preparation method thereof. The fusion protein is formedby connecting goat interleukin 2 and a goat interferon through a flexible linker. The fusion protein and a freezing and drying protecting agent are blended and frozen and dried to obtain the recombinant goat long-acting interferon gamma. The recombinant goat long-acting interferon gamma can improve the half-life period of the goat interferon remarkably. Compared with the half-life period of a common goat interferon gamma, the half-life period of the recombinant goat long-acting interferon gamma is improved by over 10 times. The recombinant goat long-acting interferon gamma also has broad spectrum antiviral action and can improve immune response of the goat.

Description

Technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to a recombinant sheep long-acting interferon gamma and a fusion protein for preparing the long-acting interferon gamma and a preparation method thereof. Background technique [0002] Animal infectious diseases caused by the virus have severely restricted the healthy development of the breeding industry in various countries and regions. China is the country with the largest number of sheep stocks, slaughters, 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 breeders, but more seriously, some zoonotic diseases also Bring potential threats to human health. [0003] At prese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/00C12R1/19
CPCC07K14/55A61P31/12A61P37/00C07K14/57C07K2319/00C12N15/70C12N2800/22
Inventor 杨建伟高耀辉徐慕珍周炜王亚男赵雨徐文俊付超
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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