Fusion protein prepared from dog interleukin 2, dog interferon gamma and dog interferon alpha and preparation method thereof

A technology of interleukin and fusion protein, applied in the field of fusion protein and its preparation, can solve the problems of short half-life, unfavorable clinical application, and high cost of interferon

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

[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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  • Fusion protein prepared from dog interleukin 2, dog interferon gamma and dog interferon alpha and preparation method thereof
  • Fusion protein prepared from dog interleukin 2, dog interferon gamma and dog interferon alpha and preparation method thereof
  • Fusion protein prepared from dog interleukin 2, dog interferon gamma and dog interferon alpha and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] A fusion protein composed of canine interleukin 2, canine interferon gamma and canine interferon alpha, the preparation method of which is as follows:

[0091] 1. Acquisition and amplification of canine interleukin 2 (IL-2), canine interferon γ (IFN-γ) and canine interferon α (IFN-α) target genes

[0092] Primer design:

[0093] The primers were designed and synthesized according to the target gene sequence reported in Genebank, as shown in Table 1. The BamHI restriction site and Linker sequence were respectively introduced into the upstream and downstream primers of canine interleukin 2, and the upstream and downstream primers of canine interleukin-γ The Linker sequence was introduced into the primers, and the Linker sequence and XhoI restriction site were respectively introduced into the upstream primer and downstream primer of canine interferon α.

[0094] Table 1 PCR amplification primers

[0095]

[0096] RT-PCR to obtain the target gene:

[0097] RNA was ext...

Embodiment 2

[0132] A fusion protein composed of canine interleukin 2, canine interferon gamma and canine interferon alpha, the others are the same as in Example 1, except that the competent cells of Escherichia coli BL21 (DE3) are replaced by BL21 with pGro7 plasmid (DE3) Competent cells. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 76.4KD 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.

[0133] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technol...

Embodiment 3

[0135] A fusion protein composed of canine interleukin 2, canine interferon gamma and canine interferon alpha, the preparation method of which is as follows:

[0136] 1. Acquisition and amplification of canine interleukin 2 (IL-2), canine interferon γ (IFN-γ) and canine interferon α (IFN-α) target genes

[0137]The canine IL-2, canine IFN-γ and canine IFN-α in Example 1 were optimized, and the target genes of canine IL-2, canine IFN-γ and canine IFN-α were artificially synthesized. After optimization, the nucleosides of the three The acid sequences are shown in SEQUENCE LISTING 400, SEQUENCE LISTING 400 and SEQUENCE LISTING 400, respectively.

[0138] 1.1 Codon optimization

[0139] 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 (in...

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Abstract

The invention discloses fusion protein prepared from dog interleukin 2, dog interferon gamma and dog interferon alpha and a preparation method thereof. The fusion protein is formed by dog interleukin2, dog interferon gamma and dog interferon alpha through the connection by flexible linkers. A recombinant dog pegylated interferon can be obtained through freeze drying after the mixing of the fusionprotein and freeze drying protection agents. The recombinant dog pegylated interferon can obviously prolong the dog interferon half life; the half life is prolonged by 15 times or more through beingcompared with that of ordinary dog interferon; the broad-spectrum antiviral effects are achieved; the immune response of the dog per se can be improved.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a fusion protein composed of canine interleukin 2, canine interferon gamma and canine interferon alpha 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 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...

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
CPCC07K14/57A61P31/12A61P37/04C07K14/55C07K14/56C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 赵雨单雪芹徐慕珍杨建伟高耀辉周炜郭志燕
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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