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Fusion protein composed of canine albumin, canine interferon gamma and canine interleukin 2 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 small molecular weight of interferon, unfavorable clinical application, short half-life, etc.

Inactive Publication Date: 2017-10-20
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 caused by the small molecular weight of interferon at the level of molecular weight
[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 composed of canine albumin, canine interferon gamma and canine interleukin 2 and preparation method thereof
  • Fusion protein composed of canine albumin, canine interferon gamma and canine interleukin 2 and preparation method thereof
  • Fusion protein composed of canine albumin, canine interferon gamma and canine interleukin 2 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0092] Primer design:

[0093] 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 albumin, respectively, and introduce the upstream primer and downstream primer of canine interferon gamma The Linker sequence was introduced respectively, and the Linker sequence and the XhoI restriction site were respectively introduced into the upstream primer and the downstream primer of canine interleukin 2.

[0094] Table 1 PCR amplification primers

[0095]

[0096]

[0097] RT-PCR to obtain the target gene:

[0098] RNA was extracted...

Embodiment 2

[0133] A fusion protein composed of canine albumin, canine interferon gamma and canine interleukin 2, the others are the same as in Example 1, except that the Escherichia coli BL21 (DE3) competent cells are replaced with BL21(DE3) 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 122.7KD 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.

[0134] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology C...

Embodiment 3

[0136] A fusion protein composed of canine albumin, canine interferon gamma and canine interleukin 2, the preparation method of which is as follows:

[0137] 1. Acquisition and amplification of canine albumin (Alb), canine interferon gamma (IFN-gamma) and canine interleukin 2 (IL-2) target genes

[0138]The canine Alb, canine IFN-γ and canine IL-2 in Example 1 were optimized, and the target genes of canine Alb, canine IFN-γ and canine IL-2 were artificially synthesized. After optimization, the nucleotide sequences of the three were respectively as follows SEQUENCE LISTING 400, SEQUENCELISTING 400 and SEQUENCE LISTING 400.

[0139] 1.1 Codon optimization

[0140] 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, yeas...

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Abstract

The invention discloses a fusion protein composed of canine albumin, canine interferon gamma and canine interleukin 2 and a preparation method thereof. The fusion protein is composed of canine albumin, canine interferon gamma and canine interleukin 2. Formed by flexible linker connection, the fusion protein is mixed with a lyoprotectant and freeze-dried to obtain the recombinant canine peginterferon. The recombinant canine peginterferon can significantly increase the half-life of canine interferon, which is more than 18 times higher than that of common canine interferon, has broad-spectrum antiviral effect and can improve the immune response of canine itself.

Description

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

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/04C12R1/19
CPCC07K14/57C07K14/55C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽符修乐王利利夏兵兵徐慕珍朱亚召付超杨建伟
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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