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Fusion protein with porcine albumin, porcine interferons gamma and porcine interleukins 2 and method for preparing fusion protein

A technology for interleukin and fusion protein, applied in the field of fusion protein and its preparation, can solve the problems of high cost of interferon, unfavorable clinical application, small molecular weight of interferon, etc.

Inactive Publication Date: 2017-11-21
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

Method used

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  • Fusion protein with porcine albumin, porcine interferons gamma and porcine interleukins 2 and method for preparing fusion protein
  • Fusion protein with porcine albumin, porcine interferons gamma and porcine interleukins 2 and method for preparing fusion protein
  • Fusion protein with porcine albumin, porcine interferons gamma and porcine interleukins 2 and method for preparing fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] A fusion protein composed of porcine albumin, porcine interferon gamma and porcine interleukin 2, and its preparation method is as follows:

[0090] 1. Porcine albumin (Alb), porcine interferon γ (IFN-γ) and porcine interleukin 2 (IL-2) target gene acquisition and amplification

[0091] Primer design:

[0092] Design and synthesize the primers according to the target gene sequence reported in Genebank, see Table 1. The NcoI restriction site and Linker sequence were introduced into the upstream primer and downstream primer of porcine albumin, respectively, in the upstream primer and downstream primer of porcine interferon γ The Linker sequence was introduced separately, and the Linker sequence and HindⅢ restriction site were introduced into the upstream and downstream primers of porcine interleukin-2, respectively.

[0093] Table 1 PCR primers

[0094]

[0095]

[0096] Obtain the target gene by RT-PCR:

[0097] RNA was extracted from pig liver tissue, and the target genes of pig ...

Embodiment 2

[0132] A fusion protein consisting of porcine albumin, porcine interferon gamma and porcine interleukin 2. The others are the same as in Example 1, except that the competent cells of E. coli BL21 (DE3) are replaced with BL21 (with pGro7 plasmid). DE3) Competent cells. The SDS-PAGE electrophoresis results of the fusion protein were compared with Example 1. The dominant expression band at about 119.8KD in the supernatant was thicker, indicating that after the introduction of molecular chaperone pGro7, the expression of the target protein in the supernatant was better. The amount of fusion protein is higher. Most of the proteins expressed by E. 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.

[0133] The BL21(DE3) competent cell with pGro7 plasmid was purchased from Shanghai Nearshore Technology Co., Ltd. / Xinbainuo Bio, catalog number V205....

Embodiment 3

[0135] A fusion protein composed of porcine albumin, porcine interferon gamma and porcine interleukin 2, and its preparation method is as follows:

[0136] 1. Porcine albumin (Alb), porcine interferon γ (IFN-γ) and porcine interleukin 2 (IL-2) target gene acquisition and amplification

[0137] The porcine Alb, porcine IFN-γ and porcine IL-2 in Example 1 were optimized, and the target genes of porcine Alb, porcine IFN-γ and porcine IL-2 were artificially synthesized. After optimization, the nucleotide sequences of the three were as follows: SEQUENCE LISTING 400 , SEQUENCE LISTING 400 and SEQUENCE LISTING 400 are shown.

[0138] 1.1 Codon optimization

[0139] 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 commonly used for protein expression or production (including E. coli, yeast, mammal...

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Abstract

The invention discloses a fusion protein composed of porcine albumin, porcine interferon gamma and porcine interleukin 2 and a preparation method thereof. The fusion protein is composed of porcine albumin, porcine interferon gamma and porcine interleukin 2 through a flexible linker The recombinant porcine peginterferon can be obtained after the fusion protein is mixed with a freeze-drying protective agent and freeze-dried. The recombinant porcine long-acting interferon can significantly increase the half-life of porcine interferon, which is more than 23 times higher than that of common porcine interferon, has broad-spectrum antiviral effect and can improve the immune response of pigs themselves.

Description

Technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to a fusion protein composed of porcine albumin, porcine interferon gamma and porcine interleukin 2 and a preparation method thereof. Background technique [0002] In recent years, large-scale and intensive pig farms have rapidly emerged in my country. The number of live pigs and total pork production in my country have been ranked first in the world. Traditional pig disease prevention methods are far from being adapted to the control of infectious diseases in large-scale intensive pig production. In the past 20 years, nearly 20 kinds of infectious diseases of livestock and poultry have appeared in our country, and the original animal diseases have caused huge economic losses to our country's breeding industry. According to incomplete statistics, the mortality rate of livestock and poultry caused by various viral diseases in my country is as high as 15%-...

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
CPCC07K14/57C07K14/55C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 王明丽韩国祥符修乐王利利夏兵兵徐慕珍朱亚召付超
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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