Porcine albumin-interferon alpha-interleukin 2 fusion protein, preparation method as well as coding gene thereof, and porcine pegylated interferon

A technology of fusion protein and interferon α, applied in interferon, biochemical equipment and methods, cytokines/lymphokines/interferon, etc., can solve problems such as short half-life, unfavorable clinical application, and high cost of interferon

Inactive Publication Date: 2018-11-20
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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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  • Porcine albumin-interferon alpha-interleukin 2 fusion protein, preparation method as well as coding gene thereof, and porcine pegylated interferon
  • Porcine albumin-interferon alpha-interleukin 2 fusion protein, preparation method as well as coding gene thereof, and porcine pegylated interferon
  • Porcine albumin-interferon alpha-interleukin 2 fusion protein, preparation method as well as coding gene thereof, and porcine pegylated interferon

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] The preparation method of porcine albumin-interferon alpha-interleukin 2 fusion protein comprises the following steps:

[0091] 1. Acquisition and amplification of porcine albumin (Alb), porcine interferon-α (IFN-α) and porcine 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 EcoRI restriction site into the upstream primer of porcine albumin, introduce Linker sequence into the downstream primer, and introduce the upstream primer and downstream primer of porcine interferon α The Linker sequence was introduced into the upstream primer of porcine interleukin 2 respectively, and the Linker sequence was introduced into the downstream primer of SalI restriction site.

[0094] Table 1 PCR amplification primers

[0095]

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

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

Embodiment 2

[0134] A preparation method of porcine albumin-interferon α-interleukin 2 fusion protein, the preparation method is the same as in Example 1, except that Escherichia coli BL21 (DE3) competent cells are replaced by BL21 (DE3) competent cells with pGro7 plasmid cell. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 126.4KD 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.

[0135] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Lt...

Embodiment 3

[0138] A preparation method of porcine albumin-interferon alpha-interleukin 2 fusion protein, its preparation method is as follows:

[0139]1. Acquisition and amplification of porcine albumin (Alb), porcine interferon-α (IFN-α) and porcine interleukin-2 (IL-2) target genes

[0140] The pig Alb, pig IFN-α and pig IL-2 in Example 1 are optimized, and the target genes of pig Alb, pig IFN-α and pig IL-2 are artificially synthesized. After optimization, the nucleotide sequences of the three are respectively as follows SEQUENCE LISTING 400, SEQUENCELISTING 400 and SEQUENCE LISTING 400.

[0141] 1.1 Codon optimization

[0142] 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...

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Abstract

The invention discloses a porcine albumin-interferon alpha-interleukin 2 fusion protein, a preparation method as well as a coding gene thereof, and porcine pegylated interferon. Porcine albumin, porcine interferon alpha and porcine interleukin 2 are connected through a flexible linker to obtain the porcine albumin-interferon alpha-interleukin 2 fusion protein, the coding gene of the fusion proteinis designed to be optimized, and the recombinant porcine pegylated interferon is prepared finally; the porcine pegylated interferon can significantly improve a half-life period of porcine interferon,which is 27 times or more than 27 times of the half-life period of ordinary porcine interferon, has a 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 biogenetic engineering, and in particular relates to a porcine albumin-interferon alpha-interleukin-2 fusion protein, a preparation method and a coding gene thereof, and a porcine peginterferon. Connect porcine albumin, porcine interferon-α and porcine interleukin-2 through a flexible linker to obtain porcine albumin-interferon-α-interleukin-2 fusion protein, design and optimize its coding gene, and finally prepare recombinant porcine peginterferon. Background technique [0002] In recent years, large-scale and intensive pig farms have emerged rapidly in my country. The number of pigs on hand and the total amount of pork production in my country rank first in the world. Traditional methods of prevention and control of pig diseases are far from being able to adapt to the control of infectious diseases in large-scale intensive pig production. Nearly 20 kinds of livestock and poultry infectious diseases have...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C07K14/56C12R1/19
CPCC07K14/55C07K14/56C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 高耀辉鲍可兵徐慕珍杨建伟付超周炜凡玉芳徐文俊
Owner ANHUI JIUCHUAN BIOTECH
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