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Chicken albumin-interferon alpha-interleukin 2 fusion protein, preparation method of fusion protein, coding genes thereof and chicken pegylated interferon

A technology of fusion protein and interferon α, applied in interferon, biochemical equipment and methods, cytokines/lymphokines/interferon, etc., can solve the problems of small molecular weight of interferon, unfavorable clinical application, short half-life, etc.

Inactive Publication Date: 2017-11-28
ANHUI JIUCHUAN BIOTECH
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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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  • Chicken albumin-interferon alpha-interleukin 2 fusion protein, preparation method of fusion protein, coding genes thereof and chicken pegylated interferon
  • Chicken albumin-interferon alpha-interleukin 2 fusion protein, preparation method of fusion protein, coding genes thereof and chicken pegylated interferon
  • Chicken albumin-interferon alpha-interleukin 2 fusion protein, preparation method of fusion protein, coding genes thereof and chicken pegylated interferon

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The preparation method of chicken albumin-interferon alpha-interleukin 2 fusion protein includes the following steps:

[0092] 1. Acquisition and amplification of the target genes of chicken albumin (Alb), chicken interferon alpha (IFN-α) and chicken interleukin 2 (IL-2)

[0093] Primer design:

[0094] Design and synthesize primers according to the target gene sequence reported in Genebank, see Table 1. Introduce EcoRI restriction site in the upstream primer of chicken albumin, introduce Linker sequence in the downstream primer, and in the upstream primer and downstream primer of chicken interferon α Introduce Linker sequence in the chicken interleukin-2, the Linker sequence in the upstream primer of chicken interleukin 2, and the XhoI restriction site in the downstream primer.

[0095] Table 1 PCR primers

[0096]

[0097]

[0098] Obtain the target gene by RT-PCR:

[0099] RNA was extracted from chicken liver tissue, and the target genes of chicken Alb, chicken IFN-α and chick...

Embodiment 2

[0136] A preparation method of chicken albumin-interferon alpha-interleukin 2 fusion protein. The preparation method is the same as in Example 1, except that the competent cells of Escherichia coli BL21 (DE3) are replaced with BL21 (DE3) competent cells with pGro7 plasmid State cell. The SDS-PAGE electrophoresis results of the fusion protein were compared with those in Example 1. The dominant expression band at around 125.3KD 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 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.

[0137] The BL21(DE3) competent cell with pGro7 plasmid was purchased from Shanghai Nearshore Technology Co., Ltd. / ...

Embodiment 3

[0140] A preparation method of chicken albumin-interferon alpha-interleukin 2 fusion protein, the preparation method is as follows:

[0141] 1. Acquisition and amplification of the target genes of chicken albumin (Alb), chicken interferon alpha (IFN-α) and chicken interleukin 2 (IL-2)

[0142] The chicken Alb, chicken IFN-α and chicken IL-2 in Example 1 were optimized, and the target genes of chicken Alb, chicken IFN-α and chicken 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.

[0143] 1.1 Codon optimization

[0144] 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 chicken albumin-interferon-α-interleukin-2 fusion protein, a preparation method and its coding gene, a chicken long-acting interferon, and chicken albumin, chicken interferon-α and chicken interleukin-2 are connected through a flexible flexible linker , obtain chicken albumin-interferon-α-interleukin-2 fusion protein, design and optimize its coding gene, and finally prepare recombinant chicken long-acting interferon, which can significantly increase the half-life of chicken interferon, which is 20 times higher than that of common chicken interferon Above, and has broad-spectrum antiviral effect and can improve the immune response of chicken itself.

Description

Technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to a chicken albumin-interferon alpha-interleukin 2 fusion protein, a preparation method and its coding gene, and a chicken long-acting interferon. The chicken albumin, chicken interferon alpha and chicken interleukin 2 are connected through a flexible linker to obtain a chicken albumin-interferon alpha-interleukin 2 fusion protein, and the coding gene is designed and optimized, and finally recombinant chicken interferon is prepared. Background technique [0002] In recent years, with the scale of the breeding industry and the rapid development of the circulation of livestock and poultry and its products, our country's poultry industry has made great progress and formed a huge industry with an annual output value of more than 100 billion yuan. However, due to my country's weak animal epidemic prevention system, poultry disease is still a serious problem ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61P31/12A61P37/04C12R1/19
CPCC07K14/56C07K14/55C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 赵俊李树启高耀辉戚仕梅赖鹏飞周炜蒋敏之刘家炉
Owner ANHUI JIUCHUAN BIOTECH
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