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Pig albumin-interferon alpha-interleukin 2 fusion protein as well as preparation method and encoding gene thereof, and long-acting interferon

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

Inactive Publication Date: 2017-11-24
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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  • Pig albumin-interferon alpha-interleukin 2 fusion protein as well as preparation method and encoding gene thereof, and long-acting interferon
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  • Pig albumin-interferon alpha-interleukin 2 fusion protein as well as preparation method and encoding gene thereof, and long-acting 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 includes the following steps:

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

[0092] Primer design:

[0093] 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 porcine albumin, introduce Linker sequence in the downstream primer, and in the upstream primer and downstream primer of porcine interferon α The Linker sequence was introduced into the porcine interleukin-2, the Linker sequence was introduced in the upstream primer of porcine interleukin 2, and the SalI restriction site was introduced in the downstream primer.

[0094] Table 1 PCR primers

[0095]

[0096]

[0097] Obtain the target gene by RT-PCR:

[0098] RNA was extracted from pig liver tissue, and the target genes of pig Al...

Embodiment 2

[0135] A preparation method of porcine albumin-interferon alpha-interleukin 2 fusion protein. The preparation method is the same as that in Example 1, except that the E. coli BL21 (DE3) competent cells are replaced with the BL21 (DE3) competent cells with pGro7 plasmid cell. The SDS-PAGE electrophoresis results of the fusion protein were compared with Example 1. The dominant expression band at about 126.4KD 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.

[0136] The BL21(DE3) competent cell with pGro7 plasmid was purchased from Shanghai Nearshore Technology Co., Ltd. / Xinbainuo Bio, cata...

Embodiment 3

[0139] A preparation method of porcine albumin-interferon alpha-interleukin 2 fusion protein, the preparation method is as follows:

[0140] 1. Acquisition and amplification of porcine albumin (Alb), porcine interferon alpha (IFN-α) and porcine interleukin 2 (IL-2) target genes

[0141] The porcine Alb, porcine IFN-α and porcine IL-2 in Example 1 were optimized, and the target genes of porcine Alb, porcine IFN-α 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.

[0142] 1.1 Codon optimization

[0143] 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, mammalian cel...

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Abstract

The invention discloses pig albumin-interferon alpha-interleukin 2 fusion protein as well as a preparation method and an encoding gene thereof, and pig long-acting interferon. The pig albumin-interferon alpha-interleukin 2 fusion protein is obtained by connecting pig albumin, pig interferon alpha and pig interleukin 2 through a flexible linker, the encoding gene of the pig albumin-interferon alpha-interleukin 2 fusion protein is designed and optimized, and the recombinant pig long-acting interferon is finally prepared, so that the half-life period of the pig interferon can be prolonged obviously, the half-life period is prolonged by 27 times or above compared with that of the common pig interferon, the broad-spectrum antiviral action is achieved, and the autoimmune response of the pigs can be improved.

Description

Technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to a porcine albumin-interferon alpha-interleukin 2 fusion protein, a preparation method and its coding gene, and a porcine long-acting interferon. Porcine albumin, porcine interferon α and porcine interleukin 2 are connected through a flexible linker to obtain a porcine albumin-interferon α-interleukin 2 fusion protein, and its coding gene is designed and optimized, and finally recombinant porcine long-acting interferon is prepared. 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 ranked first in the world. Traditional methods of prevention and control of swine diseases are far from adapting to the control of infectious diseases in large-scale intensive pig production. In the past 20 years, nearly 20 kinds of infecti...

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 赵俊韩国祥李树启高耀辉戚仕梅赖鹏飞周炜凡玉芳
Owner ANHUI JIUCHUAN BIOTECH
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