Bovine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and bovine long-acting interferon

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

Inactive Publication Date: 2018-11-20
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

[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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  • Bovine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and bovine long-acting interferon
  • Bovine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and bovine long-acting interferon
  • Bovine albumin-interferon alpha-interleukin 2 fusion protein, preparation method and coding gene thereof, and bovine long-acting interferon

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0092] 1. Acquisition and amplification primer design of bovine albumin (Alb), bovine interferon-α (IFN-α) and bovine interleukin-2 (IL-2) target genes:

[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 bovine albumin, introduce Linker sequence into the downstream primer, and introduce the upstream primer and downstream primer of bovine interferon α The Linker sequence was introduced into the bovine interleukin 2 upstream primer, and the Linker sequence was introduced into the downstream primer, and the XhoI restriction site was introduced into the downstream primer.

[0094] Table 1PCR amplification primers

[0095]

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

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

Embodiment 2

[0133] A preparation method of bovine albumin-interferon α-interleukin 2 fusion protein, the preparation method is the same as in Example 1, except that E. coli BL21 (DE3) competent cells are replaced by BL21 (DE3) competent cells with pGro7 plasmid state cells. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 122.6KD in the supernatant is relatively thick, 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

[0137] A kind of preparation method of bovine albumin-interferon alpha-interleukin 2 fusion protein, its preparation method is as follows:

[0138] 1. Acquisition and amplification of bovine albumin (Alb), bovine interferon-α (IFN-α) and bovine interleukin-2 (IL-2) target genes

[0139]Bovine Alb, bovine IFN-α, and bovine IL-2 in Example 1 were optimized, and bovine Alb, bovine IFN-α, and bovine IL-2 target genes were artificially synthesized. After optimization, the nucleotide sequences of the three were as follows: SEQUENCE LISTING 400, SEQUENCELISTING 400 and SEQUENCE LISTING 400.

[0140] 1.1 Codon optimization

[0141] 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 in...

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Abstract

The invention discloses a bovine albumin-interferon alpha-interleukin 2 fusion protein, a preparation method and a coding gene thereof, and a bovine long-acting interferon. Bovine albumin, bovine interferon alpha and bovine interleukin 2 are connected by a flexible linker to obtain the bovine albumin-interferon alpha-interleukin 2 fusion protein. The coding gene of the fusion protein is designed and optimized to finally obtain a recombinant bovine long-acting interferon, which can significantly prolong the half-life of the bovine interferon by more than 20 times in comparison with a common bovine interferon, has a broad-spectrum antiviral effect and can improve the immune response of cattle.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a bovine albumin-interferon alpha-interleukin-2 fusion protein, a preparation method and a coding gene thereof, and a bovine peginterferon. Bovine albumin, bovine interferon-α and bovine interleukin-2 were connected through a flexible linker to obtain bovine albumin-interferon-α-interleukin-2 fusion protein, and its coding gene was designed and optimized to finally prepare recombinant bovine peginterferon. Background technique [0002] Cattle are one of the important livestock species in my country. With the continuous development of intensive and large-scale breeding, the incidence of bovine viral infectious diseases is increasing year by year. Many infectious diseases of cattle have been prevalent in domestic large-scale pastures for a long time. Because of the rapid spread of diseases, the high morbidity and mortality seriously restrict the healthy d...

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
CPCC07K14/56C07K14/55C07K2319/31C12N15/70C12N2800/22
Inventor 高耀辉徐慕珍杨建伟周炜蒋敏之刘家炉徐文俊郭志燕
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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