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Goat albumin-interferon tau-interleukin 2 fused protein, preparation method and encoding gene thereof and goat long-acting interferon

A technology of fusion protein and interferon, which is applied in the direction of interferon, biochemical equipment and methods, cytokines/lymphokines/interferon, etc., and can solve problems such as unfavorable clinical application, high cost of interferon, and small molecular weight of interferon

Inactive Publication Date: 2017-10-17
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

[0009] 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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  • Goat albumin-interferon tau-interleukin 2 fused protein, preparation method and encoding gene thereof and goat long-acting interferon
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  • Goat albumin-interferon tau-interleukin 2 fused protein, preparation method and encoding gene thereof and goat long-acting interferon

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] The preparation method of ovine albumin-interferon tau-interleukin 2 fusion protein comprises the following steps:

[0093] 1. Acquisition and amplification of sheep albumin (Alb), sheep interferon τ (IFN-τ) and sheep interleukin 2 (IL-2) target genes

[0094] Primer design:

[0095] Synthetic primers were designed and synthesized according to the target gene sequence reported in Genebank, as shown in Table 1. The EcoRI restriction site was introduced into the upstream primer of ovine albumin, the Linker sequence was introduced into the downstream primer, and the upstream primer and downstream primer of ovine interferon τ The Linker sequence was introduced into the upstream primer of sheep interleukin 2, and the Linker sequence was introduced into the downstream primer, and a SalI restriction site was introduced into the downstream primer.

[0096] Table 1 PCR amplification primers

[0097]

[0098]

[0099] RT-PCR to obtain the target gene:

[0100] RNA was ex...

Embodiment 2

[0137] A method for preparing ovine albumin-interferon τ-interleukin 2 fusion protein, the preparation method is the same as in Example 1, except that the Escherichia 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. The dominant expression band at about 125.9KD 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.

[0138] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Techn...

Embodiment 3

[0141] A method for preparing ovine albumin-interferon tau-interleukin 2 fusion protein, the preparation method is as follows:

[0142]1. Acquisition and amplification of sheep albumin (Alb), sheep interferon τ (IFN-τ) and sheep interleukin 2 (IL-2) target genes

[0143] Goat Alb, goat IFN-τ and goat IL-2 in Example 1 were optimized, and goat Alb, goat IFN-τ and goat 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.

[0144] 1.1 Codon optimization

[0145] 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 (opτimal codons), and those that are not frequently used are called rare or low-usage codons (rare or low-usage codons). Virtually every organism commonly used for protein expression or production (including E. coli, yeast, mammalian cells, plan...

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Abstract

The invention discloses ovine albumin-interferon τ-interleukin-2 fusion protein, a preparation method and its coding gene, a kind of ovine long-acting interferon, and ovine albumin, ovine interferon τ and ovine interleukin-2 are connected through a flexible linker , obtain ovine albumin-interferon τ-interleukin-2 fusion protein, design and optimize its coding gene, and finally prepare recombinant ovine long-acting interferon, which can significantly increase the half-life of ovine interferon, which is 17 times longer than that of ordinary ovine interferon Above, and has broad-spectrum antiviral effect and can improve the immune response of sheep itself.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a sheep albumin-interferon tau-interleukin 2 fusion protein, a preparation method and a coded gene thereof, and a sheep peginterferon. The ovine albumin, ovine interferon τ and ovine interleukin 2 were connected through a flexible linker to obtain an ovine albumin-interferon τ-interleukin 2 fusion protein, and its coding gene was designed and optimized to finally prepare the recombinant ovine long-acting interferon. Background technique [0002] Animal infectious diseases caused by viruses have seriously restricted the healthy development of the breeding industry in various countries and regions. China is the country with the largest sheep stock, slaughter and mutton production in the world. The mutton sheep industry is also one of the important pillar industries of my country's animal husbandry. . With the continuous development of the sheep breeding in...

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
CPCC07K14/555C07K14/55C07K2319/31C12N15/62C12N15/70C12N2800/22
Inventor 赵俊沈咏舟李树启高耀辉戚仕梅赖鹏飞周炜刘家炉
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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