Human recombinant interferon fusion gene obtained based on gene modification, fusion protein and preparation method thereof

A technology of fusion protein and fusion gene, which is applied to the preparation method of peptides, methods based on microorganisms, cytokines/lymphokines/interferons, etc., and can solve the problems of no biological activity, affecting production efficiency and production cost, and renaturation. Problems such as difficulty in purification and purification, to achieve the effect of simplified purification steps and high purity

Inactive Publication Date: 2020-05-19
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The target protein usually has no biological activity after being expressed as inclusion body, and its renaturation and purification are very difficult
At present,

Method used

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  • Human recombinant interferon fusion gene obtained based on gene modification, fusion protein and preparation method thereof
  • Human recombinant interferon fusion gene obtained based on gene modification, fusion protein and preparation method thereof
  • Human recombinant interferon fusion gene obtained based on gene modification, fusion protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0041] Example 1

[0042] The histidine tag and the glutamic acid lysine repeat unit were introduced into the original human recombinant interferon IFN-β1b (GenScript Biotech Corporation) gene sequence by PCR amplification to obtain a recombinant human interferon IFN-β based on genetic modification The nucleotide sequence of the fusion protein is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0043] The PCR amplification reaction system is as follows: first, 2.5 μL of the gene sequence SEQ ID NO. 3 of 50 repeating KE amino acid units is used as the template DNA, 0.5 μL of the gene sequence SEQ ID NO. 4: CATATGGAAAACCTGTATTT as the upstream primer, and 0.5 μL of the gene sequence of SEQ ID NO .5: CTTTTTCTTAAG CTACTA CTATTT is used as the downstream primer, and distilled water is made up to 50μL. The reaction process is as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 5°C for 30s, extension at 72°C for 40s,...

Example Embodiment

[0044] Example 2

[0045] The IFN-KE50 fusion gene and 2 μg PET-28a(+) plasmid (Solarbio|Cat. No.: P3110) obtained in Example 1 were digested with restriction enzymes NdeI and NotI, respectively. The reaction system is: 1.5μL Nde Ⅰ enzyme, 1.5μL Not Ⅰ enzyme, 5μL 10×Buffer, 2-5μg pET-28a(+) plasmid or IFN-KE50 fusion gene, ddH 2 Make up to 50μL of O and bath at 37°C for 30min. The digested IFN-KE50 fusion gene and pET-28a(+) plasmid were recovered from agarose gel, and the IFN-KE50 fusion plasmid was obtained by ligating at 16°C for 4hrs with T4 ligase, and then the IFN-KE50 fusion plasmid was transfected Into the competent cells of E. coli (BL21(DE3)), spread on an LB plate containing 50μg / mL kanamycin, and invert the culture overnight at 37°C. Pick colonies on the plate and place them in 50 mL of kanamycin-containing LB liquid medium, with a kanamycin concentration of 50 μg / mL, and culture at 37°C for 12 hours to expand the culture to obtain the transfected IFN-KE50 fusion pla...

Example Embodiment

[0046] Example 3

[0047] Take 3 mL of E. coli transfected with IFN-KE50 fusion gene in a volume ratio of 1 to 100 in 300 mL of 2×YT medium, add kanamycin to a concentration of 50 μg / mL, and place it at 37°C After culturing for 2.5 hours, add the IPTG inducer to make the concentration in the culture medium 0.1mM / L. After culturing and expressing for 12 hours at a temperature of 16°C, an E. coli broth containing IFN-KE50 fusion protein is obtained.

[0048] After adding the IPTG inducer according to the above steps, culture and express at 37°C for 12h, and the rest of the steps remain unchanged to obtain the E. coli strain containing the IFN-KE50 fusion protein induced at 37°C.

[0049] The SDS-PAGE electrophoresis of the IFN-KE50 fusion protein is as figure 1 As shown, the expression under two different temperature conditions was investigated, among which 1 is Marker; 2 is 16℃IFN-KE50 supernatant; 3 is 16℃IFN-KE50 precipitation; 4 is 37℃IFN-KE50 supernatant ; 5 is 37°C IFN-KE50 prec...

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Abstract

The invention discloses a human recombinant interferon fusion gene obtained based on gene modification, a fusion protein and a preparation method thereof, and belongs to the technical field of biological products. The gene of the human recombinant interferon IFN-beta is obtained based on gene modification, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The amino acid sequence ofthe fusion protein encoded by the gene is shown as SEQ ID NO. 2. The human recombinant interferon IFN-beta genetic sequence is amplified and connected with plasmid pET-28a (+) to obtain a fusion gene, and then the fusion gene is transfected into escherichia coli BL21 (DE3) to be amplified and expressed to obtain soluble fusion protein. The problem that soluble interferon cannot be obtained from escherichia coli is solved. The invention further provides a purification method for obtaining the high-purity IFN-KE50 protein from the stable cell line, due to the fact that the protein is provided with the histidine tag, the target protein can be effectively purified by conducting affinity chromatography through immobilized metallic nickel, and the purity of the purified protein can reach 94.8%.The subsequent purification steps are simplified, and higher purity can be obtained.

Description

technical field [0001] The invention belongs to the technical field of biological products, and in particular relates to a fusion gene, fusion protein and preparation method of human recombinant interferon based on gene modification. [0002] technical background [0003] Interferon is a kind of glycoprotein with multiple functions produced by inducers to stimulate reticuloendothelial cells, macrophages, lymphocytes, etc., and has genera specificity. They have broad-spectrum antiviral activity and immunomodulatory functions. According to the gene type, structural characteristics and functional role of interferon, as well as the type of receptor on the cell surface, interferon is currently divided into three types. Type I interferons include IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-ω, IFN-κ, IFN-ζ and IFN-τ. Among them, IFN-α has more than 20 subtypes; IFN-β has only one subtype; type II interferon is IFN-γ; type III interferon is IFN-λ, which can be divided into IFN-λ1, IFN- λ2 and...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/70C12N1/21C07K19/00C07K1/22C12R1/19
CPCC07K14/555C07K14/56C07K14/565C07K2319/00C07K2319/21C12N15/70
Inventor 何炜孙彬玉程昉李明洋董继程刘波
Owner DALIAN UNIV OF TECH
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