Preparation method and use of high antiviral active recombination chicken gamma-interferon crCHIFN-gamma

An interferon and virus technology, applied in the field of the preparation of broad-spectrum high-efficiency antiviral genetic engineering products, can solve the problems of low rChIFN-γ activity and no antiviral activity, etc.

Inactive Publication Date: 2005-08-24
YANGZHOU UNIV
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Problems solved by technology

The laboratory also successfully cloned the ChIFN-γ gene, and expressed and applied it in Escherichia coli and COS cells. It was found that the prokaryotic expressed rChIFN-γ had no antiviral activity, and the rChIFN-γ transiently expressed in COS cells had a higher activity than Low

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  • Preparation method and use of high antiviral active recombination chicken gamma-interferon crCHIFN-gamma
  • Preparation method and use of high antiviral active recombination chicken gamma-interferon crCHIFN-gamma
  • Preparation method and use of high antiviral active recombination chicken gamma-interferon crCHIFN-gamma

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Embodiment Construction

[0015] 1. Method

[0016] 1.1 Construction of baculovirus recombinant chicken γ-interferon transfer vector pFASTBAC1-ChIFN-γ

[0017] The eukaryotic plasmid pcDNA-ChIFN-γ was double-digested with restriction endonucleases EcoR I and Not I (purchased from Treasure Biotech Dalian Co., Ltd.), the ChIFN-γ fragment was recovered, and the recovered product of pFASTBAC1 digested with the same enzyme was ligated overnight at 4°C. DH5α competent bacteria were transformed, and the recombinant plasmid selected was named pFASTBAC1-ChIFN-γ.

[0018] 1.2 Construction and identification of recombinant baculovirus expression plasmid Bacmid-ChIFN-γ

[0019] Take DH10Bac competent cells, add 1ng pFASTBAC1-ChIFN-γ, after transposition, use SOC medium to serially dilute the culture 10 times, take 100 μl each to coat Luria plates, culture at 37°C for 24-48 hours, pick white colonies, Luria plate (containing ampicillin, kanamycin, gentamicin, tetracycline, IPTG and X-gal antibiotic plate) was scr...

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Abstract

A process for preparing the recombinant chicken gamma-interferon (rCHIFN-gamma) with high activirus activity includes such steps as cloning ChIFN-gamma gene from eukaryotic plasmid to transfer carrier by dual enzyme servenings to obtain recombinant transfer plasmid pFASTBACI-ChIFN-gamma, taking DH10Bac competent cell, adding IngpFASTBAC1-ChIFN-gamma, transposition, diluting to culture object by SOD culture medium, coating on Luria plate, culturing, choosing white clony, purifying, naming positive plasmid as Bacmid-ChIFN-gamma, extracting its DNA, and transfecting Sf9 cells. It can be used to suppress the pathogenic action of both MDV GA to CEF and AIV(H5NI) virus to cell, and the reproduction of NDV F48E8 on cells.

Description

technical field [0001] The invention relates to a preparation method of a broad-spectrum high-efficiency antiviral genetic engineering product. Background technique [0002] Chicken γ-interferon (ChIFN-γ) is a cytokine produced by activated T cells and NK cells stimulated by mitogens or specific antigens, with a molecular weight of 17-20KD. IFN-γ has biological activities such as anti-virus, anti-tumor cell growth and differentiation, and plays an important role in immune regulation. The ChIFN-γ gene was first cloned by Digby and Lowenthal in 1995, and then domestic and foreign scholars successively expressed ChIFN-γ with Escherichia coli, COS cells, baculovirus, fowl pox virus, avian adenovirus and plant tobacco mosaic virus. Biological activity has been extensively studied. The laboratory also successfully cloned the ChIFN-γ gene, and expressed and applied it in Escherichia coli and COS cells. It was found that the prokaryotic expressed rChIFN-γ had no antiviral activity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/21C12N15/09C12N15/23C12N15/63
Inventor 秦爱建许金俊孔桂美刘岳龙金文杰
Owner YANGZHOU UNIV
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