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Soluble prokaryotic expression and purification method and application of cat interferon gamma

A technology of prokaryotic expression and purification method, which is applied in the field of soluble prokaryotic expression and purification of feline interferon gamma, which can solve the problems that IFN-gamma research is rarely reported, and achieve the improvement of soluble expression level, biological activity and easy cultivation and the effect of controlling

Active Publication Date: 2021-07-16
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a new type of interferon, IFN-γ plays a strong role in anti-virus and anti-tumor. In 1995, the chicken IFN-γ gene was successfully cloned for the first time, and then successfully expressed recombinant geese, rabbits, pigs, cattle and other animals. IFN-γ and its use in clinical treatment, but the research on IFN-γ in cats is rarely reported

Method used

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  • Soluble prokaryotic expression and purification method and application of cat interferon gamma
  • Soluble prokaryotic expression and purification method and application of cat interferon gamma
  • Soluble prokaryotic expression and purification method and application of cat interferon gamma

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specific Embodiment 1

[0040] 1. Materials and reagents

[0041] 1.1 Strains, plasmids and main reagents

[0042] Competent cells DH5α and BL21 were purchased from Beijing Qingke Biotechnology Co., Ltd.; T4 DNA ligase, IPTG (isopropylthio-β-D-galactoside), and kanamycin were purchased from TaKaRa Company; Endonucleases BamHI and HindIII were purchased from New England BioLabs; PMSF (phenylmethylsulfonyl fluoride) and SUMO protease were purchased from Beijing Suolaibao Technology Co., Ltd.; Ni-NTA purification column, protein concentration column and retention column were purchased from Merck company.

[0043] 2. Soluble prokaryotic expression and purification method of feline interferon gamma

[0044] 2.1 Analysis of signal peptide sequence and physicochemical properties of FeIFN-γ protein

[0045] Signal peptides and physicochemical properties of the two proteins were predicted and analyzed through SignalP-4.0Server and Expasy online servers, among which,

[0046] SignalP-4.0Server website: htt...

specific Embodiment 2

[0082] Specific embodiment 2 feline interferon gamma antiviral activity, safety and clinical application

[0083] 1. Antiviral Activity Assay

[0084] Taking feline parvovirus as a model virus, the antiviral activity of the feline interferon gamma obtained in the present invention is determined by a microcytopathic inhibition method.

[0085] Inoculate cat kidney cells F81 in 96-well cell plates and store at 37°C, 5% CO 2 Cultivate in an incubator until the cell monolayer, add 2-fold diluted feline interferon gamma to act for 12 hours, and then add 100TCID to each well 50 At the same time, a negative control group and a known cat interferon ω2 positive control group were set up, and the cytopathic situation was observed after 24 hours. The results are shown in Table 1.

[0086] Table 1 Results of recombinant feline interferon gamma antiviral activity

[0087]

[0088]

[0089] Note: (+) means no cytopathic (that is, antiviral activity), (-) means cytopathic (that is, ...

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Abstract

The invention provides a soluble prokaryotic expression and purification method and application of cat interferon gamma, and belongs to the field of biological genetic engineering. The method comprises the following steps: (1) analyzing a signal peptide sequence and physicochemical properties of the FeIFN-gamma protein; (2), performing construction of a prokaryotic expression plasmid pET28a-SUMO-FeIFN-gamma; (3) inducible expression of BL21 bacteria containing recombinant plasmids and solubility analysis of recombinant proteins; (4) screening optimal expression conditions of the SUMO-FeIFN-gamma fusion protein; (5) carrying out mass expression and purification on the SUMO-FeIFN-gamma fusion protein; and (6) carrying out enzyme digestion and purification on the SUMO-FeIFN-gamma fusion protein. A mature protein gene sequence not containing a signal peptide is cloned, a prokaryotic expression plasmid is constructed, a BL21 competent cell is transformed for expression and purification, and a pET28a-SUMO expression vector with an SUMO hydrotropy tag is used, so that soluble expression of a target protein is realized, the soluble expression level of a foreign protein is high, the biological activity is very high , and the application prospect is wide. The purification steps are simple and convenient, and a foundation is laid for later-stage development of antiviral drugs.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, in particular to a soluble prokaryotic expression and purification method and application of cat interferon gamma. Background technique [0002] Interferon (interferon, IFN) is a kind of glycoprotein produced by the body's immune cells and secreted by the cells after the induction agent acts on the living cells, which has various biological activities such as anti-virus, anti-tumor and immune regulation. According to the source, biological properties and activity of interferon, interferon can be divided into type I and type II. Type I interferon mainly plays antiviral and antitumor effects, including IFN-α, IFN-β, IFN-ω, IFN-ε, IFN-κ, IFN-τ, IFN-δ and IFN-ζ, type II interferon Only the IFN-γ isoform is present in , which plays a key role in host defense against intracellular pathogens. In 1992, Nakamura et al. from Japan Toray Co., Ltd. isolated the feline interferon gene for the first tim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/57C12N15/70C12N15/62C12N15/64C12N1/21C07K1/22C12R1/19
CPCC07K14/57C12N15/70C07K2319/21C07K2319/95C07K2319/02
Inventor 魏衍全张勇成述儒武小椿任丽君
Owner GANSU AGRI UNIV
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