CHO细胞表达重组猪干扰素-γ及其抗病毒活性

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity

为利用中国仓鼠卵巢(Chinese hamster ovarian,CHO)细胞表达系统制备重组猪干扰素-γ(recombinant porcine interferon gamma,rPoIFN-γ),并分析其体外抗病毒活性,本研究首先构建rPoIFN-γ真核表达质粒pc DNA3.1-PoIFN-γ,转染悬浮培养的CHO细胞,进行上清分泌表达,利用亲和层析纯化目的蛋白,并进行SDS-PAGE和Western blotting鉴定;通过CCK-8实验分析rPoIFN-γ对细胞的毒性,用VSV/PK-15系统检测其抗病毒活性效价;最后分析rPoIFN-γ对塞内卡病毒A(Seneca virus A,SVA)的抗病毒活性及其对细胞内干扰素刺激基因(interferon-stimulated genes,ISGs)和细胞因子的诱导作用。结果显示,本研究利用CHO悬浮细胞表达系统成功制备了纯化的rPoIFN-γ,该rPoIFN-γ对细胞无毒性,VSV/PK-15系统检测其活性效价为5.59×10^(7) U/mg;此外,rPoIFN-γ可诱导细胞内多种ISGs和细胞因子的表达,并显著抑制SVA的复制。总之,本研究成功利用CHO表达系统制备了高活性的rPoIFN-γ,并在体外完成其对SVA的抗病毒作用分析,为rPoIFN-γ的功能及抗病毒制剂的研究提供了实验材料和基础。

The aim of this study was to produce recombinant porcine interferon gamma(rPoIFN-γ)by Chinese hamster ovarian(CHO)cells expression system and to analyze its antiviral activity.Firstly,we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ.The rPoIFN-γwas purified by affinity chromatography and identified with SDS-PAGE and Western blotting.Subsequently,the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test,and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15(virus/cell)test system.Finally the anti-Seneca virus A(SVA)of rPoIFN-γ activity and the induction of interferon-stimulated genes(ISGs)and cytokines were also analyzed.The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells.CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells.The biological activity of rPoIFN-γ was 5.59×10^(7) U/mg in VSV/PK-15 system.Moreover,rPoIFN-γ could induced the expression of ISGs and cytokines,and significantly inhibited the replication of SVA.In conclusion,the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system,which showed strong antiviral activity on SVA.This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.