鹅α干扰素基因在COS-7细胞中的瞬时表达及抗病毒功能初探  

Transient expression of goose IFN-α gene in COS-7 cells and primary investigation of its antiviral function

作　　者：刘菲[1,2] 唐卫杰 程安春[1,2] 汪铭书[1,2] 赵婷婷[1] 高丽芹[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]四川农业大学动物医学院动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《浙江大学学报（农业与生命科学版）》2011年第4期380-386,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：四川省教育厅重点资助项目(2006A012);教育部"长江学者和创新团队发展计划"创新团队资助项目(IRT0848);现代农业产业技术体系建设专项资金资助项目(nycytx-45-12)

摘　　要：为了研究鹅α干扰素(goIFN-α)基因的特性和功能,将天府肉鹅的α干扰素基因克隆到pcDNA3.1(+)真核表达载体上,构建重组质粒pcDNA3.1-goIFN-α,脂质体介导将其转入COS-7细胞中;应用间接免疫荧光、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和免疫印迹(Western-blot)法检测goIFN-α基因在COS-7细胞中的转录和表达情况,并对COS-7细胞表达上清液进行抗病毒活性检测.结果表明:目的蛋白在转染12 h后就可以被检测出,36 h前目的蛋白在上清液中快速增加,之后增加速度变慢;重组质粒能够在COS-7细胞内得到高效表达,表达的蛋白分子质量为38ku,比预测的分子质量(约21 ku)大;间接免疫荧光显示,细胞内出现黄绿色荧光,胞核附近最亮;表达产物经细胞病变抑制法测定,显示出具有明显的抗猪水泡性口炎病毒(VSV)及小鹅瘟病毒(GPV)的活性.To elucidate the character and functions of goose interferon-alpha gene（IFN-α）,the gene IFN-α of Tianfu meat goose open reading frame（ORF） was cloned into eukaryotic expression vector pcDNA3.1（＋） to generate the recombinant plasmid pcDNA3.1-goIFN-α,which was then transfected into the COS-7 cells by lipofectin transfection.Then,its expression and localization in COS-7 cells were determined by indirect immunofluorescence,enzyme-linked immunosorbent assay and Western-blot assay,and the antiviral activity of supernatant from transfected COS-7 cells was also determined.The results indicated that goIFN-α protein was fist detected at hour 12 post-transfection,which was rapid increase in the supernatant before 36 h,thereafter it became slow.The recombinant plasmid could be highly expressed in COS-7 cells,and a protein of 38 ku in transfected COS-7 cells was also detected,which was larger than the predicted molecular mass of 21 ku.The indirect immunofluorescence showed that the cells appeared yellow-green fluorescence,the fluorescent intensity in which was the most highest nearby nucleus.The antiviral activities of supernatant against vesicular stomatitis virus（VSV） and gosling plague virus（GPV） were obvious,as verified by method of cytopathic effect inhibition test.

关 键 词：鹅α干扰素 COS-7细胞 免疫印迹 间接免疫荧光 细胞病变抑制法 

分 类 号：Q786[生物学—分子生物学] S852.4[农业科学—基础兽医学]