黑头软口鲦上皮瘤细胞ifn-1基因的表达及抗病毒活性  

Expression and antiviral activity of ifn-1 gene in epithelioma papulosum cyprini cells of Pimephales promelas

作　　者：孙杰 郭雅娜 戴彩姣 陈孝煊[1] 李莉娟[1] 袁军法[1,2] SUN Jie;GUO Yana;DAI Caijiao;CHEN Xiaoxuan;LI Lijuan;YUAN Junfa(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Hubei Engineering Research Center for Aquatic Animal Diseases Control and Prevention,Wuhan 430070,China)

机构地区：[1]华中农业大学水产学院,湖北武汉430070 [2]湖北省水生动物病害防控工程技术研究中心,湖北武汉430070

出　　处：《水产学报》2020年第9期1448-1456,共9页Journal of Fisheries of China

基　　金：国家自然科学基金(31872598)。

摘　　要：为了体外表达黑头软口鲦上皮瘤细胞(EPC)I型干扰素(IFN-1),本实验通过RTPCR从EPC中扩增ifn-1基因,构建重组表达质粒pET-32a-IFN-1,并转化到感受态细胞Transetta(DE3),体外纯化后检测其抗病毒活性。结果显示,ifn-1编码区大小为552 bp,编码184个氨基酸,与草鱼干扰素1(CiIFN1)亲缘关系最近。通过SDS-PAGE分析,重组表达质粒pET-32a-IFN-1在宿主菌中可明显表达约35 ku的融合蛋白条带,且部分呈可溶性表达,进而通过亲和纯化可溶性重组IFN-1(rIFN-1),免疫新西兰大白兔获得效价较高的抗IFN-1多克隆抗体,可用于检测细胞内源性的IFN-1。定量PCR显示rIFN-1与EPC细胞孵育可以诱导抗病毒蛋白Mx1的表达,并抑制鲤春病毒血症病毒(SVCV)引起的细胞病变(CPE)及SVCV的复制,表明rIFN-1具有抗病毒活性。To clone Type I interferon(IFN-1)from epithelioma papulosum cyprini cells(EPC)of Pimephales promelas,the ifn-1 gene was amplified from EPC by RT-PCR,and the recombinant plasmid of pET-32a-IFN-1 was constructed and transformed into the host strain Transetta for expression.Sequence analysis showed that the coding region of ifn-1 gene was 552 bp in length and encoded 184 amino acids,which was closely related to grass carp interferon 1(CiIFN1).Recombinant soluble IFN-1(rIFN-1)was induced in Transetta by IPTG and obtained by Niaffinity purification.Then,anti-rIFN-1 polyclonal antibody was prepared by immunizing New Zealand white rabbits with rIFN-1 and could be used to detect endogenous IFN-1 in EPC cells.Quantitative PCR showed that the antiviral protein Mx1 was induced and spring viremia of carp virus(SVCV)replication as well as the production of cytopathic effect(CPE)was significantly inhibited when EPC was pre-incubated with rIFN-1,which indicated that rIFN-1 possessed antiviral activity in EPC cells.

关 键 词：黑头软口鲦 ifn-1 上皮瘤细胞 鲤春病毒血症病毒 原核表达 

分 类 号：Q786[生物学—分子生物学] S941.41[农业科学—水产养殖]