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鲤疱疹病毒Ⅱ型ORF4基因的克隆、表达与免疫学检测方法
引用本文:周勇,范玉顶,徐进,马杰,江南,刘文枝,曾令兵.鲤疱疹病毒Ⅱ型ORF4基因的克隆、表达与免疫学检测方法[J].淡水渔业,2017,47(1):61-65.
作者姓名:周勇  范玉顶  徐进  马杰  江南  刘文枝  曾令兵
作者单位:中国水产科学研究院长江水产研究所,武汉,430223
基金项目:国家“现代农业产业技术体系建设专项资金”,中国水产科学研究院基本科研业务费专项课题,武汉市科技计划项目
摘    要:根据鲤疱疹病毒II型(Cyprinid herpesvirus Ⅱ,Cy HV-2)ORF 4基因序列(Gen Bank:JQ815364.1)设计特异性引物,PCR扩增得到ORF 4基因编码框全长序列1 041 bp,将其克隆至原核表达载体p ET-32a(+)中,构建了重组原核表达载体p ET-32a-ORF 4。将p ET-32a-ORF 4重组载体转化大肠杆菌BL21(DE3),经IPTG诱导得到融合表达的重组蛋白,融合表达的重组蛋白主要以包涵体的形式存在,其分子质量约为57 ku,与预期大小一致。将纯化的重组蛋白免疫日本大耳兔,制备了多克隆抗体,ELISA检测抗体效价大于1∶50 000,Western blot检测显示该抗体可以特异性识别重组蛋白。间接免疫荧光检测结果表明:该多克隆抗体可与由Cy HV-2感染引起细胞病变的异育银鲫脑组织细胞(GICB)发生特异性的结合。

关 键 词:鲤疱疹病毒Ⅱ型  ORF  4  原核表达  多克隆抗体  免疫荧光检测

Cloning,expression and immunological assay of ORF 4 encoding protein of Cyprinid herpesvirus II
ZHOU Yong,FAN Yu-ding,XU Jin,MA Jie,JIANG Nan,LIU Wen-zhi,ZENG Ling-bing.Cloning,expression and immunological assay of ORF 4 encoding protein of Cyprinid herpesvirus II[J].Freshwater Fisheries,2017,47(1):61-65.
Authors:ZHOU Yong  FAN Yu-ding  XU Jin  MA Jie  JIANG Nan  LIU Wen-zhi  ZENG Ling-bing
Abstract:The specific primer pairs were designed according to the ORF 4 gene sequence of Cyprinid herpesvirus II ( Cy-HV-2) and the 1041bp full-length sequence of ORF4 gene coding frame was amplified by PCR, and then the full-length CyHV-2 ORF 4 gene was cloned into prokaryotic expression vector pET-32a( +) to construct the recombinant ex-pression vector pET-32a-ORF 4. The recombinant expression vector pET-32a-ORF 4 was successfully expressed in E. coli BL21 (DE3) as a fusion protein with a size of 57 ku after induction by isopropyl-β-D-thiogalactoside (IPTG). The recombinant protein was presented mainly in inclusion body. The purified recombinant proteins were used to immune Japanese white rabbit to produce polyclonal antibody. Titer of polyclonal antibodies was above 1∶50 000. Western blot test demonstrated that the recombinant protein was recognized by the polyclonal antibody.
Keywords:Cyprinid herpesvirus II ( CyHV-2 )  ORF 4  prokaryotic expression  polyclonal antibodies  immunological assay
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