鳗鲡疱疹病毒ORF8基因的克隆表达及转录时相分析  

Cloning,prokaryotic expression and transcription profile analysis of Anguillid herpesvirus ORF8 gene

作　　者：陈曦[1] 李英英[1] 陈强[1] 宋铁英[1] 葛均青[1] CHEN Xi;LI Yingying;CHEN Qiang;SONG Tieying;GE Junqing(Institute of Biotechnology, Fujian Academy of Agricultural Science, Fuzhou 350003, China)

机构地区：[1]福建省农业科学院生物技术研究所,福建福州350003

出　　处：《大连海洋大学学报》2021年第1期10-15,共6页Journal of Dalian Ocean University

基　　金：国家自然科学基金(31101933);福建省公益类科研院所基本科研专项(2017R1019-5,2018R1019-5);福建省农业科学院项目(AC2017-1,A2016-3)。

摘　　要：为研究鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)ORF8基因的特性,根据GenBank中AngHV参考株(NC_013668)的ORF8序列设计引物,采取PCR方法从分离的AngHV福建病毒株(AngHV-FJ)基因组中扩增出特异性片段,克隆至pMD19-T载体,经酶切和测序验证,获得了ORF8的基因序列。结果表明:经生物信息学分析,ORF8编码的蛋白稳定性较好,存在跨膜结构且抗原表位多,符合病毒囊膜蛋白的特征;经转录时相分析,ORF8是病毒的早期基因(early gene,E),可能在病毒感染的早期阶段就发挥作用;将ORF8克隆至表达载体pET-32a,转化入大肠杆菌E.coli BL21中,以IPTG诱导蛋白表达,通过SDS-PAGE及Western blot检验,实现了ORF8在大肠杆菌中的高效表达,进一步通过割胶的方法获得了相对分子质量约为44000的高纯度融合表达蛋白。本研究为下一步评价该表达蛋白的免疫效果和制备抗体,以及开发免疫诊断试剂和疫苗提供了研究基础。The ORF8 sequence of Anguillid herpesvirus(AngHV)was amplified from the genome of AngHV-FJ strain by PCR with primers based on the sequence of AngHV reference strain(NC_013668)in GenBank,and then the target fragment was cloned into pMD19-T vector and verified by restriction enzyme digestion and sequencing.Bioinformatics analysis indicated that ORF8 protein might be anchor on the viral envelop with good stability,transmembrane structure,and numbers of antigen epitopes.The analysis of transcription profiles revealed that ORF8 gene was the early gene of AngHV and probably had an active role in the early stage of viral infection.ORF8 gene was then cloned into the expression vector pET-32a and transformed into Escherichia coli BL21(DE3).The encoded protein was highly expressed under the induction of IPTG and followed by SDS-PAGE and Western blot verification.The expressed high-purity ORF8 protein with relative molecular mass of 44000 was obtained by following gel extraction purification.The findings provide fundamental data on evaluation of the immunity effect of the expressed protein and preparation of specific ORF8 antibody,as well as on development of immunodiagnostic test reagent and vaccine preparation of the virus.

关 键 词：鳗鲡疱疹病毒 ORF8基因 转录时相 克隆 原核表达 

分 类 号：S941.41[农业科学—水产养殖]