茶尺蠖小RNA病毒全长cDNA克隆的构建  

Construction of the full-length cDNA clone of Ectropis oblique picorna-like virus

作　　者：林美娟[1] 谢键[1] 张珈敏[1] 叶姗[1] 胡远扬[1] 

机构地区：[1]武汉大学生命科学学院病毒学国家重点实验室,武汉430072

出　　处：《昆虫学报》2010年第7期818-823,共6页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(30670084)

摘　　要：为了初步研究茶尺蠖小RNA病毒(Ectropis oblique picorna-like virus,EoPV)的复制机制,从被EoPV感染致死的茶尺蠖幼虫中分离并纯化病毒粒子,提取病毒RNA,根据已公布的EoPV核苷酸序列,利用基因组上单一的酶切位点,设计特异性引物,应用RT-PCR扩增出5个覆盖全长的片段。随后采用融合PCR将5个片段拼接,最终将全长定向克隆到低拷贝质粒载体上,成功构建cDNA全长克隆p-EoPV。双酶切及测序鉴定证明全长克隆构建成功。与原序列比较发现,该克隆在氨基酸水平上有8个突变和1个缺失。本研究为深入探讨EoPV病毒生物学特性、病毒复制机理等奠定了基础。The viral total RNA was extracted from dead larvae of the tea looper, Ectropis oblique infected by E. oblique picorna-like virus （EoPV）. Five pairs of primers were designed according to the published sequence of EoPV to amplify 5 overlapping fragments covering EoPV genome. These fragments were subcloned to pMD18-T vector and assembled. The full-length cDNA of p-EoPV was cloned to pET-28a and proved by sequencing and restrictive digestion. Comparing to the published sequence, this clone possessed an 8-amino acid mutation and a 1-amino acid deletion. This study represents the first step towards understanding the mechanism of EoPV replication.

关 键 词：茶尺蠖小RNA病毒 全长CDNA克隆 传染性软化病毒科 反向遗传学 

分 类 号：Q966[生物学—昆虫学]