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

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.