| 茶尺蠖小RNA病毒全长cDNA克隆的构建 |
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| 引用本文： | 林美娟,谢键,张珈敏,叶姗,胡远扬. 茶尺蠖小RNA病毒全长cDNA克隆的构建[J]. 昆虫学报, 2010, 53(7): 818-823 |
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| 作者姓名： | 林美娟 谢键 张珈敏 叶姗 胡远扬 |
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| 作者单位： | 武汉大学生命科学学院病毒学国家重点实验室,武汉,430072 |
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| 基金项目： | 国家自然科学基金项目 |
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| 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
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| 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 |
Construction of the full-length cDNA clone of Ectropis oblique picorna-like virus |
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| LIN Mei-Juan,XIE Jian,ZHANG Jia-Min,YE Shan,HU Yuan-Yang. Construction of the full-length cDNA clone of Ectropis oblique picorna-like virus[J]. Acta Entomologica Sinica, 2010, 53(7): 818-823 |
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| Authors: | LIN Mei-Juan XIE Jian ZHANG Jia-Min YE Shan HU Yuan-Yang |
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| Abstract: | 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. |
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| Keywords: | Ectropis oblique picorna-like virus full-length cDNA clone Iflaviridae reverse genetics manipulation technique |
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