一株中华绒螯蟹呼肠孤病毒RNA1 cDNA文库构建及其RNA聚合酶基因部分序列分析

在中华绒螯蟹体内分离一株呼肠孤病毒（命名为EsRV905株），采用Trizol试剂提取病毒核酸，经聚丙烯酰胺凝胶电泳，碎胶法回收基因组各节段。随机引物法合成第一节段的cDNA文库，胶回收试剂盒去除小片段，平端连接于载体，化学转化，利用蓝白斑筛选阳性克隆子，酶切鉴定重组质粒。从基因组第一节段的重组质粒中选择2个插入片段为1.5kb的质粒测序，结果得到包括RNA聚合酶主要特征性结构的一段序列。结果说明，这株蟹呼肠孤病毒的RNA聚合酶定位于基因组第一节段。

cDNA Library Construction of a Chinese mitten crab reovirus RNA1 and Partial Sequence Analysis of its RNA Polymerase Gene

A reovirus (designated as EsRV905), was isolated from Chinese Mitten Crab (Eriocheir sinensis). Viral genome was extxacted with Trizol reagent and individual segment was recovered separately from gel after 6.5% PAGE. The cDNA library of RNA1 was synthesized using random primer method and short fragments less than 200 bp were discarded using gel recovery kit. The cDNA was ligated to blunt-end plasmid and transformed to competent cell. Positive clones were screened by blue and white colonies and checked by enzyme digestion. Two plasmids containing around 1.5kb inserts from the first segment were selected for sequencing. One of them contained the main motif of RNA polymerase(RdRp). The RdRp of EsRV905 is located in the first segment of EsRV905 genome.