草鱼出血病病毒基因组的cDNA合成、克隆及部分序列分析  

cDNA Synthesis, Cloning and Partial Nucleotide Sequence of Grass Carp Hemorrhage Virus (GCHV)

作　　者：田静[1] 邹桂平[1] 方勤[1] 

机构地区：[1]中国科学院武汉病毒研究所

出　　处：《中国病毒学》1999年第1期87-92,共6页Virologica Sinica

基　　金：国家自然科学基金

摘　　要：采用差速离心的方法纯化感染草鱼出血病病毒的细胞悬液。提纯的病毒粒子经蛋白酶Ｋ处理和酚氯仿抽提，在１％琼脂糖凝胶电泳条件下分离得到１１条ｄｓＲＮＡ，利用柱离心式胶回收试剂盒纯化各基因片段。纯化的ｄｓＲＮＡ溶于９０％的ＤＭＳＯ中，７０℃变性１５ｍｉｎ，然后采用随机引物法反转录合成各基因片段的ｃＤＮＡ，并平头连接于ｐＺＥｒＯ２．０载体的ＥｃｏＲＶ位点，电转化ＴＯＰ１０感受态细胞。重组质粒经酶切、ＰＣＲ扩增得到大小不等的插入片段，ｃＤＮＡＲＮＡ斑点杂交的结果进一步证实其插入为目的基因片段。采用循环ＰＣＲ测序的方法，对其插入片段进行了序列测定，对其中第１１片段的部分序列作了报道。Grass Carp Hemorrhage Virus (GCHV) was purified from infected fish cell culture by using different centrifugation. Its total genome was isolated by proteinase K treatment following phenol/chloroform extraction. The individual segment of GCHV dsRNA was separated by running 1% agarose gel electrophoresis and recovered by Gel Extraction Kit. Each segment was denatured in 90% DMSO by heating at 70℃ for 15min. The cDNAs of several segments were synthesized with random hexamers method and cloned into EcoRV site of pZErO 2.0 vector, then transformed into TOP10 E.coli competent cell by electroporation transformation. It was showed that the recombinant plasmids contained GCHV RNA genome of interest by dot hybridization besides both restriction analysis and PCR amplification. The partial DNA sequence of GCHV segment 11 was analysed.

关 键 词：草鱼出血病 病毒 CDNA克隆 序列分析 

分 类 号：S941.411[农业科学—水产养殖] S943.112[农业科学—水产科学]