TIAN Jing, JU Gui-Beng and FANG Qi. cDNA Synthesis,Cloning and Partial Nucleotide Sequence of Grass Carp Hemorrhage Virus(Gcnv)[J]. Virologica Sinica, 1999, 14(1): 87-92.
Citation: TIAN Jing, JU Gui-Beng, FANG Qi. cDNA Synthesis,Cloning and Partial Nucleotide Sequence of Grass Carp Hemorrhage Virus(Gcnv) .VIROLOGICA SINICA, 1999, 14(1) : 87-92.

草鱼出血病病毒基因组的cDNA合成、克隆及部分序列分析

  • 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。

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

  • 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.

  • 加载中
  • 加载中

Article Metrics

Article views(3979) PDF downloads(982) Cited by(0)

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

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

    • 1. 

    Abstract: 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.

    Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return