轮状病毒VP4基因的克隆与核苷酸序列分析

Gene Cloning and Sequencing of Rotavirus VP4

用反转录聚合酶链式反应 (RT- PCR)技术 ,从轮状病毒感染的细胞中扩增了 816 bp的 VP4片段中抗原表位区。通过 T4 DNA连接酶将其直接连接于克隆载体质粒 p GEM- T上 ,转化至受体菌 DH5 α中。提取质粒经 PCR扩增、酶切鉴定 ,证明重组质粒 p T- V4中含有轮状病毒的 VP4基因片段。经核苷酸序列分析 ,表明克隆了轮状病毒主要保护性抗原

The antigenic determinants of Rotavirus VP4 genes were amplyfied from cell infected by rotavirus by reverse transcription polymerase chain reaction(RT PCR), the lenth of target genes was 816bp and. The products of RT PCR were ligated with plasmid pGEM T and transformated to E.coli DH5α.By the analysis of restriction endonuclease and PCR ,the fragments of VP4 were cloned and the recombinant plasmids PT V4 were constructed. The results of nucleotid sequencing showed that the inserted genes had positive reading frame.