绵羊痘病毒RPO30基因的克隆及序列分析

Sheeppox Virus RPO30 Gene Cloning and Sequence Analysis

选择羊痘病毒RNA聚合酶RPO30进行分析,为进行RNAi羊痘病毒研究,进行目的基因的初步筛选。PCR扩增RPO30基因,连入pMD18-T simple载体并转化DH5a大肠杆菌。经蓝白斑筛选挑选白斑制备质粒,经双酶切、PCR及测序鉴定。结果成功克隆了RPO30基因,克隆的SSPV RPO30基因ORF为585bp,编码193个氨基酸,阅读框内有一个HindIII酶切位点,测序结果与Gen Bank数据库中不同痘病毒毒株之间同源性存在着明显区别。进一步经过生物学软件分析表明RPO30蛋白质氨基酸4—12、18—26、50—61、68—92,176—190位之间区域形成活性中心的可能性较大,选择这些区域进行作用可能会造成该基因产物酶的失活,从而高效抑制病毒的复制。

To research the goat pox virus by RNAi,the RNA polymerase 30 gene （RPO30） as a target sequence was analyzed.In the study,RPO30 was cloned by PCR,and sequencing shows that the open reading frame is 585 bp that can code 193 amino acids,and there is a HindIII enzyme cut sites located during 110 to 117 bp.The RPO30 sequence we cloned has different homology between different pox virus strains in the GenBank database.More bio-informatics software analyzed show that the amino acids site 4-12aa,18-26aa,50-61aa,68-92aa and 176-190aa have more possibility of forming enzyme activity center.So,we should select these sites to inference.