丙型肝炎病毒NS5B区基因的扩增、克隆及序列分析

Detection,cloning and sequencing of the NS5B gene of hepatitis C virus

目的 对丙型肝炎病毒 (HCV)NS5B区基因克隆并测序 ,研究其变异状况。方法 从HCVRNA阳性血清中抽提病毒RNA ,利用巢式RT PCR对NS5B区基因进行扩增 ,将扩增获得的靶cDNA克隆至PKPL 3a质粒载体中 ,并以内引物为测序引物进行序列测定分析。结果 构建了已克隆HCVNS5B区基因的重组质粒KHC5 1 ;序列分析表明 ,与HCV BDS株同源性最高 ,属于主要流行于我国的HCV 1b基因亚型。结论 证实了在NS5B区 ,与RNA依赖的RNA聚合酶 (RDRP)活性密切相关的基元结构Gly Asp

Objective To clone and sequence HCV NS5B gene,and to investigate the mutation of the gene.Methods HCV RNA was extracted from a HCV positive serum and amplified with RT nested PCR.The PCR product was cloned into P KPL 3a vector plasmid.Then the cloned gene was identified as HCV NS5B gene by Southern blot and sequenced.Results It was demonstrated that the recombinant plasmid KHC5 1 included NS5B gene.The nucleotide sequence of NS5B gene has high homology with HCV BDS isolate,and was 1b HCV gene subtype which was popular isolate in China.Conclusion That the characteristic Gly Asp Asp motif in HCV NS5B is highly conserved is demonstrated.