2a/Ⅲ型丙型肝炎病毒基因组非结构4区全长序列的扩增、克隆及分析

Amplifying, cloning and sequence analyzing the gene of full-length NS4 region of genotype 2a/Ⅲ hepatitis C virus by using long nested PCR

目的 获得 2a/Ⅲ型丙型肝炎病毒 (HCV)基因组全长非结构 (NS) 4区克隆并作序列分析。方法 用限制性片段长度多态性分析 (RFLP)基因分型方法筛选出 2a/Ⅲ型HCV一株。为了获得该毒株全长NS4区基因序列 ,依据代表株序列的NS3区之 3′末端及NS5区之 5′末端相对保守区域合成引物 ,以RT -nested -PCR方法扩增一段 1.3kb的片段cDNA ;将此片段插入pUC19质粒载体。结果 克隆了丙型肝炎基因组非结构NS4区全长基因 ,构建了pUC -NS4重组体 ,对重组体进行酶切鉴定 ,克隆的cDNA与预计的片段大小相符 ,并作序列测定。结论 获得全长NS4区基因克隆 ,经序列分析表明 ,与日本HC -J6有较高的同源性 (92 .1%)。

Objective To clone and sequence the gene of full-length nonstructural 4 region of genotype 2a/Ⅲ hepatitis C virus. Methods A strain of genotype 2a/Ⅲ hepatitis C virus was screened by RFLP. In order to obtain the gene of full-length NS4 region, the primers based on the conservation region from 3′ end of NS3 region and 5′ end of NS5 region of the representative strain were designed and synthesized. A long nested polymerase chain reaction assay was established to amplify the interested cDNA (1.3 kb). The PCR product,the interested cDNA,was cloned into pUC19 plasmid. Results The clone of the gene of full-length NS4 region was obtained and sequenced. The recombinant pUC-NS4 was identified by RFLP analysis. Conclusion Analysis of the sequence of the clone of full-length NS4 region gene indicated that the nucleotide identity is high (92.1%) as compared with HC-J6 from Japan.