我国西部1a型丙肝病毒结构区的cDNA克隆和序列分析

Cloning and sequencing of hepatitis C virus construct genomes from western China

克隆我国西部丙型肝炎病毒全部结构区基因,为探讨HCV的变异和致癌作用,研制丙肝疫苗作准备。从1名西安市丙肝感染者血液中,用Trizol试剂裂解HCV病毒颗粒,用糖原与RNA共沉淀提取HCVRNA,用AMV逆转录酶和随机引物逆转录为cDNA,用RT-PCR方法扩增目的片段并转化到pGEM-T质粒,得到pGEM-T-HCVc、pGEM-T-HCVe1和pGEM-T-HCVe2克隆。将以上3个克隆用特定的酶降解,用连结酶进行连接,构建了HCV结构区的完整克隆。将克隆好的质粒pGEM-T-HCVjg从两端双向测序,得到完整的HCV结构基因cDNA核苷酸序列,总长度为2238bp,与已发表的HCV序列长度一致,未发现缺失或移码突变,序列中间亦未发现转录终止子。本序列与HCV1a、1b序列核苷酸的同源性分别为91.8%、84.5%;氨基酸的同源性分别为94.2%、86.3%;应为HCV1a亚型。以上结果说明我国西部地区存在HCV1a亚型,所克隆HCV结构区cDNA克隆可以用于基因工程表达。

To construct and sequence hepatitis C virus construct genomes from western China. HCV RNA was extracted from a Xi'an HCV infected patient and reverse transcripted to cDNA by AMV reverse transcriptase. HCV core, E1, E2 cDNA were ampliphicated and cloned into pGEM-T vectors respectively. The clone pGEM-T-HCVjg containing whole HCV construct genomes(core, E1, E2) was constructed by digesting and rejoining the three above plasmids. By sequencing pGEM-T-HCVjg, the nucleic acid sequence of HCV construct genome was abtained. The homology percents between pGEM-T-HCVjg and HCV1a and 1b from GenBank were 91.8% and 84.5%(nucleic acid), 94,2% and 86.3%(amino acid),our HCV clone seems belong to HCV1a genotype. The whole HCV construct cDNA clone was abtained. There is HCV1a genotype in western China.