我国丙肝病毒结构区的cDNA克隆  

Clone and detection of construct genomes in a hepatitis C virus from western China

作　　者：祁欣[1] 贾文祥[1] 杨春[1] 杨远[1] 曾蔚[1] 陈恬[1] 

机构地区：[1]四川大学华西医学中心基础医学与法医学院微生物学教研室,成都610045

出　　处：《药品评价》2004年第1期29-33,共5页Drug Evaluation

摘　　要：目的克隆我国西部丙型肝炎病毒全部结构区基因并进行序列分析,为研究丙肝病毒和基因工程表达做准备。方法从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。结果将克隆好的质粒从两端双向测序,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。本文序列与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%。本文序列与HCV1a核苷酸及氨基酸的同源性均大于90%,应为HCV1a亚型。结论我国西部地区存在HCV1a亚型,所克隆HCV结构区cDNA克隆可以用于丙肝病毒研究和基因工程表达。Object To clone and determine construct genomes of hepatitis C virus from western China. Method HCV RNA was extracted from a Xiaan HCV infected patient and reverse transcripted to cDNA by AMV reverse transcriptase. HCV core, E1, E2 cDNA were amplified 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. Results BY sequencing pGEM-T-HCVjg, the nucleic acid sequence of HCV construct genome was obtained. The homology percents between pGEM-T-HCVjg and HCV 1a and 1b from Genbank were 91.8% and 84.5%(nucleic acid),94.2% and 86.3%(amino acid),the HCV clone seems belong to HCV 1a genotype. Conclusion The whole HCV construct cDNA clone was obtained. There is HCV 1a genotype in western China.

关 键 词：肝炎病毒 丙型 基因结构 病毒 病毒蛋白质类 克隆 分子 序列分析 

分 类 号：R346[医药卫生—基础医学]