丙型肝炎病毒 HCV E1区基因的真核表达载体构建及序列分析  

Construction of mammalian cell expression vector of HCV E1 gene and analysis of its sequence

作　　者：高建恩[1] 蒋栋[1] 陶其敏[1] 纪和平[1] 季颖[1] 冯百芳[1] 

机构地区：[1]北京医科大学人民医院肝病研究所

出　　处：《北京医科大学学报》1999年第2期113-116,共4页Journal of Peking University(Health Sciences)

基　　金：国家自然科学基金!（３９４００１１６）;美国中华医学会!（ＣＭＢ．９３５８２）基金

摘　　要：建立丙型肝炎病毒包膜区基因的真核表达载体，并通过对其序列分析阐明其作为基因接种的可能性。方法：经常规ＲＴ－ＰＣＲ法从ＨＣＶ ＲＮＡ阳性病人的血清中扩增出ＨＣＶ Ｅ１区的基因片段，经ＥｃｏＲⅠ和ＸｂａⅠ双酶发后将其克隆到真核表达载体ＰｃＤＮＡ３中，阳性克隆经ＳｍａⅠ和ＸｂａⅠ双酶切鉴定以脱氧终止法测序，同时利用计算机软件对其推定的氨基酸序列进行分析。To construct the mammalian cell expression vector and to analyze the character of the sequence of E1 both in DNA and in the putative glycoprotein. Methods: RT-PCR was performed in the conventional way to amplify the HCV E1 gene. The product of the amplification was linked into the mammalian expression vector PcDNA3 after the digestion with the restricted endonuclease of EcoR 1 and Xba 1. The sequence of the inserted fragment was sequenced by the dideoxidation terminal methods and the computer software of Goldkey was used to analyze it. Results: There appeared the product of 489 hp after PCR amplification and there also appeared the product of 144 bp after the digestion of the plasmid extracted from the ampicilin resistant bacteria colony with the restricted endonuclease Sma Ⅰ and Xba Ⅰ. The homology ratio of the inserted fragment with that of HCV Ⅰ, Ⅱ, Ⅲ and Ⅳ were 72. 8%, 93. 25%, 61. 96% and 56. 44% in nucleotides and 77. 3%, 95. 7 %, 54. 6 % and 51.35 % in amino acids, respectively. The N-terminal of the inserted fragment was a signal peptide sequence. And it had one transmembrane peptide sequence in the middle and three antigenic determinants at the two sides of the trans-membrane sequence. Conclusion: the genotype of the sequence of HCV E1 gene cloned here was genotype Ⅱ. It has the character of genotype specific, and may become a candidate region for the research of DNA inoculation and T cell immunization of HCV.

关 键 词：丙型肝炎病毒 基因表达 遗传载体 序列分析 

分 类 号：R373.21[医药卫生—病原生物学] R512.63[医药卫生—基础医学]