【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 EcoR Ⅰ和Xba Ⅰ双酶切后将其克隆到真核表达载体 PcDNA3中,阳性克隆经 Sma Ⅰ和 Xba Ⅰ双酶切鉴定;用双脱氧终止法测序,同时利用计算机软件对其推定的氨基酸序列进行分析。结果:RT-PCR扩增产物经酶切过夜后与经过同样双酶切的真核表达载体 PcDNA3连接,阳性克隆经 Sma Ⅰ和 Xba Ⅰ酶切后产生了预期大小的 144 bp的片段。测序后其核苷酸序列与已经报道的HCVⅠ、Ⅱ、Ⅲ、Ⅳ型的同源性分别为72.8%、93.25%、61.96%和56.44%;其推定的氨基酸序列的同源性分别为 77. 3%、95. 7%、54 6%和 51. 35%;与已经报道的中国株的同源性为 95. 06%和93. 25%;属 HCV Ⅱ型;经计算机辅助分析表明该片段内含有4个可能的糖基化位点, l个跨膜区,其氨基端为信号肽序列,在跨膜区的两端分别含有2个和1个抗原决定簇,而每一个抗原决定簇内均含有1个糖基化位点,该片段内还含有 2个 T细胞识别位点。结论:本实验克隆的 HCV EI基因属Ⅱ型,为我国流行的基因型,其基因的一级结构具备进行有关 HCV核酸免疫的研究条件,其真核表达载体的构建,为进一步研究 HCV EI基因的功能及 HCV基因免疫的研究创造了条件。更多还原

【Abstract】 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.更多还原