Ⅲ型中国株丙型肝炎病毒E2/NS1基因的克隆与序列分析

Cloning and sequencing of E2/NS1 gene from a Chinese genotype Ⅲ isolate of hepatitis C virus

目的构建插入Ⅲ型中国株丙型肝炎病毒Ｅ２／ＮＳ１基因片段的真核表达载体，分析该基因片段的核苷酸一级结构。方法利用逆转录套式ＰＣＲ扩增Ⅲ型中国株丙型肝炎病毒（ＨＣＶ）Ｅ２／ＮＳ１基因片段，通过定向克隆将其克隆到真核表达载体ｐｃＤＮＡ３上，采用双脱氧链终止法测定其核苷酸序列。结果构建出含有Ⅲ型中国株丙型肝炎病毒Ｅ２／ＮＳ１基因片段的克隆，该区基因片段的核苷酸一级结构和Ⅲ型日本株ＨＣＶ（ＨＣ－Ｊ６）的同源性为８８．３７％，Ⅱ型中国株ＨＣＶ（ＨＣ－Ｃ２）的同源性为７０．６９％，其推定的氨基酸同源性分别为８９．２９％和７５．５５％。结论基因的核苷酸一级结构在同一基因型中有较高的同源性，而不同基因型之间的同源性则相对较低。对不同基因型之间Ｅ２／ＮＳ１基因一级结构的研究将有助于ＨＣＶ疫苗的研制。

Objective To clone E2/NS1 gene from a Chinese genotype Ⅲ isolate of hepatitis C virus into mammalian expression vector and analyze its primary nucleotide structure.Methods Reverse transcription nested PCR methods were employed for amplification E2/NS1 gene which was cloned into mammalian expression vector pcDNA3 by directed clone. Sequence of inserted gene was analyzed by Sanger′s method.Results E2/NS1 gene from a Chinese genotype Ⅲ isolate of hepatitis C virus was cloned into eukaroytic expression vector effectively. Sequence of E2/NS1 showed 88.37% identity in nucleotide and 89.29% in putative amino acid to that of a Japanese genotype Ⅲ isolate of hepatitis C virus. Nevertheless it showed 70.69% and 75.55% identity to that of a Chinese genotype Ⅱ isolate of hepatitis C virus.Conclusion E2/NS1 gene from a Chinese genotype Ⅲ isolate of hepatitis C virus was successfully cloned into mammalian expression vector by technique of DNA recombination. Sequence analysis showed that E2/NS1 gene has good homology intra genotype and poor homology intergenotype of hepatitis C virus. Research of primary nucleotide structure of different genotype hepatitis C virus might be helpful for the development of vaccine against the virus.