HCV非结构蛋白(NS2/NS3)基因表达载体的构建及其一级结构分析

Eukaryotic Expression Vector Construction and Primary Structure Analysis of HCV HS2 NS3 Gene

应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术，从ＨＣＶ感染者血清中扩增编码ＨＣＶ病毒蛋白酶的ＮＳ２－ＮＳ３ｃＤＮＡ片段，在其５′和３′端分别引入ＥｃｏＲⅠ和ＸｂａⅠ限制性内切酶位点，定向克隆至真核表达载体ｐｃＤＮＡ３，构建重组载体ｐｃＤＮＡ－ＮＳ２３，重组表达载体经限制性内切酶消化鉴定．用ＳＰ６和Ｔ７通用引物对目的基因片断进行序列分析．序列同源性分析结果表明，与ＨＣＶ－Ｊ、ＨＣ－Ｃ２有高度的同源性，与ＨＣＶ－１、ＨＣＶ－Ｊ６、ＨＣＶ－Ｊ８同源性差，提示所克隆的基因属ＨＣＶⅡ型．该区内重要的功能位点如Ｚｎ２＋依赖性金属蛋白酶催化中心。

HCV NS2 NS3 gene encoding viral protease was amplified by reverse transcription nested PCR(RT PCR)from serum of a patient infected with HCV and cloned into pcDNA3 between Eco RⅠ and Xba Ⅰ sites.The recombinant vector,designated as pcDNA NS23,was characterized by restriction digestion.The sequence was analysed with SP6 and T7 primers.Nucleotide sequence analysis indicated that it had well homology to reported isolates HC C2 and HCV J,poor homology to HCV 1,HCV J6 and HCV J8.Sequence analysis has suggested that it may fall into HCV genotype Ⅱ.Some sites,such as Zn 2+ dependent metalloprotease catalytic center and serine protease catalytic center,are conservative.