丙型肝炎病毒E1基因的克隆、表达、序列分析及定点突变

Cloning,Expression,Sequencing and Site-directed Mutagenesis of HCV E1 Area Genes

目的 :为了获得具有完整框架的HCVE1基因。方法 :用RT PCR从HCV(+)血清中扩增出E1基因 ,在原核系统中进行表达、测序及定点突变 ,并对表达的E1蛋白进行纯化及初步的活性鉴定。结果 :在 86株阳性克隆中 ,11株表达了不完整的HCVE1蛋白 ;使用表达出来分子量最大的蛋白作为抗原 ,在HCV(+)血清中抗 E1的检出率为 16 .7% (2 / 12 ) ,对其插入的E1基因 (HCVe118)测序 ,表明 134 7位的C→T而形成终止密码 ,用“大引物”法对HCVe118作定点突变 ,使T→C ,从而获得具正确框架的HCVE1基因。结论 :在原核表达系统中 ,去除C末端的HCVE1基因可获得高效表达 ;表达的HCVE1蛋白抗原性很弱。“大引物”法用于基因改构工作简便而又快速。

Objective: To obtain the full lenagth HCV E1 gene.Methods:Amplified by RT-PCR from HCV(+)serum,the HCV E1 genes were expressed,sequenced and mutagenesed at fixed site in prokaryotic expression system,and the expressed E1 proteins were purified and their activities were preliminavily identified.Results:11 of 86 strains of positive clones expressed the non full length HCV E1 Proteins.When using the expressed E1 protein with maximal molecular weight as an antigen,the detection rate of anti-E1 in HCV(+) serum was 16.7%(2/12).The sequencing of inserted gene(HCVe 118)showed that due to the C→T at 1347 site,the stop code was formed,Using the“ megaprimer” method,the site-directed mutagenesis of HCVe 118 was performed and T→C was accomplished,thereby the HCV E1 gene with correct length was obtained. Conclusion:In the prokaryotic expression system,the HCV E1 gene with truncated C-terminal could be expressed with high effectiveness.The expressed HCV E1 protein had low antigenicity.It was very simple and rapid to use the“megaprimer”method to change the gene′s structure.