摘要
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.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2000年第2期69-72,共4页
Chinese Journal of Blood Transfusion
基金
国家自然科学基金!资助课题 (编号 :39770 690 )
