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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 a complete framework of HCVE1 gene. Methods: The E1 gene was amplified from HCV (+) serum by RT-PCR and expressed in prokaryotic system. Sequencing and site-directed mutagenesis were carried out. The expressed E1 protein was purified and its primary activity was identified. RESULTS: Of the 86 positive clones, 11 expressed incomplete HCVE1 protein; the highest expressed protein was used as antigen in the positive HCV (+) serum and the positive rate of anti-E1 was 16.7% (2 / 12). The inserted E1 gene (HCVe118) was sequenced, indicating that the C → T at position 134 7 formed a stop codon and the “big primer” method was used to make a point mutation in HCVe118 to make T → C, HCVE1 gene. CONCLUSION: In prokaryotic expression system, HCVE1 gene with C-terminal deletion is highly expressed, and the expression of HCVE1 protein is weakly antigenic. The “big primer” approach to gene engineering is quick and easy.