人乳头瘤病毒16型E6蛋白真核表达载体的构建及表达

目的构建在哺乳动物细胞中表达的人乳头瘤病毒16型早期蛋白6真核表达载体pcDNA3．1（-），E6，为研究HPV16 E6蛋白在细胞永生化作用中的机制奠定实验基础。方法用聚合酶链反应（PCR）扩增E6基因。将PCR产物纯化后与pUCm-T载体连接，经蓝白斑筛选、双酶切鉴定及序列分析后，亚克隆至pcDNA3．1（-）真核表达载体中；阳性克隆经双酶切鉴定后转染RAW264．7巨噬细胞，Western blotting鉴定其在RAW264，7细胞中的表达。结果重组载体经酶切、PCR及测序证明插入片断序列正确无误，转染RAW264．7细胞后可在细胞中表达HPV16E6蛋白。结论成功构建的HPV16E6真核表达载体pcDNA3，1（-）／E6能在RAW264．7细胞中表达E6蛋白。

Construction of Human Papillomavirus Type 16 E6 Eukaryotic Expression Vector and its Expression in Eukaryotic Cells

Objective To construct the eukaryotic expressing vector of human papillomavirus type 16 E6(HPV16 E6) and test its expression in RAW264.7 cells.Methods HPV16 E6 gene was amplified from pGEX1/E6 by PCR.Then the product was cloned into pUCm-T,sequenced and then subcloned into eukaryotic expressing vector pcDNA3.1(-).The recombined vector was transfected into RAW264.7 cells with lipofectin and the expression of E6 gene was detected by Western blotting.Results The eukaryotic expressing vector encoding HPV16 E6 was successfully constructed and its expression in RAW264.7 cells could be detected.Conclusion The eukaryotic expressing vector encoding HPV16 E6 was constructed and it expressed E6 protein correctly in RAW264.7 cells.