人乳头瘤病毒11型E7蛋白基因重组腺病毒载体的构建及表达  

Construction and Expression of Human Papillomavirus Type 11 E7 Gene with Recombinant Adenovirus Vectors in Eukaryotic Cells

作　　者：王飞[1] 毕志刚[1] 李光富[2] 吴海玮[2] 王群[3] 刘丰[2] 王新军[2] 张兆松[2] 

机构地区：[1]南京医科大学第一附属医院皮肤科,210029 [2]南京医科大学分子生物研究所 [3]广东省人民医院皮肤科

出　　处：《中华皮肤科杂志》2005年第5期276-278,共3页Chinese Journal of Dermatology

基　　金：江苏省重点学科基金资助项目(135-3)

摘　　要：目的构建人乳头瘤病毒11型(HPV11)E7蛋白基因腺病毒载体,并在真核细胞表达。方法用PCR法,扩增出HPV11E7基因,定向克隆至pENTR-TOPO载体形成重组质粒TOPO-E7。TOPO-E7与腺病毒载体pAD/CMV/V5-DESTTM重组反应,E7基因被重组到腺病毒载体上。该载体经PacI酶切,脂质体法转染人胚肾293A细胞,获得重组腺病毒载体pAD-E7。pAD-E7转染人HaCaT细胞,激光共聚焦显微镜分析E7蛋白表达情况。结果经酶切及序列分析鉴定,重组质粒TOPO-E7成功构建。重组子pAD-E7转染293A细胞后,获得滴度为1.4×107pfu/mL的重组腺病毒载体。该载体转染HaCaT细胞,48h后激光共聚焦显微镜下可见细胞内E7蛋白表达。结论重组腺病毒载体能有效介导HPV11E7基因在真核细胞的表达。Objective To construct and express human papillomavirus type 11(HPV11) E7 gene with recombinant adenovirus vectors. Methods HPV11 E7 gene was amplified by PCR and directionally cloned into vector pENTR-TOPO to form TOPO-E7 plasmid. E7 gene was transferred into the pAD/CMV/V5-DESTTM gateway vector by LR recombination reaction with pAD/CMV/V5-DESTTM gateway vectors and TOPO-E7 plasmid. The recombination vector was digested by Pac I enzyme and transfected into 293A cell by Lipofectamine method to obtain recombinant adenovirus vectors pAD-E7. Expression of E7 on HaCaT cells infected with pAD-E7 vectors was analyzed by confocal microscopy. Results The recombinant plasmid TOPO-E7 was identified and confirmed with enzyme digestion and sequencing. Recombinant adenovirus vectors pAD-E7 were generated efficiently with a titer of 1.4 × 107 pfu/mL in transfected 293A cells. E7 protein could be identified in HaCaT cells with confocal microscope 48 h after infected with recombinant adenovirus vector. Conclusions The results indicate efficient expression of HPV11 E7 gene in eukaryotic cells by recombinant adenovirus mediated transfer, which facilitates further research of its function.

关 键 词：人乳头瘤病毒11型 基因重组腺病毒载体 HaCaT细胞 激光共聚焦显微镜 HPV11 E7蛋白基因 真核细胞表达 E7基因 重组质粒 PCR法 定向克隆 PacI 脂质体法 表达情况 分析鉴定 蛋白表达 A细胞 转染 CMV pAD 人胚肾 重组子 细胞内 

分 类 号：R392[医药卫生—免疫学]