中华皮肤科杂志 ›› 2005, Vol. 38 ›› Issue (5): 276-278.

• 论著 • 上一篇    下一篇

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

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   

  1. 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
  • 收稿日期:2004-05-27 出版日期:2005-05-15 发布日期:2005-05-15
  • 通讯作者: 毕志刚,E-mail:eltonbi@21cn.com E-mail:eltonbi@21cn.com
  • 基金资助:
    江苏省重点学科基金资助项目(135-3)

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

WANG Fei1, BI Zhi-gang1, LI Guang-fu2, WU Hai-wei2, WANG Qun3, LIU Feng2, WANG Xin-jun2, ZHANG Zhao-song2   

  1. Department of Dermatovenerology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
  • Received:2004-05-27 Online:2005-05-15 Published:2005-05-15

PDF

195

赞

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。

关键词: 乳头状瘤病毒,人, 尖锐湿疣, 基因表达, 基因病毒, 遗传载体

Abstract: 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.

Key words: Condylomata acuminata, Papillomavirus, human, Genes, viral, Genetic vectors, Gene expression