中华皮肤科杂志 ›› 2005, Vol. 38 ›› Issue (2): 115-117.

• 论著 • 上一篇    下一篇

聚合酶链反应诱导HPV16E7C端基因突变和突变蛋白在真核细胞表达

左亚刚, 王家璧, 刘方, 闫言, 朱铁山, 马东来, 王宝玺   

  1. 中国医学科学院中国协和医科大学北京协和医院皮肤科 北京 100730
  • 收稿日期:2004-02-25 出版日期:2005-02-15 发布日期:2005-02-15
  • 通讯作者: 王家璧,E-mail:wangjbhe@263.net E-mail:wangjbhe@263.net

PCR-induced Modification of C Terminus of HPV-16 E7 and Expression of Mutational E7 in Eukaryotic Cells

ZUO Ya-gang, WANG Jia-bi, LIU Fang, YAN Yan, ZHU Tie-shan, MA Dong-lai, WANG Bao-xi   

  1. Department of Dermatology, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China
  • Received:2004-02-25 Online:2005-02-15 Published:2005-02-15

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摘要： 目的 聚合酶链反应(PCR)诱导人乳头瘤病毒(HPV16)早期基因E7C端锌指结合基序基因突变,评价突变对抗原稳定性的影响。方法 采用PCR技术对HPV16E7第58、91位氨基酸进行突变,构建野生型(pcDNA3.1/16E7)和突变型(pcDNA3.1/16ME7)重组体,对其蛋白表达进行比较。结果 HPV16E7C端第58、91位氨基酸突变后真核细胞表达成功,免疫荧光检测在质粒转染COS-7细胞24h后两种重组体均有阳性细胞出现,但在48h时突变型重组体阳性细胞消失。免疫印迹技术在24h和48h时,突变型重组体均未见阳性条带。结论 HPV16E7C端锌指结构对维持蛋白稳定性起重要作用,突变后蛋白稳定性下降。

关键词: 乳头状瘤病毒, 人, 突变, 基因表达

Abstract: Objective To induce the mutation of HPV-16 E7 in two zinc-binding motifs near the C terminus by polymerase chain reaction (PCR) and evaluate the effect of this mutation on the antigen-specific immunity of HPV-16 E7. Methods HPV-16 E7 fragment was amplified by PCR and cloned into pGEM-3zf vector. Two site mutations at 58 and 91 animo acid sites in the open reading frame of HPV-16 E7 were induced by PCR, and then the molecular clones of HPV-16 E7 wild type (pcDNA3.1/E7) and mutant (pcDNA3.1/ME7) were successfully reconstructed. Western blot and immunofluorescence were used to detect the expression of E7 protein. Results Intracellular fluorescence signals were observed in the cells transfected with pcDNA3.1/E7 and pcDNA3.1/ME7 24 hours after transfection, but the signals in the cells transfected with pcDNA3.1/ME7 disappeared 48 hours after tansfection. Twenty-four and 48 hours after transfection with pcDNA3.1/ME7, E7 protein was not detected by Western blot. Conclusions The stability of HPV-16 E7 protein is reduced by mutations (C58G, C91G) near two zinc-binding motifs. It is suggested that the zinc-binding motifs near the C terminus of HPV-16 E7 may be important for maintaining the stability of E7 protein.

Key words: Papillomavirus, human, Mutation, Gene expression