K14和CMV启动子驱动裸鼠体内HPV16 E6/E7基因表达的差异  

Different Expression of HPV E6/E7 Gene Driven by K14 and CMV Promoters in Nude Mice

作　　者：潘巍巍[1,2] 徐营[1] 曹利仙[1] 沈忠飞[1] 李平[1] 郭连军[1] 邬维娅[1] 宋方洲[1,3] 

机构地区：[1]嘉兴学院医学院生物教研室,浙江嘉兴314001 [2]重庆医科大学生物化学与分子生物学教研室,重庆400016 [3]重庆医科大学临床检验诊断学省部共建教育部重点实验室,重庆400016

出　　处：《中国生物化学与分子生物学报》2009年第7期620-626,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.30371479);浙江省实验动物科研计划项目(No.2008F80025);嘉兴市科技局科研重点项目(No.2007AZ2013);嘉兴市科技局科研项目(No.2008AY2039-3;No.2008AY2039-2)~~

摘　　要：人乳头瘤病毒(human papillomavirus,HPV)是一种无包膜的环状闭合双链DNA病毒,具有严格的嗜人组织的特性.为探讨不同启动子驱动HPVE6/E7癌蛋白在裸鼠体内不同组织的表达效率,构建了带有角蛋白(K14)启动子和带有巨细胞病毒(CMV)启动子的E6/E7腺病毒载体(pAd-K14-E6/E7和pAd-CMV-E6/E7),pAd-K14-E6/E7和pAd-CMV-E6/E7、以及作为对照的重组腺病毒空载体pAdtrack-K14和pAd-CMV同源重组后,分别在293细胞中包装,收集重组病毒Ad-K14-E6/E7、Ad-CMV-E6/E7、Adtrack-K14和Ad-CMV,通过尾缘静脉注射到随机分组的裸鼠体内,并每d向裸鼠腹腔注射0.05 mg雌激素.采用RT-PCR和Western免疫印迹检测不同实验组E6/E7 mRNA和蛋白表达水平,免疫组化法检测P53和Bcl-2蛋白表达.结果显示,注射病毒Ad-K14-E6/E7(实验组1)裸鼠子宫体中E6/E7 mRNA、E6蛋白质、P53和Bcl-2蛋白高表达,而其它组织中低表达;注射病毒Ad-CMV-E6/E7(实验组2)裸鼠各组织E6/E7 mRNA、E6蛋白质、P53和Bcl-2蛋白均低表达.研究表明,在裸鼠体内角蛋白K14启动子可以调控E6/E7在子宫体中表达,CMV启动子未能诱导E6/E7在子宫体中表达.Human papillomaviruses （HPVs）, one kind of double-stranded covalently closed circular DNA （cccDNA） viruses, have a specific infection property in human tissues. To explore the expression ability of different promoters on HPV E6/E7 oncoproteins in different tissues of nude mice, the vectors of pAd-K14-E6/ E7 containing K14 promoter and pAd-CMV-E6/E7 containing CMV promoter, were constructed, respectively. The replication-deficient adenovirus Ad-K14-E6/E7, Ad-CMV-E6/E7, Adtraek- K14 and Ad-CMV were generated and amplified after the vectors were transfected into HEK293 ceils. The virus supernatant was collected and injected into nude mice grouped randomly by trail vein. The Ad-K14 -E6/E7 and Ad-CMV-E6/ E7 were as experimental groups, while the Ad-CMV and Adtraek-K14 were as control groups . These nude mice were treated with 0.05 mg estrogen by intraperitoneal injection every day. E6 and E7 mRNA and protein expression of different groups were detected through RT-PCR and Western blot, and P53 and Bel-2 protein expression were detected by immunohistoehemistry. The results showed that E6/E7 mRNA, E6 protein, P53 and Bcl-2 proteins were all highly expressed in uterine body of experimental group 1 （injected Ad-K14-E6/E7 ＋ estrogen）, while they showed low expression in other tissues. In experimental group 2（injected Ad-CMV- E6/E7 ＋ estrogen）, those proteins and mRNA were expressed at quite low levels in all tissues. The conclusion was that the K14 cytokeratin promoter could drive E6/E7 expression, in uterine body of nude mice, while the CMV promoter could not.

关 键 词：人乳头瘤病毒 腺病毒 E6/E7基因 裸鼠 CMV启动子 角蛋白K14启动子 

分 类 号：R373.39[医药卫生—病原生物学]