K14和CMV启动子驱动裸鼠体内HPV16 E6/E7基因表达的差异

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HPV E6/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-6/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．

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

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, Adtrack K14 and Ad-CMV were generated and amplified after the vectors were transfected into HEK293 cells. 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 Adtrack 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 Bcl-2 protein expression were detected by immunohistochemistry. 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.