构建皮肤组织中特异表达HPV16-E6基因的小鼠模型  

Modeling of Transgenic Mice Specifically Expressed HPV16- E6 in Skin

作　　者：汤俊明[1] 赵彦平[1] 刘奇[1] 盛青松 吴黎明[1] 乔国洪 

机构地区：[1]宜兴市人民医院,宜兴214200 [2]无锡市申瑞生物制品有限公司,无锡214200

出　　处：《中国生物工程杂志》2015年第10期27-31,共5页China Biotechnology

基　　金：江苏省自然基金资助项目(BK2012564)

摘　　要：目的:构建特异在皮肤表达乳头瘤病毒(HPV16)E6基因的真核表达载体,并鉴定其在转基因小鼠体内的表达。方法:通过PCR方法扩增皮肤特异启动子p INV及HPV16-E6,将以上片段通过酶切连接,插入去掉CMV启动子的pc DNA3.1(-)载体,获得dpc DNA3.1(-)-p INV-E6载体;并显微注射制备其转基因小鼠,利用RT-PCR、Western blot及免疫组化技术检测获得的阳性小鼠体内E6的表达水平。结果:dpc DNA3.1(-)-p INV-E6载体测序正确;经鉴定31只实验小鼠中,有2只小鼠携带外源基因,将其与野生型小鼠交配获得的F1代中又有2只阳性小鼠;且在获得阳性小鼠的皮肤组织中RT-PCR检测有E6的转录本,Western blot检测有E6蛋白表达,且免疫组化检测结果显示有E6在皮肤表达且引起皮肤微增生。结论:成功构建了p INV-E6转基因模型小鼠,HPV16-E6基因在小鼠皮肤中特异表达,为进一步研究HPV16-E6在癌症中的作用奠定了基础。Objective： To construct the eukaryotic vector for E6 gene of papilloma virus （HPV16） specifically expressed in skin and to identify the mRNA or protein of E6 in transgenic mice. Methods ： pINV promoter and HPV16- E6 were amplified by polymerase chain reaction （PCR）, and the obtained segments were sequentially digested and inserted to pcDNA3. 1 （ - ） vector removed CMV promoter, acquiring dpcDNA3. 1 （ - ）-pINV-E6 vector. The transgenic mice were prepared by micro-injection, while the RT-PCR, Western blot and immunochemistry were used to detect the expression of E6 in transgenic mice. Results： The dpcDNA3. 1 （ -）-pINV-E6 vector was correctly connected by sequencing. Then two positive mice carrying exogenous gene were produced among 31 experimental mice. The FI generation was got by transgenic mice mating with the wild type mice. The E6 mRNA or protein was specifically positive in skin of the transgenic mice by RT-PCR, Western blot or immunochemistry. Conclusion ： The pINV-E6 transgenic model mice were successfully built with E6 gene specifically expressed in mouse skin. This would lay a solid foundation for the further study of the role of E6 in cancer.

关 键 词：乳头瘤病毒(HPV) 转基因小鼠历基因 特异表达 

分 类 号：Q789[生物学—分子生物学]