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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 an eukaryotic expression vector specifically expressing E6 gene of papilloma virus (HPV16) on the skin and to identify its expression in transgenic mice. Methods: The skin specific promoter p INV and HPV16-E6 were amplified by PCR. The above fragment was inserted into the pcDNA3.1 (-) vector without CMV promoter by digestion and ligated to obtain dpc DNA3.1 (-) - p INV-E6 vector. The transgenic mice were prepared by microinjection. The expression of E6 in positive mice was detected by RT-PCR, Western blot and immunohistochemistry. Results: The dpc DNA3.1 (-) - p INV-E6 vector was sequenced correctly. Two out of 31 experimental mice were identified as carrying the exogenous gene and the F1 generation obtained by mating with wild-type mice There were 2 positive mice; E6 transcripts were detected by RT-PCR in the skin tissues of the positive mice, E6 protein was detected by Western blot, and the results of immunohistochemistry showed that E6 was expressed on the skin and caused Micro-hyperplasia of the skin. CONCLUSIONS: The mouse model of p INV-E6 transgenic mice was constructed successfully. The HPV16-E6 gene was specifically expressed in mouse skin, which laid the foundation of further study on the role of HPV16-E6 in cancer.