摘要
目的构建HPV18型E6E7反义荧光真核表达载体,并观察其对宫颈癌HeLa细胞中HPV18 E6和E7基因表达的影响,探索反义技术用于治疗临床HPV感染及宫颈癌的可能性。方法以HPV18型全基因质粒为模板,PCR法扩增HPV18型E6E7区716bp片段,利用基因重组技术将目的片段反向插入荧光真核表达载体pEGFP-C1,EcoR I酶切并测序鉴定;采用脂转法将重组质粒pEGFP-HPV18 E6E7as(EGFP-18AS)转染宫颈癌HeLa细胞株,通过RT-PCR及western blot检测细胞中E6、E7 mRNA和蛋白的表达。结果成功构建HPV18E6E7反义荧光真核表达载体EGFP-18AS,经脂质体转染HeLa细胞,48h后在荧光倒置显微镜下可见明显的绿色荧光,且细胞中E6、E7 mRNA及蛋白表达水平均明显下调。结论反义荧光真核表达载体可以有效的抑制HPV18E6、E7癌基因的表达,为治疗HPV感染和宫颈癌提供了一种新的方法。
Objective In order to seek for a therapeutic approach to the diseases caused by HPV or cervix cancer, we constructed antisense eukaryotic fluorescent expression vector of human papillomavirus （HPV） 18 E6E7 and detected their expression in HeLa cervical carcinoma cells. Methods HPV18 E6E7 716bp was amplified the by PCR with the full-length HPV18 cDNA clone vector as the template. Then the PCR product was inserted into eukaryotic fluorescent expressing vector pEGFP in antisense orientation by DNA recombinant technology and further transfected into HeLa cervical carcinoma cells. Expression of HPVI8 E6/E7 in transfectants was examined by the fluorescence microscope, RT-PCR and Western blot. Results The fluorescence eukaryotic expression vector carrying antisense HPV18 E6E7 gene fragment was successfully constructed. At 48h after transfection of recombinant plasmid, bright green fluorescence was visible and the E6/E7 mRNA and proteins were obviously down regulated in the HeLa cervical carcinoma cells. Conclusion Delivery of antisense HPV18 E6E7 can effectively suppress the expression of E6/E7 oncogenes and is potentially a new approach to treat HPV infection and cervix cancer.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2007年第6期402-404,共3页
Cancer Research on Prevention and Treatment
基金
国家自然科学基金资助项目(30528012
30672227)
国家重点基础研究发展计划(973)资助项目(2002CB513100)
