HPV18E6真核表达载体的构建及其在HaCaT细胞的表达  

Construction of HPV16E6 eukaryotic expression vector and its expression in HaCaT cells

作　　者：李兵[1] 冯定庆 李跃波[1] 伍娇娇[1] 张红丽[1] 尤青叶 朱园园 凌斌[1] 

机构地区：[1]安徽医科大学附属省立医院妇产科,安徽合肥230001 [2]安徽医科大学附属省立医院分子实验室

出　　处：《中国妇幼保健》2014年第11期1739-1742,共4页Maternal and Child Health Care of China

基　　金：国家自然基金课题(81372779;81372777);安徽省自然科学基金课题(110406M176;110406M178)

摘　　要：目的:构建pEGFP-C2-HPV18E6真核表达载体,观察其在HaCaT细胞中的表达。方法:采用RT-PCR法从HeLa细胞中扩增出带有XhoⅠ、EcoRⅠ酶切位点的HPV18E6基因片段,双酶切技术将HPV18E6基因全长片段克隆到真核表达载体pEGFP-C2上,PCR、双酶切、测序鉴定重组质粒;将重组质粒pEGFP-C2-HPV18E6通过脂质体瞬时转染HaCaT细胞,RT-PCR及融合蛋白绿色荧光观察,检测目的基因的表达。结果:双酶切及测序结果表明真核表达载体pEGFP-C2-HPV18E6构建成功,转染HaCaT细胞48h后可见绿色荧光蛋白的表达,RT-PCR可扩增目的基因条带。结论:成功构建了pEGFP-C2-HPV18E6真核表达载体,为进一步研究HPV18E6的生物学功能提供了帮助。Objective： To construct pEGFP- C2 -HPV18E6 eukaryotic expression vector, observe its expression m HaldaT cells. Methods： RT- PCR was used to amplify HPV18E6 gene segment with Xho I , EcoR I restriction enzyme cutting sites in HeLa cells, the total HPV18E6 gene segment was cloned into eukaryotic expression vector pEGFP - C2. PCR, double enzyme cutting, and DNA sequencing were used to identify the recombined plasmid, pEGFP - C2 - HPV18E6 was transfected into HaCaT cells through liposomes. RT - PCR and fusion protein green fluorescent protein observation were used to detect the expression of target gene. Results： Double enzyme cutting and DNA sequencing verified that pEGFP - C2 - HPV18E6 eukaryotic expression vector was successfully constructed. The visible expression of green fluorescent protein was detected after the reeombined plasmid transfecting into HaCaT cells for 48 hours, RT - PCR amplified the target gene stripe. Conclusion： pEGFP- C2 -HPV18E6 eukaryotic expression vector is successfully constructed, which provides assistance for further studying the biological function of HPV18E6.

关 键 词：宫颈癌 HPV18E6 基因表达 

分 类 号：R730.7[医药卫生—肿瘤]