HPV16E6真核表达载体的构建及其在CaSki细胞中的表达及定位

目的:构建pEGFP-C2-HPV16E6真核表达载体,转染CaSki细胞,检测其在CaSki细胞中的表达。方法:用RT-PCR法从CaSki细胞中扩增出带有HindⅢ、XbaⅠ酶切位点的HPV16E6基因片段,将HPV16E6基因全长片段克隆到增强型绿色荧光蛋白(EGFP)基因真核表达载体pEGFP-C2上,通过脂质体将pEGFP-C2-HPV16E6转染入CaSki细胞。结果:pEGFP-C2-HPV16E6真核表达载体构建成功,转染CaSki细胞48h后经RT-PCR及Western-blot检测可见HPV16E6蛋白的高表达,激光共聚焦显微镜观察融合蛋白主要在CaSki细胞核内表达。结论:成功构建了pEGFP-C2-HPV16E6真核表达载体,为进一步对HPV16E6的研究奠定了基础。

Construction of HPV16E6 eukaryotic expression vector and its expression and location in CaSki cells

Objective:To construct pEGFP-C2-HPV16E6 eukaryotic expression vector,transfect CaSki cells,and detect its expression in CaSki cells.Methods:RT-PCR was used to amplify HPV16E6 gene segment in CaSki cells with HindⅢ and XbaⅠ restriction enzyme cutting sites,the total HPV16E6 gene segment was cloned into eukaryotic expression vector pEGFP-C2 encoding enhanced green fluorescent protein(EGFP).HPV16E6 was transfected into CaSki cells according to plasmid.Results:pEGFP-C2-HPV16E6 eukaryotic expression vector was successfully constructed.High expression of 16E6 protein could be detected by RT-PCR and Western blot after transfecting into CaSki cells for 48 hours,the expression of fusion protein in CaSki cell nucleus was observed under laser scanning confocal microscope.Conclusion:pEGFP-C2-HPV16E6 eukaryotic expression vector is successfully constructed,which provide a foundation for further investigation of HPV16E6.