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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。
OBJECTIVE: To construct the eukaryotic expression vector pEGFP-C2-HPV16E6 and transfected into CaSki cells to detect its expression in CaSki cells. Methods: The HPV16E6 gene fragment with Hind Ⅲ and Xba Ⅰ restriction sites was amplified from CaSki cells by RT-PCR. The full-length HPV16E6 gene fragment was cloned into the eukaryotic expression vector pEGFP of enhanced green fluorescent protein (EGFP) -C2, pEGFP-C2-HPV16E6 was transfected into CaSki cells by liposomes. Results: The eukaryotic expression vector pEGFP-C2-HPV16E6 was constructed successfully. The expression of HPV16E6 protein was detected by RT-PCR and Western-blot 48 h after transfected with CaSki cells. The expression of HPV16E6 protein was mainly observed in CaSki cells by laser confocal microscopy. Conclusion: The eukaryotic expression vector pEGFP-C2-HPV16E6 has been successfully constructed, which lays the foundation for the further research on HPV16E6.