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 eukaryotic expression vector pEGFP-C2-HPV18E6 and observe its expression in HaCaT cells. Methods: HPV18E6 gene fragment with XhoⅠand EcoRⅠ restriction sites was amplified by RT-PCR from HeLa cells. The full-length fragment of HPV18E6 gene was cloned into eukaryotic expression vector pEGFP-C2 by double enzyme digestion, The recombinant plasmid pEGFP-C2-HPV18E6 was transiently transfected into HaCaT cells by lipofectamine. The expression of the target gene was detected by RT-PCR and green fluorescence analysis of the fusion protein. Results: The results of double enzyme digestion and sequencing showed that the eukaryotic expression vector pEGFP-C2-HPV18E6 was constructed successfully. After transfection with HaCaT cells for 48h, the expression of green fluorescent protein was observed. RT-PCR amplified the target gene band. CONCLUSION: The eukaryotic expression vector pEGFP-C2-HPV18E6 has been successfully constructed, which helps to further study the biological function of HPV18E6.