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目的构建p3×flag-myc-CMV-24-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HindⅢ、XbalⅠ酶切位点的HPV16E6基因片段,将HPV16E6基因全长片段克隆到真核表达载体p3×flag-myc-CMV-24上,通过脂质体将p3×flag-myc-CMV-24-HPV16E6转染入Caski细胞。结果构建的p3×flag-myc-CMV-24-HPV16E6的真核表达载体转染Caski细胞48h后经Western-blot可检测到融合蛋白高表达。结论成功构建了p3×flag-myc-CMV-24-HPV16E6真核表达载体;为进一步研究HPV16E6导致子宫颈癌病变的机制奠定了基础。
Objective To construct the eukaryotic expression vector p3 × flag-myc-CMV-24-HPV16E6 and transfect Caski cells to detect its expression in Caski cells. Methods The HPV16E6 gene fragment with Hind Ⅲ and Xbal Ⅰ restriction sites was amplified from CaSki cells by RT-PCR. The full-length HPV16E6 gene was cloned into the eukaryotic expression vector p3 × flag-myc-CMV-24 Liposomes transfected p3 × flag-myc-CMV-24-HPV16E6 into Caski cells. Results The expression of fusion protein in p3 × flag-myc-CMV-24-HPV16E6 eukaryotic expression vector was confirmed by Western-blot 48h after transfection with Caski cells. Conclusion The eukaryotic expression vector p3 × flag-myc-CMV-24-HPV16E6 was successfully constructed and laid the foundation for the further study on the mechanism of HPV16E6-induced cervical cancer.