人乳头瘤病毒18型E6蛋白真核表达载体的构建及其在子宫颈癌细胞株Hela细胞中的表达  

Construction of p3×flag-myc-CMV-24-HPV18E6 fusion gene expression vector and its expression in Hela Cells

作　　者：卫莹[1,2] 朱靖[1,2] 王青元[1,2] 范少君[1,2] 李敏[1,2] 肖卫华[1,2] 凌斌[1,2] 周颖[1,2] 

机构地区：[1]安徽医科大学附属省立医院妇产科分子实验室安徽省分子医学重点实验室,安徽合肥230001 [2]中国科技大学生命科学院免疫学研究所

出　　处：《中国实验诊断学》2010年第2期159-161,共3页Chinese Journal of Laboratory Diagnosis

基　　金：安徽省自然科学基金项目(编号:090413117)

摘　　要：目的构建并鉴定p3×flag-myc-CMV-24-HPV18E6真核表达载体,并转染Hela细胞后,检测其在Hela细胞中的表达。方法用RT-PCR法从Hela细胞中扩增出带HindⅢ、SalⅠ酶切位点的HPV18E6基因片段,将HPV18E6基因片段连接到pMD-18T载体上,通过限制性内切酶HindⅢ、SalⅠ酶切,T4连接酶连接到含有上述酶切位点的p3×flag-myc-CMV-24真核表达载体上,通过脂质体将p3×flag-myc-CMV-24-HPV18E6转染入Hela细胞48h后,Western-blot检测HPV18E6及3×flag表达情况。结果p3×flag-myc-CMV-24-HPV18E6真核表达载体构建成功,构建的真核表达载体可以被限制性内切酶HindⅢ、SalⅠ切开,电泳可显示相应条带,经测序其序列与设计的一致,转染到Hela细胞48h后经Western-blot检测可见HPV18E6的高表达及flag的表达。结论p3×flag-myc-CMV-24HPV18E6真核表达载体的成功构建为进一步研究HPV18E6的致癌机制奠定了基础。Objective To construct p3× flag-myc-CMV-24-HPV18E6 fusion gene in eukaryotic expression vector and test its expression in Hela cells.Methods HPV18E6 gene sequence with HindⅢ and Sal Ⅰ restriction enzyme cutting site were amplified from total RNA of Hela cell line by reverse transcription-polymerase chain reaction （RT-PCR）, then the HPV18E6 gene sequence was in： serted into pMD-18T vector. After enzyme cutting by HindⅢ and Sal Ⅰ , use T4 ligase to form the eukaryotic expression vector p3 ×flag-myc-CMV-24-HPV18E6. The plasmid p3 × flag-myc-CMV- 24-HPV18E6 were transfected into the Hela cells. Western-blot were used to test the expression levels of flag and HPV18E6 after 48 h. Results The p3 × flag-myc-CMV-24-HPV18E6 eukaryotic expression vector was successfully constructed. Design of consistent seen after transfection of the expression of flag. Conclusion The eukaryotic expressing vector encoding HPV18 E6 was constructed and it expressed E6 protein correctly in Hela cells.

关 键 词：HELA细胞 真核表达载体 HPV18E6 FLAG 

分 类 号：R737.33[医药卫生—肿瘤]