携带人VE-cadherin基因的慢病毒载体的构建及其在Sup-B15白血病细胞株的表达

Construction of Lentiviral Vector Carrying Human VE-cadherin Gene and Expression of VE-cadherin in Leukemic Cell Line Sup-B15

本研究旨在构建携带人血管内皮钙黏蛋白(VE-cadherin)基因的重组慢病毒载体及探讨VE-cadherin蛋白在Sup-B15细胞中的表达。采用RT-PCR扩增人VE-cadherin基因并克隆至pCR-Blunt载体。将VE-cadherinDNA片段连入慢病毒转移质粒pLB,生成重组慢病毒质粒pLB-VEC。用三质粒共转染法包装慢病毒,重组慢病毒感染Sup-B15细胞,在光学显微镜下观察细胞状态,用流式细胞术及Western blot法鉴定VE-cadherin蛋白表达。结果表明,成功扩增出人VE-cadherinDNA片段并克隆至pCR-Blunt载体;亚克隆构建慢病毒载体pLB-VEC。经三质粒包装系统包装后获得高滴度慢病毒颗粒,体外可有效感染Sup-B15细胞。感染后细胞发生明显的形态改变,流式细胞术及Western blot检测到VE-cadherin蛋白表达。结论 :成功构建携带人VE-cadherin基因的慢病毒载体pLB-VEC,并可在Sup-B15白血病细胞株获得有效的表达。

In order to construct a lentiviral vector carrying human VE-cadherin gene, and to express VE-cadherin in Sup-B15 cells, the VE-cadherin gene was amplified by RT-PCR from the human placenta, and then cloned into pCR-Blunt vector. The VE-cadherin DNA fragment was subcloned into pLB vector to generate a lentiviral vector pLB-VEC. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT packing cells using lipofectamine 2000. The Sup-B15 cells were transfected by the lentivirus. The post-transfected Sup-B15 cells were observed by microscopy and flow cytometry. Western blot was used to determine the expression of VE-cadhedn. The results showed that the VE-cadherin DNA fragment was amplified from human placenta and was cloned into pCR-Blunt vector, the recombinant lentiviral vector pLB-VEC was successfully constructed. High titer lentivirus was prepared by 3-plasmid packing system, and transfected into Sup-B15 cells in vitro effectively. The obviously morphological changes occurred in transfected cells, the expression of VE-cadherin protein could be detected in Sup-B15 cells via flow cytometry and Western blot. It is concluded that the lentiviral vector pLB-VEC carrying human VE-cadherin gene is successfully constructed; VE-cadherin gene is expressed in Sup-B15 cells via lentiviral vector transfection, which provides an optional tool for further study on the mechanism of VE-cadherin controlling leukemia development.