Zhang Huan-Xin, Chen Chong, Zeng Ling-Yu, Yan Zhi-Ling, Li Zhen-Yu, Xu Kai-Lin
Laboratory of Transplantation Immunology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2011 Jun;19(3):574-7.
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-cadherin. 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.
为构建携带人血管内皮钙黏蛋白（VE-cadherin）基因的慢病毒载体，并使其在Sup-B15细胞中表达，通过逆转录聚合酶链反应（RT-PCR）从人胎盘中扩增VE-cadherin基因，然后克隆至pCR-Blunt载体。将VE-cadherin DNA片段亚克隆至pLB载体以产生慢病毒载体pLB-VEC。使用脂质体2000将三种质粒共转染至293FT包装细胞中以产生重组慢病毒。用该慢病毒转染Sup-B15细胞。对转染后的Sup-B15细胞进行显微镜观察和流式细胞术检测。采用蛋白质免疫印迹法（Western blot）测定VE-cadherin的表达。结果显示，从人胎盘中扩增出VE-cadherin DNA片段并克隆至pCR-Blunt载体，成功构建了重组慢病毒载体pLB-VEC。通过三质粒包装系统制备了高滴度慢病毒，并有效转染体外培养的Sup-B15细胞。转染后的细胞出现明显的形态学变化，通过流式细胞术和Western blot可在Sup-B15细胞中检测到VE-cadherin蛋白的表达。结论：成功构建了携带人VE-cadherin基因的慢病毒载体pLB-VEC；通过慢病毒载体转染使VE-cadherin基因在Sup-B15细胞中表达，为进一步研究VE-cadherin调控白血病发生发展的机制提供了可选工具。
