Construction of lentiviral vector carrying human VE-cadherin gene and expression of VE-cadherin in leukemic cell line Sup-B15 / 中国实验血液学杂志

ABSTRACT

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.