人Semaphorin 4D慢病毒载体的构建及鉴定

Construction and Identification of a Lentiviral Vector Expressing Semaphorin 4D

目的:构建并制备能够有效表达Semaphorin 4D的重组慢病毒。方法:从人急性T细胞白血病Jurkat细胞DNA扩增人Semaphorin 4D基因,克隆至pWPI GW慢病毒载体上,与pVSVG及pSPAX质粒共转染人胚肾293T细胞,包装出重组慢病毒,将纯化后的重组病毒直接感染293T和HUVEC细胞,通过免疫印迹、免疫荧光染色和血管内皮细胞迁移分析等方法检测Semaphorin 4D的表达和诱导血管内皮细胞迁移的作用。结果:重组慢病毒介导Semaphorin 4D在293T和HUVEC内获得表达,能介导血管内皮细胞迁移。结论:成功构建了表达Semaphorin 4D的重组慢病毒载体。

Objective： To construct a lentiviral vector expressing Semaphorin 4D to investigate the role of Semaphorin 4D in angiogenesis. Methods： Semaphorin 4D sequence was amplified from the Jurkat cell cDNA, inserted into pWPI GW vector and identified by DNA sequencing. The packing cell line （human embryonic kidney 293T cell） was cotransfected with pWPI GW/Sema4D, pVSVG and pSPAX. The recombinant lentivirus was packaged and amplified, followed by infection of 293T and HUVEC cells. The expression level of Semaphorin 4D gene was analyzed by Western blot and immunofluresence staining. A Boyden chamber migration assay was performed to establish the biological significance of Semaphorin 4D. Results： The recombinant lentiviral vector expressing Semaphorin 4D was successfully constructed. Western blot and immunofluresence staining results demonstrated that Semaphorin 4D was successfully expressed in 293T and HUVEC cells infected by recombinant lentivirus expressing Semaphorin 4D. Boyden chamber migration assay results showed that enhanced endothelial cell migration is induced by Sema4D-expressing Hela cells.