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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 and prepare a recombinant lentivirus that can express Semaphorin 4D efficiently. METHODS: Human Semaphorin 4D gene was amplified from DNA of human acute T cell leukemia Jurkat cells and cloned into pWPI GW lentiviral vector. The recombinant plasmid was co-transfected into 293T cells with pVSVG and pSPAX plasmids. The recombinant lentivirus was packaged and purified. Of 293T and HUVEC cells were directly infected with recombinant virus. The expression of Semaphorin 4D and the migration of vascular endothelial cells were detected by Western blotting, immunofluorescence and vascular endothelial cell migration assay. Results: Recombinant lentivirus-mediated expression of Semaphorin 4D in 293T and HUVECs could mediate the migration of vascular endothelial cells. Conclusion: Recombinant lentiviral vector expressing Semaphorin 4D was successfully constructed.