小鼠Sema3A基因过表达慢病毒载体的构建与鉴定

摘要

目的：构建小鼠Sema3A基因过表达慢病毒载体。方法通过体外合成小鼠全长Sema3A cDNA，采用Gateway技术将其基因插入到pDown⁃mSema3A⁃IRES/EGFP质粒中，再交换重组获得重组质粒pLV/EXPNZ⁃puro⁃mSema3A⁃IRES/EGFP，经过测序鉴定后，包装与浓缩慢病毒载体pLV（Exp）⁃Puro⁃CMV⁃mSema3A⁃IRES/EGFP，最后重组慢病毒载体转染293T细胞获得病毒液。结果重组慢病毒载体11538 bp，测序结果证实Sema3A基因共2319 bp正确插入带有EGFP的载体中，成功构建小鼠Sema3A基因过表达载体。结论成功构建小鼠慢病毒载体pLV（Exp）⁃Puro⁃CMV⁃mSema3A⁃IRES/EGFP，为该基因在特定细胞内过表达株的筛选奠定基础。%Objective To construct and identify over⁃expressing lentiviral vector of mSema3A. Methods Sema3A gene of mice was amplified by PCR,then the gene was inserted into plasmids pDown⁃mSema3A⁃IRES/EGFPby Gateway technology. The plasmids pLV/EXPNZ⁃puro⁃mSema3A⁃IRES/EGFP were produced by recombination. After sequencing identification,the vector pLV(Exp)⁃Puro⁃CMV⁃mSema3A⁃IRES/EGFP was packed and condensed. Finally the recombinant vectors were used to transfect 293T cells to obtain virus pools. Results The recombinant lentiviral vectors were 11 538 bp with EGFP marker,and Sema3Agenes were inserted into the lentiviral vector correctly,indicating the over⁃expressing vector of Sema3A gene in mice was successfully constructed. Conclusion The over⁃expressing lentiviral vector of mSema3A was constructed correctly, which lay a foundation of screening of over⁃expressing strains of such gene in specific cells.