用Gateway 系统构建携带Grb2-SH2 基因重组慢病毒载体

目的:构建携带Grb2-SH2基因重组慢病毒载体。方法:把Grb2-SH2基因从合成的工程载体PUC-57中导到入门载体pentr-3C中,再利用LR反应重组到目的载体plenti,通过酶切和测序分析对比验证Grb2-SH2基因后,将plenti-Grb2-SH2质粒利用慢病毒转染系统转染人胚胎肾上皮细胞株293T细胞获得携带Grb2-SH2慢病毒载体。结果:构建的重组质粒经酶切及测序和分析比对鉴定正确,目的基因片段大小约为500bp;该质粒与包装质粒共转染293T细胞获取的慢病毒,转染效率约为90%。结论:成功构建转载质粒plenti-Grb2-SH2及携带Grb2-SH2基因慢病毒载体。

Construction of Grb2-SH2 genetic restructuring-slow virus carrier with Gateway system

Objective: Construct Grb2-SH2 genetic recombination slow virus carrier.Methods: Put the Grb2-SH2 genes into introductory carrier pentr-3 C from synthetic engineering carrier PUC-57,reuse LR reaction to recombinate into the purpose plenti carrier,after comparing and testing gene Grb2-SH2 through the enzyme cut and sequencing analysis and comparison,use slow virus carrying system to transfect human embryonic renal epithelial cell lines 293 T cells acquring Grb2-SH2 slow virus carrier.Results: The restructuring plasmid proved to be correct by the enzyme cut and sequencing analysis and the purpose genetic fragments is about 500 bp;The efficiency of the plasmid and packaging plasmid transfect 293 T cells get slow is about 90%.Conclusion: Successful construction reproduced plasmid plenti-Grb2-SH2 and Grb2-SH2 gene slow virus carrier.