小鼠Akt基因真核表达载体的构建及鉴定

Construction and identification of eukaryotic expression vector of Akt gene in mouse

目的构建含有小鼠Akt基因以及绿色荧光蛋白报告基因的真核表达载体pDoubleEx-EGFPAkt1。方法以小鼠卵巢RNA为模板,通过RT-PCR扩增Akt,并将其克隆到pDoubleEx-EGFP载体中。通过酶切和测序鉴定,构建真核表达载体pDoubleEx-EGFP-Akt1。之后转染人卵巢癌上皮细胞SKOV-3。结果经测序鉴定,构建的Akt基因序列与野生型Akt序列完全相符。将pDoubleEx-EGFP-Akt1转染进入人卵巢癌上皮细胞SKOV-3,观察到稳定转染后的细胞有明显绿色荧光蛋白表达。结论成功构建含有小鼠Akt基因的真核表达载体pDoubleEx-EGFP-Akt1,为进一步研究Akt基因的功能提供初步的实验基础。

Objective To construct an eukaryotic expression vector p Double Ex-EGFP-Akt1 containing mouse Akt gene and green fluorescent protein reporter gene. Methods Using RNA of mouse ovary as template,Akt c DNA was amplified by RT-PCR and cloned into vector p Double Ex-EGFP. It was confirmed by enzyme digestion and sequencing before making recombinant plasmid p Double Ex-EGFP-Akt. The correct recombinant plasmid was transfected into SKOV-3 cells. Results Akt gene sequence contained in p Double Ex-EGFP-Akt1 recombinant plasmid was verified by enzyme digestion as well as sequencing results. After being transfected into SKOV-3 cells,it showed stable and highly transfection with green fluorescent protein expression. Conclusion p Double Ex-EGFP-Akt recombinant plasmid has been constructed successfully with high efficiency in SKOV-3 cells,which may provide an useful tool to analyze the function of Akt gene in reproductive.