小鼠nNOS(AA1-133)基因慢病毒载体的构建及功能初步检测

Construction and detection of lentiviral vector carrying mouse nNOS(AA1-133)

目的:构建含nNOS(AA1-133)基因的慢病毒载体,检测其表达和功能。方法:采用RT-PCR扩增小鼠nNOS(AA1-133)基因,构建真核表达载体pIRES2-EGFP/nNOS(AA1-133)。鉴定正确后将目的基因克隆入慢病毒载体pGC-FU,得重组载体pGC-FU/nNOS(AA1-133),采用Lipofectamine 2000将其转染293T细胞,包装慢病毒颗粒后感染原代神经元,检测目的片段的表达和功能。结果:成功扩增小鼠nNOS(AA1-133)基因片段,测序证明重组慢病毒载体pGC-FU/nNOS(AA1-133)构建成功,包装后的慢病毒颗粒可以感染神经元,目的片段可在神经元中表达并与PSD95结合。结论:慢病毒载体pGC-FU/nNOS(AA1-133)构建成功,目的片段在原代神经元中可表达并发挥功能。

Objective：To construct the lentiviral vector carrying nNOS（AA1-133）and detect its expression and function.Methods：Mouse nNOS（AA1-133）was amplified by RT-PCR and then eukaryotic expression vector of pIRES2-EGFP / nNOS（AA1-133）was constructed.After DNA sequence analysis,nNOS（AA1-133）was cloned into lentiviral vector pGC-FU to construct recombinant vector pGC-FU / nNOS（AA1-133）.pGC-FU / nNOS（AA1-133）was transfected into 293T cells by Lipofectamine 2000 mediation to package lentiviral particles.The particles were transfected into primary neurons,and the expression and function of nNOS（AA1-133）were detected.Results：The coding sequence of mouse nNOS（AA1-133）was successfully amplified and the analysis of DNA sequence approved that the recombinant lentiviral vector contained nNOS（AA1-133）.nNOS（AA1-133）expressed and combined with PSD95 in the infected neurons.Conclusion：The lentiviral vector of pGC-FU/nNOS（AA1-133）was constructed successfully and the infected neurons could express nNOS（AA1-133）.