【摘要】 目的:构建编码大鼠神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)全长基因的慢病毒载体,并检测其表达效率和催化活性功能。方法:采用RT-PCR提取nNOS的cDNA,同时改造真核表达载体pCDH-GFP。鉴定nNOS的cDNA碱基序列正确后,将目的基因克隆入慢病毒载体pCDH-GFP,得重组载体pCDH-GFP/nNOS,采用Lipofectamine 2000将其及慢病毒包装辅助质粒转染293T细胞,超速离心纯化后,感染神经干细胞和海马齿状回神经元进行感染能力鉴定,并检测nNOS蛋白表达和催化功能。结果:成功构建编码nNOS全长cDNA,测序证明重组慢病毒载体pCDH-GFP/nNOS构建成功。包装慢病毒颗粒LV-nNOS-GFP可以感染离体神经干细胞和在体神经元。Western blot检测证明nNOS蛋白成功表达。一氧化氮浓度检测表明表达的nNOS具有催化活性。结论:慢病毒载体LV-nNOS-GFP构建成功,可以表达功能性全长nNOS蛋白。更多还原

【Abstract】 Objective:This study aims to construct the lentiviral vector carrying the cDNA encoding neuronal nitric oxide synthase(nNOS)of rat and to detect its expression and catalytic function. Methods:The cDNA of nNOS was extracted by RT-PCR followed by eukaryotic expression vector of CDH-GFP for cloning. After confirming the cDNA of nNOS by sequencing,cDNA of nNOS was cloned into the lentiviral vector pCDH-GFP for constructing recombinant vector pCDH-GFP/nNOS.The plasmid pCDH-GFP/nNOS was then transfected into 293 T cells in the presence ofhelper plasmids by Lipofectamine 2000 mediation for packing lentiviral particles LV-nNOS-GFP. The cultured neuronal stem cells(NSCs)were used to verify whether LV-nNOS-GFP can infect NSCs by coincubation with LV-nNOS-GFP Afterwards,LV-nNOS-GFP was injected into the dentate gyrys(DG)of the hippocampus to measure the expression of nNOS and the generation of nitric oxide(NO). Results:The full length cDNA of nNOS could besuccessfully amplified and constructed into the recombinant lentiviral vector pCDH-GFP/nNOS. The packaged LV-nNOS-GFP successfully infected cultured NSCs and neurons in the hippocampal DG. The expressed nNOS was catalytic effective in catalyzing NO generation. Conclusion:The lentiviral vector of LV-nNOS-GFP was constructed successfully with functional nNOSexpression after infection.更多还原