文章摘要
朱贤慧,张 蕾,杜紫薇,徐 楚,孙 楠,周亚萍,张 宇,周其冈.大鼠神经元型一氧化氮合酶基因慢病毒载体的构建及功能测定[J].南京医科大学学报,2019,(6):841~845.861
大鼠神经元型一氧化氮合酶基因慢病毒载体的构建及功能测定
Construction of lentiviral vector carrying neuronal nitric oxide synthase and detection of the function
投稿时间:2018-12-07  
DOI:10.7655/NYDXBNS20190610
中文关键词: 神经元型一氧化氮合酶  慢病毒  神经干细胞  一氧化氮
英文关键词: neuronal nitric oxide synthase  lentivirus  neuronal stem cells  NO
基金项目:国家自然基金面上项目(81871065);江苏省自然科学基金(BK20140905)
作者单位
朱贤慧 南京医科大学药学院江苏 南京 211166 
张 蕾 南京医科大学药学院江苏 南京 211166 
杜紫薇 南京医科大学药学院江苏 南京 211166 
徐 楚 南京医科大学药学院江苏 南京 211166 
孙 楠 南京医科大学药学院江苏 南京 211166 
周亚萍 南京医科大学药学院江苏 南京 211166 
张 宇 南京医科大学药学院江苏 南京 211166 
周其冈 南京医科大学药学院江苏 南京 211166 
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中文摘要:
      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。
英文摘要:
      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 293T 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 co? incubation 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.
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