文章摘要
王国栋,郭连瑞,谷涌泉,李建新,汪忠镐,张建.pEGFP-N1-heNOS重组质粒的构建与鉴定[J].南京医科大学学报,2007,(7):651~655
pEGFP-N1-heNOS重组质粒的构建与鉴定
Construction and verification of pEGFP-N1-heNOS recombinant plasmid
投稿时间:2007-05-12  
DOI:10.7655
中文关键词: 人内皮型一氧化氮合酶基因  绿色荧光蛋白  真核表达质粒  基因重组
英文关键词: endothelial nitric oxide synthase  green fluorescent protein  eukaryotic expression recombinant plasmid  gene recombination.
基金项目:国家自然科学基金资助项目(30471708),北京市自然科学基金资助项目(7072031)
作者单位
王国栋 首都医科大学宣武医院血管外科,北京 100053 
郭连瑞  
谷涌泉  
李建新  
汪忠镐  
张建  
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中文摘要:
      目的:构建pEGFP-N1-heNOS真核表达重组质粒?方法:应用基因重组手段,根据人内皮型一氧化氮合酶(human endothelial nitric oxide synthase,heNOS)基因序列和表达载体pEGFP-N1质粒上的多克隆位点设计了1对引物,对含有heNOS基因的质粒pBluescript Ⅱ SK-heNOS进行了PCR扩增,得到了约3.6 kbp的目的片段?将其插入到载体启动子下游,与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因融合,重组质粒经菌液PCR?限制性内切酶酶切和测序进行鉴定?结果:用PCR方法成功从质粒pBluescript Ⅱ SK-heNOS中扩增出heNOS基因片段,经菌液PCR?酶切及测序鉴定,heNOS基因成功插入到载体pEGFP-N1质粒中,插入目的基因的真核表达载体的酶切图谱与预期一致,并且heNOS基因与Genbank所提供的序列完全一致?结论:成功构建了pEGFP-N1-heNOS真核表达重组质粒?
英文摘要:
       Objective:To construct a EGFP(enhanced green fluorescent protein)-labled eukaryotic expression recombinant plasmid of heNOS(human endothelial nitric oxide synthase)gene. Methods:According to the gene of heNOS and the multiple clone site of the expression vector pEGFP-N1 plasmid,a specific pair of primers were designed and synthesized. PCR amplification of heNOS gene from the pBluescript Ⅱ SK-heNOS plasmid was performed,and an approximate 3.6 kbp objective fragment was achieved. The objective fragment was inserted into the downstream of the carrier promoter and combined with the report gene EGFP. The recombinant plasmid was verified by PCR of the bacterium liquid,restriction enzyme digestion and gene sequencing. Results:The heNOS fragment from pBluescript Ⅱ SK-heNOS plasmid were amplified and inserted into the pEGFP-N1 carrier. The enzyme digesting spectra of the recombinant plasmid was correct,and the heNOS gene sequence was completely in accordance with the sequence provided by the Genbank. Conclusion:The pEGFP-N1-heNOS eukaryotic expression recombinant plasmid were successfully constructed.
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