pEGFP-N1-heNOS重组质粒的构建与鉴定  

Construction and verification of pEGFP-N1-heNOS recombinant plasmid

作　　者：王国栋[1] 郭连瑞[1] 谷涌泉[1] 李建新[1] 汪忠镐[1] 张建[1] 

机构地区：[1]首都医科大学宣武医院血管外科,北京100053

出　　处：《南京医科大学学报（自然科学版）》2007年第7期651-655,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：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)

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3.6kbp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pBluescriptⅡSK-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。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 cartier. 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-NI-heNOS eukaryotic expression recombinant plasmid were successfully constructed.

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 

分 类 号：Q784[生物学—分子生物学]