摘要
目的:构建人RIP2基因启动子的绿色荧光蛋白表达载体。方法:根据特定限制性内切酶位点,以人基因组DNA为模板,PCR扩增含人RIP2基因启动子不同长度2段序列,构建含人RIP2基因启动子驱动的绿色荧光蛋白载体pEGFP-C2-RIP2(750 bp)wt、pEGFP-C2-RIP2(941 bp)wt,用VspⅠ和NheⅠ双酶切鉴定重组质粒,进行DNA序列分析,重组质粒经阳离子聚合物JetPeiTM介导转染HEK293细胞48 h后观察。结果:酶切鉴定和序列测定证实目的基因已插入重组质粒;细胞转染结果表明,重组质粒转染HEK293细胞均能表达绿色荧光,其中构建的pEGFP-C2-RIP2(750 bp)wt重组质粒绿色荧光表达强于pEGFP-C2-RIP2(941 bp)wt。结论:成功构建2段不同长度的人RIP2基因启动子绿色荧光蛋白表达载体。
Aim：To construct a specific GFP expression vector driven by promoter of human RIP2 gene. Methods： Two different DNA segments of RIP2 gene promoter were amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-C2 which had cut out promoter by restriction enzyme.Plasmids were identified by VspⅠand NheⅠ double digestion,and sequence analysis.Recombinant plasmids were transfected into cell line HEK293 by JetPeiTM.At 48 hours post-transfection,cells were observed under the inverted fluorescence microscope.Results： Two recombinant plasmids were the same as the design confirmed by restriction digestion and sequence analysis.The results of cell transient transfection indicated that the GFP could be observed in HEK293 by two plasmids transfection.The GFP expression of constructed pEGFP-C2-RIP2（750 bp）wt was stronger than that of pEGFP-C2-RIP2（941 bp）wt.Conclusion：The GFP expression vector driven by human RIP2 gene promoter is successfully constructed.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2010年第2期140-144,共5页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
广东省自然科学基金项目(06025159)
广东省教育厅自然科学研究项目(2005-126)
