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目的构建含绿色荧光蛋白(GFP)基因与人胰岛素基因的重组真核表达载体。方法将人胰岛素基因插入到真核表达载体pIRES2-EGFP的EcoRⅠ和BamHⅠ位点中,构建重组质粒pIRES2-EGFP-INS。酶切鉴定,测序证实。结果重组真核表达载体pIRES2-EGFP-INS经限制性内切酶EcoRⅠ和BamHⅠ酶切,电泳后显示360bp的INS的目的片段和5.3kb的pIRES2-EGFP载体片段,进一步测序并证明重组质粒连接正确。结论成功构建了pIRES2-EGFP-INS重组质粒,为简便快速了解胰岛素基因的转染效率奠定了基础。
Objective To construct a recombinant eukaryotic expression vector containing green fluorescent protein (GFP) gene and human insulin gene. Methods The human insulin gene was inserted into EcoRI and BamHI sites of eukaryotic expression vector pIRES2-EGFP to construct recombinant plasmid pIRES2-EGFP-INS. Restriction enzyme digestion, sequencing confirmed. Results The recombinant eukaryotic expression vector pIRES2-EGFP-INS was digested with restriction endonucleases EcoRⅠand BamHⅠ. After electrophoresis, the target fragment of 360bp and the pIRES2-EGFP vector fragment of 5.3kb were displayed. The sequencing results showed that the recombinant plasmid was correctly ligated . Conclusion The recombinant plasmid pIRES2-EGFP-INS was successfully constructed and laid the foundation for simple and rapid understanding of the transfection efficiency of insulin gene.