人钙调磷酸酶-B真核表达质粒的构建

Construction of eukaryotic expressing plasmid of human calcineurin B

目的为研究钙振荡现象,构建人钙调磷酸酶-B(CNB)基因的真核表达质粒。方法采用RT-PCR技术,从MCF-7细胞中扩增出人CNB基因序列;利用分子克隆技术将其插入到真核表达载体pcDNA3.0中,并进行酶切鉴定和DNA测序。结果琼脂糖凝胶电泳分析显示RT-PCR产物为预期分子量大小,酶切鉴定图谱正确,DNA测序结果经核实完全符合。结论成功构建人CNB基因的真核表达质粒,为进一步研究钙振荡现象打下了基础。

Objective To investigate calcium oscillations phenomenon,the eukaryotic expressing plasmid of human CNB was constructed.Methods The human CNB gene was amplified from MCF-7 cells by using RT-PCR techniques and cloned into the eukaryotic expressing vector pcDNA3.0 with molecular cloning techniques.The recombinant plasmid was identified with the assay of restriction enzyme digestion and DNA sequencing.Results The agarose gel electropboreis analysis showed that the production of RT-PCR was just what expected and the map of restriction enzyme digestion of the recombinant plasmid was correct.Furthermore,the recombinant plasmid was confirmed by DNA sequencing.Conclusion The eukaryotic expressing plasmid of human CNB was successfully constructed,which provides a foundation for further calicum oscillations researches.