Construction and Identification of Recombinant Plasmid of p3XFLAG-CMV7-NICD1 / 中国医科大学学报

ABSTRACT

Objective To construct the eukaryotic expression vector of Notch1intracellular domain,p3XFLAG?CMV7?NICD1,so as to prepare for the further research and exploration of effect of Notch1 on promoting epithelial?mesenchymal transition of human lens epithelial cells. Methods The cDNA fragment was reversely transcribed by RT?PCR from total RNA extracted from the SRA01/04 cells and was encoded with the specific am?plification?targeted NICD1was obtained from the SRA01/04 cells,then the cDNA fragment was inserted into p3XFLAG?CMV7 to transcribe Esche?richia coli DH5α. And the recombinant plasmid was extracted after bacterial screening by LB plating medium and confirmed by the restriction endo?nuclease digestion and DNA sequencing. Results The target gene obtained had the same molecular size as predicted. It was indicated that recom?bined p3XFLAG?CMV7 plasmid contained correct recombinant human Notch1 sequences and p3XFLAG?CMV7?NICD1 was constructed success?fully. The western blotting showed protein NICD1 expressed in SRA01/04 cells transfected with p3XFLAG?CMV7?NICD1. Conclusion The suc?cessful construction of p3XFLAG?CMV7?NICD1 will provide a foundation for a further study studies in on the effect relationship of Notch signaling pathway and in posterior capsular opacification(PCO)after cataract extraction.