Construction of the eukaryotic expression vector pEGFP-N1-ND CyclinB1 and its expression in SGC-7901 cells

Aim:To construct an eukaryotic expression vector pEGFP-N1 Non-degrable CyclinB1,and to observe its expression in human gastric cancer cell lines SGC-7901 and effects on SGC-7901 cells. Methods: ND-CyclinB1was amplified by reverse transcription polymerase chain reaction(RT-PCR).The eukaryotic expression vector pEGFP-N1-ND CyclinB1 was constructed by introducing ND-CyclinB1 DNA fragment into the sites of Xho I and BamH I in pEGFP-N1 vector.The plasmid was transfected into SGC-7901 cells by using lipofectamine.The expressed EGFP was observed by fluorescent microscope.Then the CyclinB1 was analyzed by Western blotting,and the apoptosis index was detected under a FACScan flow cytometry.Results: Identification of pEGFP-N1-ND CyclinB1 by enzyme digestion and PCR showed that the recombinant vector was correctly constructed.The EGFP was observed 24 hours after transfection,the expression of CyclinB1 was increased,and the apoptosis index was found to be increased too.Conclusion:The eukaryotic expression plasmid pEGFP-N1-ND CyclinB1has been successfully constructed and it can be expressed in SGC-7901 cells.