WWOX基因真核表达载体的构建及其在SMMC-7721中的表达

摘要

Objective:To construct eukaryotic expression plasmid pEGFP-N1-WWOX and transiently express it in SMMC-7721 cells.Methods:Total mRNA was extracted from normal human liver tissue.RT-PCR was used to amplify the aimed segments WWOX cONA which was then digested with Hindlll and BamHl and inserted into a eukaryotic expression plasmid pEGFP-N1 to construct pEGFP-N1-WWOX.The constructed plasmid was transfected into SMMC-7721 cells by lipofectamine 2000-mediated transfer method.The expression of WWOX in transfected SMMC-7721 cells was detected 24.36 and 48 h post-transfection with fluorescence microscope and the expression level of WWOX mRNA in transfected SMMC-7721 cells was assay by using RT-PCR.The change of WWOX expression and cell proliferation rates were detected by immunocyto-chemistry and MTT methods respectively.Results:The results showed pEGFP-N1-WWOX was successfully constructed and expressed transiently in SMMC-7721 cells.At 48th hour post-transfection.the number of positive cells was jncreased significantly and much brighter green fluorescence could be detected,while no green fluorescence was detected in the control group.In SMMC-7721 cells transfected with pEGFP-NI-WWOX a high level of porcine WWOX was detected.WWOX ex-pressed by transfected cells could significantly inhibit che proliferation of SMMC.7721 cells.Conclusion:pEGFP-N1-WWOX was expressed successfully in SMMC-7721 cells,which suggested that might be used as a new therapeutic method for liver cancer.