Research On The Cloning Of KLF6Tumor Suppressor Gene And Plasmid Construction For Transfection Into Bladder Cancer BIU-87Cells

Objective：Building cells with transfected pEGFP-C₁-KLF6plasmid vector andtransfected into bladder cancer BIU-87cells，and studied its transfectionto contribute to the conduct of follow-up experiment，having a choice ingene therapy of bladder cancer.Methods:1、Acquisition of the target gene and plasmid construction：RNA extractionand design of a KLF6the primer sequence by RT-PCR was used to clonethe gene, BamH I and EcoR I double digestion KLF6plasmid vector pEGFP-C₁after ligation construct containing the target gene KLF6the recombinantplasmid of pEGFP-C₁-KLF6；2、Plasmid amplification and transfection：the reorganization of pEGFP-C₁-KLF6plasmid was amplified, and purified in order to achieve number ofexperiment.The mediated by cationic liposomes containing or without KLF6plasmid was transfected into BIU-87cells, respectively as thetransfected group and empty vector group，the BIU-87cells untransfectedcontrol group selection；3、RT-PCR and Western blot detect after transfection of KLF6mRNA andprotein expression levels.Results:1、Success of target genes and build of KLF6plasmid of pEGFP-C₁-KLF6forcell transfection and experimental.2、 The recombinant plasmid after amplification， extraction andpurification，electrophoresis test for the same location before amplified reorganization of pEGFP-C₁-KLF6of plasmid.3、Experimental control group and empty vector group no fluorescenceobserved by fluorescence microscopy, transfection group about60％greenfluorescent cells visible under a fluorescence microscope.4、RT-PCR and Western experiments could be detected in turn transfectedgroup of KLF6mRNA and protein expression, while the control group andempty vector group, no significant expression.Conclusion:1、 Successfully constructed pEGFP-C₁-KLF6of plasmid with cellstransfected with the characteristics.By competent bacteria oftransformation、amplification、separation and purification of recombinantpEGFP-C₁-KLF6of plasmid DNA， to obtain sufficient recombinantpEGFP-C₁-KLF6of plasmid for experiment.2、Lipofectamine2000mediated by the recombinant plasmid was transfectedinto bladder cancer BIU-87cells，and to lay the foundation for furtherstudy.