Construction of pEGFP-N3-APC vectors carrying various APC functional domains and their expression in HCT-II6 cells

Objective To construct recombinant plasmids containing various functional domains of APC protein and detect their expression in HCT-116 cells. Methods Five APC gene fragments were amplified by PCR with whole APC gene as template and primers designed according to APC cDNA sequence and mutation cluster domain. The five obtained fragments were cloned into eukaryotic expression vector pEGFP-N3 to generate recombinant pEGFP-N3-APC1-5. Sequence of the inserted gene was identified and analyzed after restriction enzyme digestion. Liposome-mediated recombinant plasmid pEGFP-N3-APC was transfected into HCT 116 cells and identified by green fluorescence. RT-PCR was employed to validate the expression of recombinant vectors in cells. Results Recombinant pEGFP-N3-APC1-5 were confirmed by restriction enzyme digestion and sequence analysis. The plasmids could be expressed in HCT-116 cell line detected by fluorescence microscope. Results of RT-PCR made clear that vectors constructed could be expressed in HCT-116 cells. Conclusion The relative efficient expression of five recombinant expressive vector in HCT-116 cell line may provide an experimental basis for selecting specific therapy peptide for colorectal cancer. Key words: Gene,APC;  Recombinant expressive vector, Polymerase chain reaction;  Transfection