Abstract:

objective The eukaryotic expression plasmid pcDNA3.1（+）/β-catenin was constructed and transfected into CNE2 nasopharyngeal carcinoma cells，and β-catenin overexpression was verified in transfected cells. Methods The β-catenin gene was amplified by RT-PCR，gel-purified and cloned into pGEM-T Easy，then transformed into E.coli. Positive clones were screened by digesting extracted plasmid with Kpn I/Xba I and by analyzing with PCR. The subcloned β-catenin gene was inserted into the eukaryotic expression vector pcDNA3.1/Hygro（+） using T4 DNA ligase， resulting in the recombinant eukaryotic expression plasmid pcDNA3.1（+）/β-catenin. This construct was transformed into competent DH5α cells and positive clones were identified using PCR，Kpn I/Xba I digestion and DNA sequencing. Purified pcDNA3.1（+）/β-catenin was transfected into CNE2 cells using FuGENE HD transfection reagent，and transfectants were selected using hygromycin B. Expression of β-catenin in transfected cells was assessed using real-time fluorescence quantitative PCR and Western blotting. Results The eukaryotic expression plasmid pcDNA3.1（+）/β-catenin was constructed and confirmed by PCR，double enzyme digestion and DNA sequencing. Overexpression of β-catenin in transfected CNE2 cells was confirmed using real-time fluorescence quantitative PCR and Western blotting （P<0.01）. Conclusion  The eukaryotic expression plasmid pcDNA3.1（+）/β-catenin was constructed and can be transfected into CNE2 cells to up-regulate β-catenin. These transfected cells may provide an in vitro model for studying the role of β-catenin in nasopharyngeal carcinoma.

Key words: Nasopharyngeal neoplasm, Eukaryotic expression vector, β-catenin, Cell model