Abstract:

objective To construct the eukaryotic expression plasmid pIRES2-Zs-Green1-LMP2A encoding green fluorescent protein，and to transfect it into CNE2 nasopharyngeal carcinoma cells. Methods The LMP2A sequence from EBV-positive B95-8 cells was cloned into the vector pIRES2-Zs-Green1. The identity of the resulting pIRES2-Zs-Green1-LMP2A was confirmed using restriction enzymes and sequencing. The expression plasmid was transfected into CNE2 cells using a liposome-based method. The resulting transfectants were compared with negative controls transfected with pIRES2-Zs-Green1 and with untransfected cells in terms of expression of green fluorescent protein and LMP2A transcription. Results Restriction enzyme digestion and sequencing showed that pIRES2-Zs-Green1-LMP2A was constructed successfully. Transfection of pIRES2-Zs-Green1-LMP2A or empty vector pIRES2-Zs-Green1 led to expression of green fluorescent protein，and fluorescence microscopy indicated transfection efficiency of approximately 75%. Only cells transfected with the complete plasmid pIRES2-Zs-Green1-LMP2A produced LMP2A mRNA. Conclusion The eukaryotic expression plasmid pIRES2-Zs-Green1-LMP2A has been successfully constructed，and it can drive steady expression of LMP2A in CNE2 cells，making this system useful for studies of nasopharyngeal carcinoma.

Key words:  Nasopharyngeal neoplasm, EBV latent membrane protein 2A, Sequence clone, Vector pIRES2-Zs-Green1, Eukaryotic expression vector pIRES2-Zs-Green1-LMP2A, Gene transfection, Gene expression