Generation and expression of Epstein-Barr Virus Latent membrane protein 2A recombinant adenovirus

Objective:To generate Epstein-Barr virus(EBV) Latent Membrane Protein 2A(LMP2A) recombinant adenovirus,and provide for further investigation on the therapy vaccine against EBV associated malignancies.Methods:Full length cDNA of encoding LMP2A of EBV had been amplified by reverse transcription-PCR and cloned into pGEM-T vector.The encoding cDNA of LMP2A was inserted into E1,E3-substituted adenovirus vector pAX1CW,then the LMP2A recombinant adenovirus vector was contransfected into 293 cells togetherwith EcoT221 digested Ad5-TPC.The LMP2A recombinant adenovirus was generated by homologous recombination,and primarily identificated by ClaI enzyme digestion.The expression of LMP2A on CV1 cells infected with recombinant adenovirus analyzed by fluorescence-activated cell sorting(FACS) and confocal microscope.Results:The replication-deficient LMP2A recombinant adenovirus was generated efficiently with the titers of 2.3×10 8 pfu/ml.The LMP2A could be seen on CV1 cells membrane with confocal microscope 48 h post infected with recombinant adenovirus and the percentage of CV1 cells expressing LMP2A was 94.4% by means of FACS analysis.Conclusion:These suggested that LMP2A could be expressed efficiently by recombinant adenovirus mediated transfer,and it was the foundation of further researching in its function and developing the suitable genetic engineering vaccine against EBV associated malignancies.