Construction of Small Interfering RNA System Targeting EBV Latent Gene EBNA2

[Purpose] To construct a recombinant retroviral vector pSUPER-EBNA2 that targets EBV nuclear antigen 2 (EBNA2) and then to sift a stable virus-producing cell line. [Methods] The 60nt nucleiotide sequence encoded targeting EBNA2 shRNA was cloned into a retroviral vector pSUPER. retro with DNA recombinant technique, and the recombinant vector was confirmed by the restrictive enzyme analysis and DNA sequencing. The packaging cell Pheonix A was transfected with recombinant plasmid using liposome-based transfection method and the stable intergrant was selected by using G-418. [Results] The restrictive enzyme analysis and electrophoresis showed two DNA strips which were 7167bp and 281bp, respectively. The result of DNA sequencing demonstrated that the insertion sequence was exactly correct. The recombinant vector was transfected into the packaging cell, and then the anti-G418 positive clones which can excrete recombinant retrovirus were sieved out. The tite of the recombinant retrovirus is 2.5×104 CFU/ml. [Conclusion] A recombinant retroviral vector pSUPER-EBNA2 and a stable virus-producing cell line were constructed and sifted successfully which provide on experimenteal base to study the effect of cell transformation and anti-apoptosis in EBNA2 gene.