[Construction and identification of recombinant retroviral vector and stable cell line expressing latent membrane protein 2A of Epstein-Barr virus].

AIM

To clone EBV-LMP2A gene, construct and identify the recombinant retroviral vector and stable cell strains expressing EBV LMP2A.

METHODS

The full-length EBV LMP2A gene was generated by RT-PCR amplification from B95.8 cells which contain complement nucleotide sequence of EBV LMP2A gene. The gene was ligated to T-vector and sequenced to construct retroviral vector consisting with LMP2A. To produce retroviral virus, packing cells, 293T cells were co-transfected with recombinant retroviral expression vector pGEZ-LMP2A and two auxiliary viral vectors pHIT456 and pHIT60 by lipofectAMINE2000. Viral titration was performed according to the instructions of the manufacturer. To establish L929 cell line stable expressing LMP2A, L929 cells were infected with recombinant retrovirus three times and selected by Zeocine. The Zeocine-resistant clones (L929/LMP2A) were screened for LMP2A expression by RT-PCR and Western blot.

RESULTS

The recombinant retrovirus vector carrying LMP2A gene was constructed successfully. Transfection yield a titer of 5 x 10(8) infectious particles/L. The infected L929 cells were selected by Zeocine. Results of RT-PCR and Western blot indicated that L929 transgenetic cells could stably express EBV-LMP2A.

CONCLUSION

The L929 cell line stably expressing LMP2A provides suitability for extraction of the LMP2A protein and preparations of the vaccine for the therapy of EBV-associated diseases.