Construction and identification of recombinant adenovirus Ad5F35-LMP2 containing LMP2 gene

Objective:To construct recombinant adenovirus expressing Epstein-Barr Virus(EBV)-LMP2 in vector Ad5F35.Methods:Restriction fragment of LMP2 from pSH-LMP2 was inserted in the vector of pDC316. The insertion and the direction of the insert were confirmed by polymerase chain reaction(PCR) and restriction enzyme digestion. The constructed pDC316-LMP2 was co-transfected with adenovirus backbone pBHG-fiber5/35 into 293 cells to establish the recombinant adenovirus Ad5F35-LMP2, which was confirmed by PCR and indirect immune-fluorescence test.Results:The construction of pDC316-LMP2 was completed and confirmed with PCR and restriction enzyme digestion. Second, the significant virus plaques was observed in the 293 cells co-transfected with pDC316-LMP2 together with pBHG-fiber5/35, which means the successful homologue recombination and virus packaging of the interest fragment in 293 cells. Third, the successful construction of the recombinant adenovirus Ad5F35-LMP2 with PCR was confirmed, and the efficient expression of LMP2 protein was observed on the cell membrane with indirect immune-fluorescence test.Conclusion:The recombinant adenovirus of Ad5F35-LMP2 which contains the full-length coding region of EBV LMP2 protein has been established that could be used in the gene therapy for nasopharyngeal carcinoma patients and to test the bio-safety and biological function of Ad5F35-LMP2 in the future.