Construction Of Recombinant Adenovirus Expression Vector Carrying GM-CSF And BZLF1 Gene

Objective The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and the EBV encoding immediate early genes (BZLF1) were fused to BZGM gene. To construct BZGM recombinant adenovirus expression vector and to play the fusion gene coding products GM-CSF enhancing anti-tumor immunity and BZLF1 inducing latent EBV to lytic replication. The construction of BZGM recombinant adenovirus expression vector could provide an experimental foundation and theoretical basis for kill EBV positive tumor cells.Methods GM-CSF cDNA were cloned from human peripheral blood mononuclear cells and BZLF1 cDNA were cloned from B95-8 cell line by RT-PCR. The fusion gene BZGM was constructed through a polypeptide linker (Gly4Ser)3 by using spliced overlap extension (SOE).The fusion gene BZGM was subcloned into shuttle plasmid pAdTrack-CMV. The shuttle plasmid and the backbone plasmid pAdEasy-1 were recombinated homologously in E.coli BJ5183 cells, then fusion gene eukaryotic expression vector pAd-BZGM were constructed. pAd-BZGM vector with antibiotic culture plate bolting were transfected into 293 cells to obtain recombinant adenovirus vAd-BZGM. RT-PCR and Western blotting were used to identify the state of human nasopharyngeal carcinoma CNE cells infected with vAd-BZGM. MTT was used to detect the proliferation of CNE cells infected with vAd-BZGM.Results GM-CSF gene and BZLF1 gene were fused to BZGM gene. The result of DNA sequencing demonstrated that BZGM has inserted in the expected site in the recombinant adenovirus expression vector and the insertion sequence was exactly correct. Recombination adenovirus vAd-BZGM was acquired by transfected with pAd-BZGM into 293 cells. Western blotting and RT-PCR results showed that the expression of BZGM gene and EBV early gene BMRFl can be detected in CNE cells infected with vAd-BZGM, which indicated that fusion gene could induce EBV from latency to lytic proliferation cycle. The results of MTT manifested the inhibition proliferation of CNE cells by vAd-BZGM was significantly different compared to the control (P<0.05).Conclusion BZGM recombinant adenovirus vector has been constructed, and could be packaged into recombinant adenovirus vAd-BZGM in 293 cells. The fusion gene BZGM was expressed stability in CNE cells infected with vAd-BZGM. vAd-BZGM could efficiently induce EBV from latency to lytic replication cycle, then inhibited the proliferation of CNE cells, which might provide an experimental foundation for further explore the function of BZGM gene.