Construction And Applification Of Z2A Recombinant Adenovirus Vector

Objective To construct Z2A recombinant adenovirus vector pAD-Z2A and study the effect of Z2A expression on EBV-positive cell.Method Both LMP2A and BZLF1 cDNA were cloned from EBV positive cell line by using RT-PCR , and the fusion gene (LMP2A-linker-BZLF1) was constructed by using a polypeptide linker (Gly₄Ser)₃ with spliced overlap extension(SOE). Z2A eukaryotic expressing vector pAdEasy-1 was constructed by inserting the Z2A fusion gene into pAdEasy-1 eukaryotic expressing plasmid. 293 cells were transfected with pAd-Z2A and the apoptosis of CNE cell was tested by flow cytometry.Result The electrophoresis of EcoRV and Bg1 â…¡ digested products showed two DNA fragments which was 31kb and 4.5kb,respectively.The result of DNA sequencing demonstrated that Z2A has inserted in the expected site and the insertion sequence was exactly correct.The results showed that Z2A positive recombinant adenovirus could express Z2A. Z2A recombinant adenovirus infected 293 cells and was duplicated in 293 cells, green fluorescent protein(GFP) was expressed. RT-PCR and Western blotting test revealed that there were gene expression, and the vAd-Z2A induced apoptosis of CNE cell by flow cytometryConclusion Z2A recombinant adenovirus has been constructed, the fusion protein was expressed in eukaryotic system, and the vAd-Z2A could induce the apoptosis of EBV positive CNE cells.