Preparation and Evaluation of Polyclonal Antibodies of Apoptin

Apoptin is coded by the VP3 gene from chicken anemia virus(CAV),and the protein can induce apoptosis of a large variety of tumor cells.The preparation of polyclonal antibodies specific to VP3 protein was explored.The VP3 gene was obtained from the eukaryotic expression vector pcDNA3.0-VP3.And then,VP3 gene was cloned into the prokaryotic expression vector pET8a.The prokaryotic expression vector pET8a-VP3 was employed to express VP3 protein in E.coli BL21 which was induced by IPTG,and the protein was extracted from the cell and purified.The purified VP3 protein was applied to immunize the New Zealand rabbits combined with the Freund's complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection.ELISA test was performed to measure the titer of the antibody after immunization.Two days after the measurement the whole blood was harvested by cardiac puncture and the serum was separated from the whole blood.The immunglobin IgG was purified by Protein A method from the antiserum.The final purified antibody titers were up to 1∶243 000.The immunological evaluation of the polyclonal antibody specific binding was assayed by using recombinant adeno-associated virus rAAV-VP3 infected different cell lines.First,it was used to detect the VP3 gene expression by immunofluorescence in human bladder cancer cells T24 and EJ,also in Vero cells.Apoptin was observed in T24,EJ cells,mainly located in the nucleus,whereas in Vero cells were localized in the cytoplasm.Secondly,the specific binding of the purified antibodies to VP3 protein in different human bladder cancer cells were detected by Western blotting.The results have shown that the polyclonal antibody have displayed good effectiveness and binding specificity to apoptin,which will provide a foundation for further clarify the molecular mechanisms of anti-tumor effect and biological characteristics of apoptin.