Generation Of Anti-Human DR5 Polyclonal Antibody And Preliminary Study On Its Effect Of Inducing Apoptosis In Tumor Cells

Induction of apoptosis of tumor cells is one of therapies for treatment of cancer. The death receptors of the TNFR superfamily have become a potential target for treatment. DR5 (Death receptor 5) is a member of the TNFR superfamily, has an intracellular death domain which can bind to its ligand, TRAIL (TNF-related apoptosis-inducing ligand), and thus trigger the TRAIL-induced apoptosis signaling pathway leading to death of tumor cells. Anti-DR5 monoclonal antibodies are found to kill tumor cell without toxic effect on normal cells, and therefore become a heated topic in the research and development of antibody-based drugs for tumor. Generation of monoclonal antibodies is too expensive for the monoclonal antibody to be produced and used widespread in the clinical treatment. In contrast polyclonal antibodies are cheaper to produce, however there is no report about effect of polyclonal antibody on tumor apoptosis or/and necrosis. Thus we generated anti-human DR5 polyclonal antibody and tested its effect on human liver cancer cell line HepG2 cell line.The extracellular fragment of human DR5 cDNA (eDR5) was amplified from human liver cancer cell line HepG2 by RT-PCR, and was ligated with the cloning vector pMD19-T to form pMD-eDR5. After verified by sequencing, pMD-eDR5 was cut with restriction endonucleases BamR I and EcoR I, and the eDR5 fragment was extracted and inserted into the prokaryotic expression vectors pGEX-4T-1, pET-Trx and pET-26b(+), respectively, to construct prokaryotic expression plasmids for expressing human eDR5 in E. coli cells. After comparison of expression efficacy, pGEX-eDR5 was selected to express the GST-eDR5 fusion protein in E. coli BL21 (DE3) with induction by IPTG. The expressed GST-eDR5 fusion protein, which accounted for about 19% of the total bacterial cellular proteins, was identified to be correct by Western blot with anti-human DR5 monoclonal antibody as the primary antibody.The recombinant fusion protein GST-eDR5 was purified by GST purification kit followed by polyacrylamide gel slicing method. The polyclonal antibody (antisera) against GST-eDR5 fusion protein, which was prepared by immunizing mouse with the purified GST-eDR5 fusion protein, is able to recognize GST, recombinant GST-eDR5 and DR5 from HepG2 cells in gel checked by Western blotting. After purified by immunoprecipitation reacting with GST protein to remove the anti-GST components, the antisera lost the ability to recognize GST, but still recognize native DR5 from HepG2 cells and the recombinant GST-eDR5 fusion protein. The titer of antisera is as high as 1 : 25600 determined by indirect ELISA.The morphology of HepG2 cells changed significantly after treatment with the purified polyclonal antisera whereas the normal fibroblast cells had no visible change. And HepG2 cells growth was obviously inhibited after treatment with the purified polyclonal antisera by MTT assay. However detections with TUNEL and Western blot of Caspase 3 for confirmation of apopotosis did not obtain clear results. Thus further experiments are needed to evaluate the efficiency and mechanism of inhibition to HepG2 cell growth by anti-DR5 polyclonal antisera.