Generation Of Anti-Human DR4 Polyclonal Antibody And Preliminary Study On Its Effect Of Inducing Apoptosis In HeLa Cells

Induction of apoptosis in tumor cells is one of therapies for treatment of cancer. The death receptors (DRs) of the TNFR superfamily have become a potential target for treatment to induce cell apoptosis. DR4 is a member of the TNFR superfamily, has an extracellular domain which can bind to its ligand, TRAIL (TNF-related apoptosis-inducing ligand), and thus triggers the TRAIL-induced apoptosis signaling pathway to lead to death of tumor cells. Anti-DR4 monoclonal antibodies are found to kill tumor cell without toxic effect on normal cells, and therefore become a hot issue in the research on and development of antibody-based drugs for tumor. However, monoclonal antibodies are too expensive to generate and to be used widespread in the clinical treatment. In contrast polyclonal antibodies are cheaper to produce. Thus we generated anti-human DR4 polyclonal antibody and tested its apoptosis-inducing effect on HeLa cell line.The extracellular fragment of human DR4 cDNA(eDR4) was amplified from human cervical cancer cell line HeLa by RT-PCR, and was ligated with the cloning vector pMD19-T to construct the pMD-eDR4. After verified by sequencing, the pMD-eDR4 was cut with restriction endonuclease BamH I and EcoRâ… , and the eDR4 fragment was extracted and inserted into the prokaryotic expression vectors pGEX-4T-1 to construct prokaryotic expression plasmid for expressing recombinant human DR4 in E. coli cells BL21(DE3) with induction by IPTG. The expressed GST-DR4 fusion protein was identified to be correct by Western blot with the anti-GST monoclonal antibody as the primary antibody.The recombinant fusion protein GST-DR4 was purified by GST purification kit followed by polyacrylamide gel slicing method. The polyclonal antibody against GST-DR4 fusion protein was prepared by immunizing mouse with the purified GST-DR4 fusion protein, and was confirmed to be able to recognize GST and recombinant GST-DR4 from E. coli cells and DR4 from the HeLa cells by Western blotting. The titer of the polyclonal antibody, which was determined by indirect ELISA, is as high as 1:12800.The morphology of the HeLa cells changed significantly after treatment with the purified polyclonal antibody whereas the normal HASMC cell line had no visible changes. And proliferation of the HeLa cells, which was measured by MTT assay, was obviously inhibited after treatment with the polyclonal antibody. Co-treatment with anti-DR4 and anti-DR5 polyclonal antibodies lead to higher inhibition to HeLa cell growth than unitary treatment with the same concentration of anti-DR4 or anti-DR5 polyclonal antibody. TUNEL assay and Western blotting analysis of the caspase 3 revealed that the mechanism underlying this inhibitory effect is apoptosis induced by treatment with the anti-DR4 polyclonal antibody. Our results provide the basic data for developing the anti-DR4 polyclonal antibody as an anti-cancer medicine.