Gene Clone And Expression Of Human DR5 Extracelluar Fragment And Development Of Anti-DR5 Monoclonal Antibody

BackgroundsDR5(death receptor 5)was a key receptor of TRAIL receptors and played a important role in TRAIL-induced apoptosis.. Most important DR5 was typeⅠtransmembrane protein that contained cytoplasmic death domain and upon ligation to TRAIL, mediated apoptosis. So DR5 was a very target of activating cell apoptosis signal. TRAIL had been tested on a number of different tumor cell lines, and results showed that a high proportion of human tumor cells had been sensitive to TRAIL-mediated apoptosis, at the same time many experiments showed TRAIL had minimal cytotoxity to most normal cells and tissues. TRAIL was supposed to be the most potential drug for cancer therapy. As the researchs go on, there had a view that TRAIL could be harmful to human liver cells. So researchers put emphasis on the development and application of anti-DR5 monoclonal antibody, and hoped they could gain a monoclonal antibody that could activate apoptosis signal upon ligation to DR5 and had no toxicity to normal hepatocytes. If we could have one, we could develop it and substituted it for TRAIL as a potential anti-cancer drug.AimsTo acquire human DR5 extracellular fragment with bioactivity and a anti-DR5 monoclonal antibody with satisfied bioactivity.MethodsTotal RNA was prepared from Jurkat cells by Trizol. Human DR5 extracellular fragment gene was amplified by RT-PCR, cloned into pGEM?-T Easy vector, and confirmed by sequence analysis. Then the gene was subcloned into expression vector pET30a with a His-tag at the amino terminus and expressed in E.coli BL21 (DE3). The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. ELISA method was used to detect its binding activity to anti-DR5 monoclonal antibody (mAb) mDRA-6. The protein was used to block the effect of TRAIL apoptosis effect to Jurkat cells.We had a hybridoma cell by hybridoma technique. MTT results showed that A6 could suppress the growth of Jurkat. G-R staining,PI/AnnexinⅤresults showed the apoptosis effect of Jurkat cells that A6 affected. We used competitive ELISA to show the site that A6 and TRAIL recognize and bind to.ResultsHuman DR5 extracellular fragment gene was successfully amplified and high level expression was obtained in E.coli BL21 (DE3) induced by 0.1 mM IPTG. The DR5 extracellular fragment protein was identified by SDS-PAGE and Western blot analysis. ELISA results showed that the purified DR5 could be recognized by mDRA-6. The DR5 extracellular protein can block the apoptosis effect of TRAIL.We had a hybridoma cell (A6) by hybridoma technique. MTT results showed A6 suppress the growth of Jurkat cells on dose-dependent and when A6 treated at 25μg/mL for 12 hours, the suppress rate was up to 90%. G-R staining result showed morphology changes of Jurkat cells, PI/AnnexinⅤanalysis showed the apoptosis rate of Jurkat cells reaches 52.46﹪when Jurkar cells were incubated with A6 for 12 hours which was in the concertration of 250 ng/mL. Competitive ELISA showed that the sites that TRAIL and A6 recognize were different.ConclusionsFirst, we gain the DR5 extracellular fragment with bioactivity. Second, we have an anti-DR5 monoclonal antibody (mAb)-A6.