The Expression And Function Identification Of TRALL Extracellular Domain And The Humanization Of Anti-DR5 Monoclonal Antibody MDRA6

TRAIL(TNF-related apoptosis-inducing ligand.)is a type II membrane protein which belongs to the TNF superfamily and can be detectable in many nomal organs and tissues. It has the ability of inducing apoptosis, which was happened rapidly in susceptible cells including both malignant and some transformed cells. Although TRAIL shows high homology in sequence with TNFαand FasL molecules, However, TNF-a and FasL have failed to be approved for clinical application owing to their severe cytotoxic effects, which could lead to either a lethal inflammatory response related to septic shock syndrome or lethal liver damage due to apoptosis of hepatocytes respectively. It is believed that TRAIL selectively induces tumor cell apoptosis that may be more important in biologic homeostasis. TRAIL is also showed a synergic effect with the traditional radio and chemotherapy. There is report that the different recombinant versions TRAIL has different effect in anti-tumor and cytoxicity .On one hand, there was continuing concern about the safety of TRAIL. TRAIL is also actively in the search for other alternatives. So, the development of and the anti-DR4 or DR5 monoclonal antibody with apoptosis inducing ability can be the ideal candidate was a good direction to overcome the side effects of TRAIL. In 2001, an anti-DR5 antibody TRA-8 was developed. TRA-8 had excellent anti-tumor activity and cause showed no toxicity to the normal tissue which induced people's passion to develop antibody to replace TRAIL. However, so far, there are is still no antibody against DR5 or DR4 was available on the medicine market. In this study, two different forms of human TRAIL extracellular domain (114-281aa) were constructed and expressed in E. coli. In order to obtain two recombinant versions TRAIL protein proteins with biological function, human TRAIL extracellular gene(114-281aa) was amplified from PBMC and cloned into pGEM-T-Easy vector for sequence analysis. The express vectors pET-30a/TRAIL-His and pET-28a/TRAIL-NH were constructed by DNA recombinant method and expressed in E.coli BL21(BL21 (DE3). The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. The proliferation inhibition function of TRAIL-His and TRAIL-NH were detected by MTT method. PI staining and Wright-Giemsa staining assays were used to detect cell apoptosis. The target protein expressed in E.coli BL21(DE3) have the same m.w as that of expected and could be recognized by anti-TRAIL Poly-Ab and anti-His mAb. The protein could also inhibit the proliferation and induced apoptosis of Jurkat cells. Recombinant human TRAIL-His and TRAIL-NH protein with biological activity was obtained. This prospective research laid solid foundations for further research on biological activity and biotheraphybiotherapy in tumor. A room oOn the other hand, the anti-human DR5 monoclonal antibody mDRA6 source transformation to expand its applications. Mining First, the method of molecular biology used chimeric antibodies transformation. RT-PCR was used to amplify variable genes from the first secretion hybridoma cell line mDRA6 and the genes were subcloned into pGEM-T-Easy vector for further sequence analysis. BLAST results showed that the antibody was a new mouse antibody. Fvs of mDRA6 were cloned into VHexpress and VKexpress, standard transfection method was used to express cmDRA6 in 293T cells. The expression of c-mDRA6 reached to 490ng/ml.