Functional Identification of a Non-Fusion TRAIL Extracellular Protein and Preparation of Its Polyclonal Antibody

Abstract

Human tumor necrosis factor related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in a variety of transformed cells and is currently being developed as a cancer therapeutic drug. Here we expressed the TRAIL protein including extracellular (114-281aa) without any tag protein named TRAIL-NT, and prepared anti-TRAIL polyclonal antibodies (Poly-Ab). The human TRAIL extracellular gene was amplified from PBMC and cloned into pGEM-T-Easy vector for sequence analysis. The expression vector pET-28a/TRAIL was constructed using the DNA recombinant method, and the recombinant protein without any tag protein was expressed in Escherichia coli BL21(DE3). The TRAIL-NT protein was purified by cation ion-exchange column and identified by SDS-PAGE and Western blot analysis. The proliferation inhibition activity of TRAIL-NT was detected by the MTT method, Wright-Giemsa staining assay, and FACS. The polyclonal antibody of TRAIL-NT was obtained after the BALB/C mice were immunized with purificated TRAIL-NT protein. Results showed that the target protein expressed in E. coli BL21(DE3) has the same molecular weight as that expected and could be recognized by anti-TRIAL Poly-Ab. The TRAIL-NT protein could also inhibit proliferation and induced apoptosis of Jurkat cells but no cytotoxicity to human liver cells and PBMC was observed. This preliminary research laid a solid foundation for further research on its biological activity and application in anti-tumor therapy.