title: 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.
authors.affiliation: Institute of Immunology, Medical College, Henan University, PR China.
authors.forename: Kunpeng
authors.initials: K
authors.lastname: Zhao
authors.forename: Xueyin
authors.initials: X
authors.lastname: Wang
authors.forename: Yugang
authors.initials: Y
authors.lastname: Wang
authors.forename: Yuanfang
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics* / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology*; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D000906:Antibodies; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue