Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Abstract

HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37 o C. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni 2+ -affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues. Cellular & Molecular Immunology. 2009; 6(4):261-268.

Expression profile of HMBOX1, a novel transcription factor, in human cancers using highly specific monoclonal antibodies

“…Previous research has confirmed that HMBOX1 is widely expressed in human tissues (4). Here, the expression profile of HMBOX1 was further identified in various human tumors.…”

“…hmBOx1 is highly conserved in humans, mice, rats, chickens and xenopus (2), and is detected in both the cytoplasm and nucleus of 10 normal human tissues, including the cerebrum, pancreas, kidney and liver (4). co-transfection of hEK-293t cells with the pm-hmBOx1 plasmid and reporter plasmid pGal4 5 tkluc indicates that the hmBOx1 protein may be a transcription repressor, while hmBOx1b only retains faint transcriptional repressive activity (2,3).…”

Preparation and functional identification of a monoclonal antibody against the recombinant soluble human NKp30 receptor

“…The colorimetric signal was measured at OD 450nm and OD 630nm . The specification and affinity constant of the monoclonal antibodies were determined by competitive ELISA as previously described [24,25].…”

HMBOX1 negatively regulates NK cell functions by suppressing the NKG2D/DAP10 signaling pathway

“…The anti-HMBOX1 monoclonal antibody used for western blot analysis was prepared by our lab. 5 Monoclonal mouse anti-human NKG2D mAb (R&D Systems) was used to stimulate NK cells.…”

“…1, 5 Chen et al speculated that HMBOX1 may be a ubiquitous transcription repressor that is expressed in most human tissues. 1 Recently, Su et al reported that HMBOX1 is a key factor in the differentiation of bone marrow stromal cells into vascular endothelial cells.…”