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

作　　者：Jun Dai Longyan Wu Cai Zhang Xiaodong Zheng Zhigang Tian Jian Zhang 

机构地区：[1]Institute of Immunopharmacology ＆ Immunotherapy, School ofPharmaceutical Sciences, Shandong University, Jinan 250012, China [2]Institute of Immunology, School of Life Sciences, University of Science ＆Technology of China, Hefei 230027, China [3]Institute of Immunopharmacology ＆ Immunotherapy, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, China

出　　处：《Cellular & Molecular Immunology》2009年第4期261-268,共8页中国免疫学杂志（英文版）

基　　金：Acknowledgements This work was supported by grants from National Natural Science Foundation of China （#30671901; #30628014）, National 863 Science Program by Ministry of Science and Technology of China （#2007AA021010, 2007AA021109）, National 973 Basic Science Project by Ministry of Science ＆ Technology of China （#2010CB911901）.

摘　　要：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℃. 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.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℃. 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.

关 键 词：HMBOX1 expression PURIFICATION RENATURATION monoclonal antibody 

分 类 号：Q55[生物学—生物化学] S852.43[农业科学—基础兽医学]