Recombinant expression of a novel human transcriptional repressor HMBOX1 and preparation of anti-HMBOX1 monoclonal antibody.
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 degrees Celsius. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni2+-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.
Dai
Jun
J
Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.
Wu
Longyan
L
Zhang
Cai
C
Zheng
Xiaodong
X
Tian
Zhigang
Z
Zhang
Jian
J
D000911:Antibodies, Monoclonal / Q000276:immunology*; D054022:Cerebrum / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000378:metabolism; D007668:Kidney / Q000276:immunology / Q000378:metabolism; D008099:Liver / Q000276:immunology / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010179:Pancreas / Q000276:immunology / Q000378:metabolism; D040081:Protein Array Analysis; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C512050:HMBOX1 protein, human; D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins