RESUMEN
AIM: To clone human high mobility guoup box1 A box (HMGB1 A box) and express it in escherichia coli effectly, investigate the inhibit effection of the purpose protern to the activation of monocytes stimulated by immunocomplex. METHODS: According to human HMGB1 gene order which was optimized by our laboratory the PCR primer was designed which containing restriction enzyme cutting site. The HMGB1 A box gene was cloned following the whole gene synthesis template of human HMGB1, then the PCR product was inserted into clone vector pMD19-T. The positive colone was identified by colony PCR, zymography analysis and DNA sequencing. Recombinant colne vector was digested by restriction enzymes Nde I and Xho I and separated by agarose gel electrophoresis, then the fragment was inserted into the corresponding sites of expression vector pQE-T7-2. The positive recombinant expression vector was identified by colony PCR and the recombinant strains was induced by IPTG, then the purpose protein was identified by SDS-PAGE and Western blot. The recombinant protein of human HMGB1 A box was purificated by Ni(2+)-NTA chromatography and the inhibit effection of the purpose protern to the activation of monocyte stimulated by immunocomplex was identified by RT-PCR. RESULTS: We acquired expression strains of recombinant human HMGB1 A box, the target protein account for up to 40% of the whole protein of E.coli. Western blot showed recombinant protein can specificly reacted with anti-human HMGB1 polyclonal antibody and anti-His-Tag polyclonal antibody.The purpose protein was found more than 90% after purified, and can effectively inhibit the production of BAFF, IFN-gamma and TNF-alpha in monocyte which were induced by IC. CONCLUSION: A recombinant bacterial strain for expressing human HMGB1A box with biological activities was constructed successfully.