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经RTPCR从人新鲜扁桃体组织中扩增人高迁移率族蛋白B1(HMGB1)中的Bbox(88～162残基)的cDNA,构建于载体pUC19,经测序后与GenBank中报道的已知序列完全一致,再构建于原核表达载体pQE80LDHFR中,表达并鉴定目的蛋白.经Ni2+亲和层析柱、多粘菌素B层析柱纯化获得高纯度的DHFRHMGB1Bbox蛋白,然后将此重组HMGB1Bbox加入到人外周血单核细胞(PBMCs)中,37℃,5%CO2下,刺激PBMCs6h,用ELISA检测PBMCs释放TNFα、IL6的量,经检测后证明纯化后的重组HMGB1Bbox能显著地刺激PBMCs释放致炎因子TNFα、IL6.HMGB1Bbox的表达及其生物活性的初步研究,为进一步研究HMGB1Bbox的作用机制以及新型抗炎制剂的研发奠定基础.
The cDNA of Bbox (residues 88-162) in human high mobility group box 1 (HMGB1) was amplified from human fresh tonsil tissue by RTPCR and was constructed on the vector pUC19 and sequenced to complete the known sequence reported in GenBank And reconstructed into the prokaryotic expression vector pQE80LDHFR to express and identify the target protein.The high purity DHFRHMGB1Bbox protein was purified by Ni2 + affinity chromatography and polymyxin B column chromatography, and then the recombinant HMGB1Bbox was added to the human peripheral PBMCs were stimulated with PBMCs for 6h at 37 ℃ and 5% CO2, and the levels of TNFα and IL6 released by PBMCs were measured by ELISA. The purified recombinant HMGB1Bbox could significantly stimulate the release of inflammatory cytokines TNFα , IL6.HMGB1Bbox expression and biological activity of a preliminary study for the further study of the mechanism of action of HMGB1Bbox and lay a foundation for the development of new anti-inflammatory agents.