重组人HMGB1 A box的表达纯化及对单核细胞的抑制作用

Expression of human HMGB1 A box in E. coli and its inhibitory effects on monocytes

目的:克隆人高迁移率族B1Abox(HMGB1 A box)基因,构建高效稳定的大肠杆菌(E.coli)表达菌株,并探讨其对免疫复合物(IC)刺激单核细胞活化的抑制作用。方法:根据本室优化合成的HMGB1基因序列设计引物,PCR扩增目的基因片段并插入克隆载体pMD19-T,菌落PCR、酶谱分析及DNA测序鉴定阳性克隆。重组克隆载体经NdeⅠ和XhoⅠ酶切,琼脂糖凝胶电泳分离目的基因,插入表达载体pQE-T7-2的相应位点,菌落PCR鉴定重组表达载体。重组菌株经IT-PG诱导,SDS-PAGE分析及Western blot鉴定重组蛋白。Ni2+-NTA层析柱纯化重组人HMGB1 A box,RT-PCR检测其对IC刺激单核细胞活化的抑制作用。结果:获得重组人HMGB1 A box的表达菌株,目的蛋白占菌体总蛋白的40%左右。Western blot显示重组蛋白能与抗人HMGB1多克隆抗体和抗His-Tag多克隆抗体特异反应。目的蛋白纯度高达90%以上,并能有效抑制IC诱导单核细胞分泌BAFF、IFN-γ及TNF-α。结论:成功构建了重组人HMGB1 A Box的表达载体,纯化的重组蛋白能有效抑制IC刺激单核细胞活化后细胞因子的分泌。

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 NdeⅠand XhoⅠand seperated 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 Ni2＋-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-γ and TNF-α in monocyte which were induced by IC. CONCLUSION：A recombinant bacterial strain for expressing human HMGB1A box with biological activities was constructed successfully.