目的:获取重组人高迁移率族蛋白B1 (HMGBl),HMGBl A box和B box的纯化蛋白,制备HMGBl的多克隆抗血清.方法:采用PCR方法扩增人HMGB1,HMGB1的Abox和B box目的基因片段,构建原核表达载体,进行原核表达与蛋白纯化,然后用HMGB1免疫新西兰大白兔,制备多克隆抗血清.采用ELISA检测抗血清效价,用免疫组化检测HMGB1在小鼠肝损伤组织中的表达.结果:成功构建了人HMGB1,HMGB1的Abox和B box原核表达载体pET28-HMGB1、pET28-Abox、pET28-Bbox,在E.coli BL21中表达,镍亲和层析柱提纯,获取纯净目的蛋白.HMGB1免疫新西兰大白兔后,抗血清效价为1∶2,000,000,具有高度特异性.免疫组化显示小鼠坏死肝组织HMGB1表达增加.结论:本研究获得了人HMGB1以及HMGB1的A box和B box的纯化蛋白,制备了人HMGB1的多克隆抗血清,为HMGB1的结构、组织表达谱及其功能的研究奠定了基础.%To obtain the purified recombinant proteins of human HMGB1, HMGB1 B box and A box and prepare HMGB1 poly-clonal anti-sera. The target genes were amplified by PCR from human PBMCs, and cloned into pET28a vectors, respectively. The sequences were confirmed by enzymes digestion and DNA sequencing. The constructed vectors were transformed into E. Coli BL21 and the expressed proteins were purified. Rabbits were immunized with the purified HMGB1 protein and the high titer antiserum was obtained. Immunohistochemical staining of mouse liver inflammatory tissue with the antiserum was performed. The vectors of pET28-HMGBl, pET28-A box, pET28-B box were successfully constructed. The proteins expressed in E. Coli BL21 were purified by Ni-NTA column. ELISA analysis showed that anti-sera were high titer against rHMGB1. Immunohistochemical staining showed that HMGB1 was increased in mouse inflammatory liver tissue. The proteins of recombinant HMGB1, B box and A box and HMGB1 anti-serum have been obtained. This work provides the means for research on the role of HMGB1 in the pathogenesis of human diseases.
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