人高迁移率族蛋白B1,高迁移率族蛋白B1 A box和B box的原核表达、纯化及高迁移率族蛋白B1多克隆抗血清的制备  

Prokaryotic Expression and Purification of Human HMGB1,A box,B box and Preparation of HMGB1 Anti-sera

作　　者：周红颜[1] 任向荣[1] 苏绍波[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广东广州510060

出　　处：《现代生物医学进展》2011年第21期4005-4009,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金(81072483)

摘　　要：目的:获取重组人高迁移率族蛋白B1(HMGB1),HMGB1Abox和Bbox的纯化蛋白,制备HMGB1的多克隆抗血清。方法:采用PCR方法扩增人HMGB1,HMGB1的Abox和Bbox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Bbox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Abox和Bbox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, HMGBI B box and A box and prepare HMGB1 polyclonal anti-sera. The target genes were amplified by PCR from human PBMCs, and cloned into pET28a vectors, respectively. The se- quences 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-HMGB1, 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 ofHMGB 1 in the pathogenesis of human diseases.

关 键 词：HMGB1 A BOX B BOX 蛋白表达纯化 多克隆抗血清 

分 类 号：Q75[生物学—分子生物学] Q78