鸡源性HMGB1蛋白的原核表达及多克隆抗体制备

摘要

从鸡胚原代细胞CEF中扩增出鸡HMGB1基因,构建重组原核表达质粒pET-28a-chHMGB1,并将pET-28a-chHMGB1转入大肠杆菌BL21(DE3)中,于37℃进行诱导表达,经SDS-PAGE分析表明该重组蛋白可以在大肠杆菌中高效表达且以可溶性蛋白的形式存在.蛋白通过Ni-NAT纯化树脂亲和纯化,并作为免疫原制备鼠抗chHMGB1多克隆抗体血清.该多克隆抗体同时具有ELISA、Western blot和IFA效价,且特异性识别禽源细胞内HMGB1蛋白.%The chHMGB1 gene was amplified from CEF cells and recombinant prokaryotic expression vector pET-28a-chHMGB1 and eukaryotic expression vectors FLAG-chHMGB1 were constructed accordingly. The recombinant protein chHMGB1 was expressed at 37℃in E.coli BL21(DE3).The SDS-PAGE analysis showed that the recombinant chHMGB1 was efficiently expressed in E coli and existed in the form of soluble protein. The recombinant chHMGB1 was then purified by affinity chromatography on Ni-NAT for preparation of polyclonal antibodies. The resulting polyclonal antibodies showed specific reaction with chHMGB1 in ELISA, Western blot and IFA.