化脓性链球菌溶血素O活性结构重组蛋白的制备

The Preparation of the Streptolysin O Active Recombinant Protein of Streptococcus pyogenes

生物信息分析化脓性链球菌溶血素O(streptolysin O,Slo)蛋白结构表明,Slo蛋白除含有由461氨基酸残基组成的溶血活性结构域Thiol_cytolysin外,在N端还有一跨膜结构域。利用pET101-GENE蛋白表达系统,成功构建出表达具有Slo活性重组蛋白的重组子,采用镍柱亲和层析分离技术,纯化目的蛋白;纯化蛋白SDS-PAGE检测分析表明,重组蛋白与预测的溶血活性结构域的分子量相一致;溶血实验显示,纯化重组蛋白具有溶血活性。以纯化的重组蛋白为免疫原,对大鼠进行4次免疫,所获得免疫血清经Elisa检测,抗Slo血清效价达到1∶12 800;Western blot检测猪链球菌、马链球菌和化脓性链球菌中的链球菌溶血素结果显示,抗Slo多克隆抗体仅能与化脓性链球菌溶血素O发生反应,表明研究制备的化脓性链球菌溶血素O活性结构重组蛋白抗原具有较好的特异性,所制备的抗原Slo可用于进一步开发抗链球菌溶血素O(ASO)试剂盒。

The SMART algorithing（ htpp：//smart.emble-heideberg.de） was used for the structural analysis of Streptolysin O（ Slo） in Streptococcus pyogenes.The location of protein domain shows that Slo contains a Thiol_ cytolysin domain which is composed of 461 amino acids and a transmembrane domain in the amino terminus.A p ET101-GENE protein expression system was used to construct genetically engineered bacteria which only express the Thiol_ cytolysin domain fusion protein.The recombinant protein was purified using Ni-NTA Resins affinity chromatography.SDS-PAGE analysis of the purified recombinant protein showed that the relative molecular weight of protein is about 50 k Da as same as we predicted by bioinformation.Hemolysis testing showed that the purified recombinant protein possessed the hemolytic activity in red blood cells.In addition,the polyclonal antibody against recombinant Slo was prepared in rats.The titre of antiserum was 1∶ 128 000 detected by ELISA.Analysis of Western blot with the polyclonal antibody showed that hemolysin was detected only in culture supermentant of Streptococcus pyogenes.This result showed that recombinant protein is a specific antigen in Streptococcus pyogenes.