为研究化脓隐秘杆菌致病机制及其病原学诊断方法,本研究克隆了编码化脓隐秘杆菌溶血素蛋白的plo基因,并构建重组表达质粒pET-plo,转化大肠杆菌Rosetta (DE3)感受态细胞中诱导表达.SDS-PAGE检测结果显示,表达的重组蛋白约为62 ku,western blot分析表明表达的重组蛋白可以与鼠抗化脓隐秘杆菌血清发生反应.采用重组蛋白免疫新西兰白兔制备的多克隆抗体效价达到1∶128 000,western blot和琼脂双扩散试验表明制备的多克隆抗体能够与天然PLO蛋白发生反应.溶血试验表明重组蛋白能够溶解红细胞,制备的多克隆抗体能有效中和重组蛋白的溶血活性.%Pyolysin (PLO),the hemolytic exotoxin expressed by Arcanobacterium pyogenes,is a major virulence factor in infections by A.pyogenes.The gene encoding PLO was amplified by PCR from A.pyogenes genome and cloned into the pET-30a for expression in E.coli Rosetta.The expressed protein was approximately 62 ku,mainly in form of inclusion body.The recombinant PLO protein (rPLO) was recognized by mouse antiserum against A.pyogenes.In addition,the polyclonal antibody against rPLO was prepared in rabbits and purified.The titre of antiserum was 1:128,000 detected by ELISA,which reacted with native PLO of A.pyogenes in western blot and agar gel diffusion test.Hemolytic assays show that rPLO possessed hemolytic activity which was completely neutralized by the antiserum.these results provide necessary basic for pathogenesis investigation and development of diagnostic method of A.pyogenes infection.
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