肺炎链球菌肺溶素的体外表达及纯化 - 第三军医大学学报.doc

肺炎链球菌溶血素的体外表达及活性鉴定 贺 潇，袁 军，王 虹，李忱炜，姜 慧，董 杰，崔 瑾，董姗姗，周爱娥，张雪梅，胥文春，尹一兵，何於娟 (400016 重庆，重庆医科大学临床检验诊断学教育部重点实验室) ［摘要］ 目的 体外扩增肺炎链球菌（Streptococcus pneumoniae，S.pn），分离培养型肺炎链球菌其DNA，采用PCR技术体外扩增ply基因，体外重组将ply克隆到原核表达载体pET28(a)测序鉴定获得重组蛋白鉴定GenBank中的数据相符，并实现了Ply蛋白的可溶表达。所获重组蛋白纯度达到95%。溶血实验显示 Ply对人红细胞具有极高的溶血活性，与对照组相比较差异有统计学意义（P0.05），并呈剂量依赖。细胞毒实验显示Ply对脐静脉内皮细胞（human umbilical vein endothelial cell,HUVEC）细胞具有抑制作用与对照组比较差异有P0.05），并呈剂量与时间依赖性，IC50（24h）为1.525 μg/ml肺炎链球菌］A In vitro expression of pneumolysin and identification of its biological activity He Xiao, Yuan Jun, Wang Hong, Li Chenwei, Jiang Hui, Dong Jie, Cui Jin, Dong Shanshan, Zhou Aie, Zhang Xuemei, Xu Wenchun, Yin Yibing, He Yujuan （, Department of Clinical Laboratory Diagnostics, Chongqing Medical University, Chongqing 400016,China）［Abstract］Objective To detect the expression of in vitro amplified pneumolysin gene in E.coli and verify its biological activity. Methods DNA was isolated from cultured S.pneumoniae D39. Pneumolysin gene was amplified in vitro by PCR and cloned into pET-28(α) expression vector. Recombinant pneumolysin protein was transferred to E.coli Rossetta(DE3) by sequencing. Over-expression of recombinant pneumolysin gene labeled with 6 infused amino acids was induced by IPTG. Biological activity and cytotoxicity of the recombinant pneumolysin gene were detected after it was purified with Ni-NTA resin and identified with SDS. Results The sequence of cloned pneumolysin gene was consistent with that in the GenBan. The pneumolysin gene was expressed a soluble manner. The purity of recombinant pneumolysin gene was 95%. Hemolysis testing showed that the hemolytic activity of pneumolysin increased in human red blood cells in a dose-dependent manner more significantly in experimental group than in control group (P0.05) . Cytotoxicity testing showed that pneumolysin inhibited the human umbilical vein endothelial cells in a dose-and-time-dependent more significantly in experimental group than in control group (P