肺炎链球菌溶血素的体外表达及活性鉴定  

In vitro expression of pneumolysin and identification of its biological activity

作　　者：贺潇[1] 袁军[1] 王虹[1] 李忱炜[1] 姜慧[1] 董杰[1] 崔瑾[1] 董姗姗[1] 周爱娥[1] 张雪梅[1] 胥文春[1] 尹一兵[1] 何於娟[1] 

机构地区：[1]重庆医科大学检验系临床检验诊断学教育部重点实验室,重庆400016

出　　处：《第三军医大学学报》2011年第3期237-240,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(30872363)~~

摘　　要：目的体外扩增肺炎链球菌(Streptococcus pneumoniae,S.pn)的溶血素(pneumolysin,ply)基因,在大肠杆菌中高效表达,并验证其生物活性。方法分离培养D39型肺炎链球菌并提取其基因组DNA,采用PCR技术体外扩增ply基因,体外重组将ply序列克隆到原核表达载体pET28(a)内,测序鉴定后将重组子转化到E.coliRosetta(DE3)中,经IPTG诱导大量表达融合6个组氨酸标签的Ply重组蛋白,经Ni-NTA树脂纯化后,获得的重组蛋白经SDS-PAGE鉴定后检测其溶血活性和细胞毒性。结果克隆的ply序列与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(P<0.05),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)细胞具有明显抑制作用,与对照组比较差异有统计学意义(P<0.05),并呈剂量与时间依赖性,IC50(24h)为1.525μg/ml。结论经测序鉴定ply基因正确扩增,并成功表达、纯化出具有高溶血活性和细胞毒性的Ply蛋白。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 Rosetta（DE3） by sequencing.Over-expression of recombinant pneumolysin gene labeled with 6 infused amino acids was induced by IPTG.Biological activity of the recombinant pneumolysin gene were detected after it was purified with Ni-NTA resin and identified with SDS-PAGE.Results The sequence of cloned pneumolysin gene was consistent with that in the GenBank.The pneumolysin gene was expressed a soluble manner.The purity of recombinant pneumolysin protein 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（P0.05）.The IC50（24 h） was 1.525 μg/ml.Conclusion The amplified pneumolysin protein can be identified by sequencing,expressed and purified with a hemolytic activity and a cytotoxicity.

关 键 词：肺炎链球菌 溶血素 原核表达 生物活性 

分 类 号：R372[医药卫生—病原生物学] R378.12[医药卫生—基础医学]