肺炎链球菌肺溶素的体外表达及纯化.docVIP

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 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%，并且具有极高的溶血活性和细胞毒性。结论 成功表达并纯化了具有高溶血活性和细胞毒性的Ply蛋白。 ［关键词］ 肺炎链球菌］A Over-expression of pneumolysin protein and identification of it’s 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 （, Chongqing Medical University, Chongqing, 400016,China）［Abstract］Objective To obtain purified pneumolysin protein produced by prokaryotic expression system and verify its haemolytic activity and cellular toxicity. Methods Template DNA was isolated from cultured S.pneumoniae D39, from which, ply gene which was cut of the signal peptide in the N termination was PCR amplified. The PCR fragments were then cloned into pET-28(α) expression vector, followed by sequencing. Recombinant protein Ply was over-expressed and purified from the host of Rossetta(DE3), and identified by SDS. Purified protein through hemolysis test and MTT to evaluate haemolytic activity and cellular toxicity of Ply. Results The ply gene was successfully cloned into pET-28(α), which was confirmed by gene sequencing. Ply protein purified by Ni-affinity resin and it was sucessfully over-expressed in soluble manner, which was confirmed by SDS. The haemolytic activity and the cellular toxicity of this protein were demonstrated. Conclusion Over-expressed recombinant Ply protein was successfully obtained from Rosseta. And it’s haemolytic activity and cellular toxicity is effective. ［Key words］ Streptococcus pneumoniae; pneumolysin; prokaryotic expression; biological activity General Program of ?National Natural Science Foundation of China . Corresponding author: He Yujuan, Tel:86-23E-mail:yjhemail@126.com [基金