In vitro expression of pneumolysin and identification of its biological activity

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

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In vitro expression of pneumolysin and identification of its biological activity

更新时间：2026-01-09

- In vitro expression of pneumolysin and identification of its biological activity
- Journal of Army Medical University Vol. 33, Issue 3, Pages: 237-240(2011)
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  CLC： R372;R378.12;R394.3
- Published：2011
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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.肺炎链球菌溶血素的体外表达及活性鉴定[J].陆军军医大学学报(原第三军医大学学报),2011,33(03):237-240.

　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.In vitro expression of pneumolysin and identification of its biological activity[J].J Amry Med Univ (J Third Mil Med Univ),2011,33(03):237-240.
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.肺炎链球菌溶血素的体外表达及活性鉴定[J].陆军军医大学学报(原第三军医大学学报),2011,33(03):237-240. DOI：

　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.In vitro expression of pneumolysin and identification of its biological activity[J].J Amry Med Univ (J Third Mil Med Univ),2011,33(03):237-240. DOI：

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.coli Rosetta（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（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）细胞具有明显抑制作用，与对照组比较差异有统计学意义（P0.05），并呈剂量与时间依赖性，IC

（24h）为1.525 μg/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。

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 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 gen

e 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 IC

(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.

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