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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (6): 51-56    DOI: 10.13523/j.cb.20160607
研究报告     
化脓性链球菌溶血素O活性结构重组蛋白的制备
杨思源, 潘敬梅, 王硕, 邓开轩, 邓强, 黄新河, 李学如
西南交通大学生命科学与工程学院 成都 610031
The Preparation of the Streptolysin O Active Recombinant Protein of Streptococcus pyogenes
YANG Si-yuan, PAN Jing-mei, WANG Shuo, DENG Kai-xuan, DENG Qiang, HUANG Xin-he, LI Xue-ru
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
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摘要：

生物信息分析化脓性链球菌溶血素O(streptolysin O,Slo)蛋白结构表明,Slo蛋白除含有由461氨基酸残基组成的溶血活性结构域Thiol_cytolysin外,在N端还有一跨膜结构域。利用pET101-GENE蛋白表达系统,成功构建出表达具有Slo活性重组蛋白的重组子,采用镍柱亲和层析分离技术,纯化目的蛋白;纯化蛋白SDS-PAGE检测分析表明,重组蛋白与预测的溶血活性结构域的分子量相一致;溶血实验显示,纯化重组蛋白具有溶血活性。以纯化的重组蛋白为免疫原,对大鼠进行4次免疫,所获得免疫血清经 Elisa检测,抗Slo血清效价达到 1 ∶12 800;Western blot检测猪链球菌、马链球菌和化脓性链球菌中的链球菌溶血素结果显示,抗Slo多克隆抗体仅能与化脓性链球菌溶血素O发生反应,表明研究制备的化脓性链球菌溶血素O活性结构重组蛋白抗原具有较好的特异性,所制备的抗原Slo 可用于进一步开发抗链球菌溶血素O(ASO)试剂盒。
关键词： 链球菌溶血素O重组蛋白;  多克隆抗体;  化脓性链球菌    
Abstract:

The SMART algorithing (htpp://smart.emble-heideberg.de) was used for the structural analysis of Streptolysin O (Slo) in Streptococcus pyogenes. The location of protein domain shows that Slo contains a Thiol_cytolysin domain which is composed of 461 amino acids and a transmembrane domain in the amino terminus. A pET101-GENE protein expression system was used to construct genetically engineered bacteria which only express the Thiol_cytolysin domain fusion protein. The recombinant protein was purified using Ni-NTA Resins affinity chromatography. SDS-PAGE analysis of the purified recombinant protein showed that the relative molecular weight of protein is about 50kDa as same as we predicted by bioinformation. Hemolysis testing showed that the purified recombinant protein possessed the hemolytic activity in red blood cells. In addition, the polyclonal antibody against recombinant Slo was prepared in rats. The titre of antiserum was 1 ∶128 000 detected by ELISA. Analysis of Western blot with the polyclonal antibody showed that hemolysin was detected only in culture supermentant of Streptococcus pyogenes. This result showed that recombinant protein is a specific antigen in Streptococcus pyogenes.
Key words: Recombinant streptolysin O protein    Polyclonal antibody    Streptococcus pyogenes
收稿日期: 2016-03-03 出版日期: 2016-06-25
ZTFLH:  Q812  
基金资助:

国家自然科学基金(31070117)、四川省科技支撑项目(2012FZ0048)、国家大学生创新性实验计划项目(201510613062)资助项目
通讯作者: 李学如     E-mail: xueruli@sina.com
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引用本文:

杨思源, 潘敬梅, 王硕, 邓开轩, 邓强, 黄新河, 李学如. 化脓性链球菌溶血素O活性结构重组蛋白的制备[J]. 中国生物工程杂志, 2016, 36(6): 51-56.

YANG Si-yuan, PAN Jing-mei, WANG Shuo, DENG Kai-xuan, DENG Qiang, HUANG Xin-he, LI Xue-ru. The Preparation of the Streptolysin O Active Recombinant Protein of Streptococcus pyogenes. China Biotechnology, 2016, 36(6): 51-56.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160607    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I6/51

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