Ⅱ型志贺样毒素B亚单位的重组表达及其受体结合活性  

The Recombinant Expression and Receptor-binding Activity of the B Subunit of Shiga-like Toxin Type Ⅱ

作　　者：包士中[1] 史晶[1] 蔡昆[1] 荫俊[1] 王慧[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《中国生物工程杂志》2008年第10期23-27,共5页China Biotechnology

摘　　要：目的:在大肠杆菌中重组表达Ⅱ型志贺样毒素B亚单位(Stx2B),并对其表达形式和受体结合活性进行分析。方法:PCR方法从肠出血性大肠杆菌(EHEC)O157:H7中钓取Stx2B编码基因,利用基因克隆技术构建重组大肠杆菌pET-stx2B/BL21,IPTG诱导目的蛋白高效表达并对表达的包涵体进行变性和复性处理,离子交换层析纯化蛋白。通过SDS-PAGE变性和非变形蛋白电泳,分析重组Stx2B的表达形式,并利用Hela细胞结合模型,评价重组Stx2B与细胞受体的结合活性。结果:构建的重组大肠杆菌pET-stx2B/BL21能高效表达Stx2B,经变性、复性及离子交换层析操作,获得高纯度的目的蛋白。SDS-PAGE变性和非变形蛋白电泳分析显示,重组Stx2B以二聚体形式存在,单体之间通过二硫键相连。细胞结合试验显示,重组Stx2B与Hela细胞具有特异结合活性。结论:成功构建表达Stx2B的基因工程菌,Stx2B的受体结合活性不依赖于五聚体形式。Objective： To express the B subunit of Shiga-like toxin type Ⅱ , and analyze its expression form and receptor-binding aetivity. Methods： The slt2b gene was obtained from EHEC O157：H7 by PC R, and cloned to the expression vector pET22b（ ＋ ）. The genetically engineered bacteria pET22b（ ＋ ）-stx2B/BL21 expressed the recombinant StxB after induced with IPTG. The renatured inclusion bodies were purified by ion exchange chromatography. The expression form of rStx2B was investigated by denaturing and native electrophoresis. The receptor-binding activity was confirmed by fluorescence detection and flow eytometer. Result： The constructed genetically engineered bacteria expressed the rStx2B at a high level. The purified protein was obtained after denaturation, renaturation and ion exchange chromatography. According to the denaturing and native electrophoresis, the rStx2B was expressed in a dimmer form, which consists of two monomers cross linked with disulfide bridge. The rStx2B showed good receptor-binding activity by Hela-binding assay. Conclusion： The genetically engineered bacteria were constructed successfully. The receptor-binding activity of rStx2B was independent of the pentamers.

关 键 词：Ⅱ型志贺样毒素B亚单位 重组表达 受体结合活性 

分 类 号：Q786[生物学—分子生物学]