Purification and functional characterization of enterohemorrhagic Escherichia coli O157: H7 Shiga toxinⅡ
- VernacularTitle:肠出血性大肠埃希菌O157:H7Ⅱ型志贺毒素的纯化及功能鉴定
- Author:
Yongjun JIAO
;
Xiaoyan ZENG
;
Xiling GUO
;
Hua WANG
;
Lunbiao CUI
;
Xian LI
;
Zhenqing FENG
;
Hui SUN
;
Jiayi WAN
;
Zhiyang SHI
- Publication Type:Journal Article
- Keywords:
Escherichia coli O157: H7;
Shiga toxin;
Vero cells;
Disease models,animal;
Electrophoresis,polyacrylamide gel;
Blotting,Protein
- From:
Chinese Journal of Infectious Diseases
2008;26(4):217-220
- CountryChina
- Language:Chinese
-
Abstract:
Objective To purify Shiga toxin Ⅱ (STX Ⅱ) of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX Ⅱ A subunit-specific antibody S1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157:H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose (CD50) for Vero cell line and 100% lethal dose (LD100) for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157:H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32 000 and 7 500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively.The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5 ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.
