| 李振军,孙强正,景怀琦,徐建国.霍乱毒素B亚单位与志贺毒素2B亚单位融合表达及抗原性检测[J].Chinese journal of Epidemiology,2008,29(4):378-382 |
| 霍乱毒素B亚单位与志贺毒素2B亚单位融合表达及抗原性检测 |
| Clone and express ctb-stx2 b fusion gene in Enterohemrrhagic escherichia coli O157:H7 Shigeai toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity |
| Received:October 11, 2007 |
| DOI： |
| KeyWord: 肠出血性大肠埃希菌O157:H7 志贺毒素2B亚单位 霍乱毒素B亚单位 抗原性 |
| English Key Word: Enterohemrrhagic escherichia coli O157:H7 Shiga toxin 2B subunit Cholera toxin B subunit Immunogenicity |
| FundProject: |
| Author Name | Affiliation | E-mail | | LI Zhenjun | State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | SUN Qiangzheng | State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | JING Hauiqi | State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | XU Jianguo | State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | xujianguo@icdc.cn |
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| Abstract: |
| 目的 克隆表达出血性大肠埃希菌(EHEC)O157:H7志贺毒素2B亚单位(Stx2B)与霍乱毒素B亚单位(CTB)的融合蛋白(CTB-Stx2B),并检测其抗原性和与神经节苷脂(GM1)结合能力.方法 设计引物扩增融合蛋白CTB-Stx2B的编码基因ctb-stx2b,T-A克隆测序验证后克隆入原核表达质粒pET30a(+)C,构建表达质粒pET30a(+)-ctb-stx2b,转化E.coliBL21(DE3),IPTG诱导表达、纯化,获得目的蛋白CTB-Stx2B,SDS-PAGE和Western-blot检测其抗原性和形成五聚体的能力;GM1-ELISA法检测其与GM1结合能力.结果 扩增出约750 bp的目的片段,测序鉴定与设计序列一致;原核表达质粒pET30a(+)-ctb-stx2b转化E.coliBL21(DE3)后,经酶切和PCR扩增鉴定正确;IPTG诱导后SDS-PAGE初步测定CTB-Stx2B单体的相对分子质量(MR)约为20×103;Western-blot检测CTB-Stx2B能与CTB单克隆抗体结合,纯化产物经复性后大多以五聚体形式存在;ELISA检测CTB-Stx2B具有结合GM1活性.结论 成功克隆、表达了融合蛋白CTB-Stx2B;表达的蛋白具有良好的CTB抗原性和GM1结合能力. |
| English Abstract: |
| Objective To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157:H7(EHEC O157:H7)Shigela toxin 2B subunit(Stx2B)and vibrio cholera toxin B subunit (CTB)as well as to detect the immunogenicity and GM1-binding ability of fusion protein.Methods To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified,then to transform constructed plasmid into E.coliBL21(DE3)induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDSPAGE and Western-blot.Results The amplified ctb-stx2b fragments appeared to he 750 bp and gene sequence was identical to designed sequence.The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about Mr20×103and the expressed protein could react to CTB monoclone anti-body.The fusion protein CTB-Stx2B could bind GM1.Conclusion CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability.This study provided information on further EHEC O157:H7 vaccine research. |
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