大肠埃希菌Ⅵ型分泌系统相关蛋白多克隆抗体制备及分泌蛋白检测

目的为研究新近发现的大肠埃希菌(E.coli)Ⅵ型分泌系统的组成和功能,制备与之相关的三种重要蛋白AaiC、CRP和OmpA的多克隆抗体,并对分泌蛋白AaiC进行检测。方法采用PCR技术扩增出目的基因,并将其连接到pET-22b或pQE80原核表达载体中,酶切及测序鉴定正确后转化E.coli BL21(DE3),经IPTG诱导表达蛋白并纯化,使用纯化蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blotting检测抗体。应用AaiC抗体检测E.coli RS218菌株分泌蛋白。结果重组质粒构建成功,并能够在E.coli中高效表达,免疫得到AaiC、CRP和OmpA高效价的特异性多克隆抗体。AaiC抗体检测证实RS218菌株能够分泌AaiC蛋白。结论成功制备了E.coli三种重要蛋白AaiC、CRP和OmpA的特异性抗体,检测出RS218菌株分泌AaiC蛋白,为进一步研究E.coliⅥ型分泌系统的生物学功能以及E.coli的致病机制奠定了基础。

Preparation of polyclonal antibodies of Escherichia coli type VI secretion system related proteins and detection of secreted proteins

Objective To prepare the polyclonal antibodies of AaiC, CRP and OmpA proteins for the research of function of Escherichia coli （E. coli） type Ⅵ secretion system, and detect the secreted protein AaiC. Methods Target genes were amplified by PCR and cloned into prokaryotic expression vector pET-22b or pQE80, and the recombinant plasmids were transformed into E. coli BL21 （DE3）. The fusion proteins were induced with IPTG, and were used to immunize New Zealand rabbits to generate polyclonal antibodies after purification. The specificity of polyclonal antibodies was evaluated by Western blotting. Secreted proteins of E. coli RS218 were detected by polyclonal antibodies of protein AaiC. Results The recombinant plasmids were successfully constructed, and the fusion proteins were highly expressed in E. coli. The specificity of polyclonal antibodies of AaiC, CRP and OmpA proteins was confirmed. AaiC antibody detection revealed that RS218 could secret protein AaiC. Conclusion The specific polyclonal antibodies of three recombinant fusion proteins AaiC, CRP and OmpA in E. coli have been successfully generated, and protein AaiC can be secreted by RS218, which can be applied in the research of function of Ⅵ secretion system of E. coli and pathogenesis of E. coli.