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

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

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。

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.T...