大肠杆菌NrfA蛋白表达、纯化及多克隆抗体的制备

Expression,Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody Against NrfA

[目的]克隆大肠杆菌NrfA基因,构建pET-28a(+)-NrfA表达载体,制备相应的多克隆抗体并对其进行鉴定。[方法]以大肠杆菌基因组DNA为模板,PCR扩增得到NrfA基因编码区,构建pET-28a(+)-NrfA表达载体;经IPTG诱导表达并纯化重组蛋白;再免疫新西兰雄兔,制备多克隆抗体;用ELISA方法检测抗体的效价,Western Blotting检测抗体的特异性。[结果]构建的表达载体pET-28a(+)-NrfA在大肠杆菌中诱导后可高效表达NrfA蛋白;免疫获得的多克隆抗体用ELISA检测,其效价为1∶204 900;经Western Blotting分析,抗体的特异性较好。[结论]成功克隆大肠杆菌的NrfA基因,并构建了其表达载体,制备的NrfA多克隆抗体具有较高的效价和良好的特异性,为研究细菌有关NrfA奠定了基础。

[Objective] This study aimed to clone the E.coli NrfA gene and construct the pET-28a（＋）-NrfA prokaryotic expression vector for preparation of polyclonal antibody against E.coli NrfA.[Method] E.coli NrfA gene was cloned from the E.coli genome DNA by PCR and inserted into the vector pET-28a（＋）to construct prokaryotic expression vector pET-28a（＋）-NrfA.E.coli NrfA protein was expressed by IPTG induction and purified.Polyclonal antibody against NrfA protein was prepared by immunizing rabbit with routine method.The specificity and titer of polyclonal antibody were confirmed by ELISA and Western Blotting.[Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG,the recombinant NrfA protein could be expressed effectively.The titer of rabbit anti-NrfA polyclonal antibody obtained after immunization and purification was about 1∶ 204 900.Western Blotting analysis indicated that the obtained polyclonal antibody against E.coli NrfA protein had high titer and high specificity.[Conclusion] E.coli NrfA gene was cloned and the prokaryotic expression vector pET-28a（＋）-NrfA was constructed successfully,and the polyclonal antibody with high titer and high specificity was prepared,which had laid the foundation for the study of NrfA in different strains of bacteria.