肺炎克雷伯菌FimA的表达纯化及多克隆抗体的制备

Expression and purification of Klebsiella pneumoniae FimA and preparation of a polyclonal antibody

目的表达及纯化肺炎克雷伯菌(Klebsiella pneumoniae)菌毛FimA蛋白并制备多克隆抗体。方法在Clustal Omega网站上比对不同细菌FimA蛋白的同源性。设计特异引物,PCR扩增肺炎克雷伯菌FimA基因,双酶切后将其连接到表达质粒pET28a,构建重组质粒pET28a-FimA。将pET28a-FimA转化表达菌BL21 star(DE3)后培养,使用IPTG诱导FimA蛋白表达。使用IEDG在线网站分析FimA蛋白的B细胞表位,然后用层析纯化的重组FimA蛋白免疫小鼠,获得免疫血清,ELISA检测IgG、IgG1、IgG2a和IgA.结果肺炎克雷伯菌的FimA与其他FimA蛋白同源性较低,为23.6%?30.6%。构建的重组质粒PET28a-FimA在大肠埃希菌中能表达21.6×10^3的重组FimA蛋白,经亲和层析后得到单一电泳条带的高纯度目的蛋白。用FimA蛋白免疫小鼠,获得高滴度的IgG和IgA,且IgG2a的A450值高于IgG1.结论成功构建了表达质粒pET28a-FimA,获得高纯度的重组蛋白FimA。FimA具有免疫原性,用该蛋白免疫小鼠获得高滴度的多克隆抗体。

Objective To express and purify Klebsiella pneumoniae fimbrial protein FimA and to prepare a polyclonal antibody against it.Methods The homology of FimA proteins from different bacteria was compared using the Clustal Omega website.PCR primers were designed to amplify the FimA gene,which was double digested and ligated into pET28a to construct the recombinant plasmid pET28a-FimA.pET28a-FimA was transformed into the expression strain E.coli,which was cultured,and expression of FimA protein was induced using IPTG.The B cell epitopes of the FimA protein were analyzed,and mice were immunized with the FimA protein to produce anti-serum.Levels of IgG,IgG1,IgG2a,and IgA in serum were analyzed using ELISA.Results The FimA of K.pneumoniae had limited similarity to the FimA protein of other intestinal bacteria,with a similarity ranging from 23.6 to 30.6%.The recombinant plasmid pET28a-FimA was successfully constructed,and a recombinant FimA protein of 21.6×10^3Ku was expressed in E.coli and purified using affinity chromatography.Mice were immunized with FimA protein to produce a high titer of anti-IgG and IgA antibodies,and the level of IgG2a was higher than that of IgG1.Conclusion The expression plasmid pET28a-FimA was successfully constructed,and a highly pure recombinant FimA protein was purified.The protein has immunogenicity.A high titer of polyclonal antibodies was harvested from mice immunized with FimA protein.