致肾盂肾炎大肠埃希菌新基因R049免疫保护作用的研究

摘要

Objective To clone and express the new gene R049 of uropathogenic Escherichia coli 132.and to investigate the immunopmtective effects of the R049 recombinant protein on mice.Methods The pmkaryotic expression system of gene R049 was constructed by directed cloning.Thereafter,the R049 recombinant protein Was expressed and purified by Ni affinity chromatography.Polyclonal antibody was pre-pared by immunizing BALB/c mice with R049 recombinant protein.The R049 recombinant protein and whole bacterial proteins of UPEC132 were analyzed by SDS-PAGE and Western blot.BALB/c mice were im-munized with R049 recombinant protein before challenged by UPECl32 through urinary tract.Then the differences of urine and renal colony counts between immunization group and control group were compared.Results The recombinant strain E coli BL21(DE3)/pET32a-R049 ORF was constructed successfully,and the relative molecular mass of the R049 recombinant protein was 66.9×103 and its purity was up to 95% af-ter purification.The titer of polyclonal antibody wag≥1:102 400 analyzed by indirect ELISA.Both of the R049 recombinant protein and whole bacterial proteins of UPECl 32 were confirmed to show specffic reactions on the antiserunl throughh Western blot.The animal experiments showed the urine and renal colony counts of immunization group were significantly lower than that of the control group(P<0.01,P<0.05).Conclu-sion The new gene R049 of uropathogenic E.coli 132 had immunopmtective effects on mice and the defini-tive mechanism would be needed to further study.%目的 克隆和表达致肾盂肾炎大肠埃希菌(UPEC)132新基因R049,研究R049重组蛋白对小鼠的免疫保护作用. 方法 利用PCR定向克隆方法构建R049基因原核表达系统,表达后通过镍亲和层析纯化获得R049重组蛋白,制备鼠多克隆抗体.重组蛋白与UPECl32全菌蛋白经SDS-PAGE和Western blot分析,继而R049重组蛋白免疫BALB/c小鼠,用同源菌对小鼠经尿道上行感染,比较免疫组与对照组小鼠尿液与肾脏细菌计数差异. 结果 成功构建重组株E.coli BL21(DE3)/pET32a-R049 ORF,重组蛋白相对分子质量(M,)约为66.9×103,纯化后其纯度达到95%,制备多克隆抗体效价为≥l:102 400.Western blot证实重组R049蛋白与UPECl32全菌蛋白均能与小鼠抗血清发生特异性反应.动物实验结果表明尿液和肾脏菌落计数在免疫组均明显低于对照组(P