中文摘要：

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UPEC132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-R049ORF，重组蛋白相对分子质量（MT）约为66．9×10^3，纯化后其纯度达到95％，制备多克隆抗体效价为≥1：102400。Western blot证实重组R049蛋白与UPEC132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〈0．01，P〈0．05）。结论UPEC132的新基因R049具有一定的免疫保护作用，其机制有待进一步研究。

英文摘要：

Objective To clone and express the new gene R049 of uropathogenic Escherichia coli 132, and to investigate the inununoprotective effects of the R049 recombinant protein on mice. Methods The prokaryotic 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 prepared 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 immunized with R049 recombinant protein before challenged by UPEC132 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 BI21 （DE3）/pET32a-R049 ORF was constructed successfully, and the relative molecular mass of the R049 recombinant protein was 66.9 × 10^3 and its purity was up to 95% after purification. The titer of polyclonal antibody was ≥1：102 400 analyzed by indirect ELISA. Both of the R049 recombinant protein and whole bacterial proteins of UPEC132 were confirmed to show specific reactions on the antiserum through 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 ）. Conclusion The new gene R049 of uropathogenic E. coli 132 had immunoprotective effects on mice and the definitive mechanism would be needed to further study.