EPEC粘附素IntiminC280抗血清的制备及其中和能力测定

发布时间：2018-06-15 18:58

本文选题：肠致病性大肠埃希菌 + 紧密素　；参考：《中国病原生物学杂志》2017年10期

【摘要】：目的制备EPEC粘附素IntiminC280抗血清,检测其中和EPEC对Hep-2细胞粘附作用。方法用基因克隆技术构建IntiminC280表达载体pET32(α)-intiminC280,转化表达宿主菌BL21,用IPTG诱导His-IntiminC280融合蛋白的表达,SDS-PAGE分析其表达形式;用NI柱亲和层析法纯化融合蛋白,并辅以佐剂免疫家兔,4次免疫后采血,分离血清,对流免疫电泳法检测抗血清效价;以该抗血清作为一抗,采用Western blot检测EPEC粘附素Intimin的表达;用不同稀释度的抗血清中和EPEC,显微镜下观察细菌被中和后对Hep-2细胞的粘附情况。结果成功构建BL21/pET32(α)-intiminC280原核表达系统,表达蛋白的分子质量单位为50ku,与预期相符。用纯化的His-IntiminC280免疫家兔获得高效价抗血清,16倍稀释后仍能有效抑制EPEC对Hep-2细胞的粘附。结论 IntiminC280抗血清具有抑制是EPEC粘附Hep-2细胞作用,可作为EPEC疫苗研发的备选抗原。
[Abstract]:Objective to prepare the antiserum of EPEC adhesion hormone IntiminC280 and to detect the adhesion of EPEC to Hep-2 cells. Methods IntiminC280 expression vector pET32 (伪 -intiminC280) was constructed by gene cloning technique and transformed into host strain BL21. The expression of His-IntiminC280 fusion protein was analyzed by SDS-PAGE induced by IPTG, and the fusion protein was purified by NI affinity chromatography. After the rabbits were immunized with adjuvant for 4 times, the serum was isolated, the titer of antiserum was detected by convection immunoelectrophoresis, and the expression of imtimin was detected by Western blot. The adherence of bacteria to Hep-2 cells was observed under microscope with different dilution of antiserum neutralization and EPECs. Results the prokaryotic expression system of BL21 / pET32 (伪 -intimin C280) was successfully constructed. The molecular weight unit of the expressed protein was 50ku. 16 times dilution of purified His-Intimin C280 immunized rabbits with high titer antiserum could effectively inhibit the adhesion of EPEC to Hep-2 cells. Conclusion the inhibition of Intimin C280 antiserum is the adhesion of EPEC to Hep-2 cells and can be used as an alternative antigen for the development of EPEC vaccine.
【作者单位】：川北医学院附属医院儿科;川北医学院基础医学院病原生物学与免疫学实验教学中心;
【基金】：四川省教育厅项目(No.15ZA0205)
【分类号】：R392

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