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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。
Objective To prepare EPEC adhesin IntiminC280 antiserum to detect the effect of neutralizing EPEC on Hep-2 cell adhesion. Methods The intiminC280 expression vector pET32 (α) -intiminC280 was constructed by gene cloning technique and transformed into host strain BL21. The expression of His-IntiminC280 fusion protein was induced by IPTG. The expressed protein was analyzed by SDS-PAGE and purified by NI column affinity chromatography The fusion protein was added to the rabbits and the rabbits were immunized with the adjuvant. After 4 immunizations, the blood was collected and the serum was separated. The titer of antisera was detected by flow cytometry. The antiserum was used as the primary antibody. Western blot was used to detect the expression of EPEC adhesin Intimin EPEC was neutralized with different dilutions of antisera and the adhesion of Hep-2 cells after neutralization was observed under a microscope. Results The prokaryotic expression system of BL21 / pET32 (α) -intiminC280 was successfully constructed. The molecular mass unit of the expressed protein was 50ku, which was in agreement with the expectation. The purified His-IntiminC280 was used to immunize rabbits to obtain high-titer antiserum, which could effectively inhibit the adhesion of EPEC to Hep-2 cells after 16-fold dilution. Conclusion The inhibition of IntiminC280 antiserum is a potential antigen for EPEC adhesion to Hep-2 cells and can be used as an antigen for EPEC vaccine development.