EPEC粘附素IntiminC300抗血清制备及其中和能力探讨

发布时间：2018-03-18 03:00

本文选题：肠致病性大肠杆菌　切入点：紧密素　出处：《川北医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:制备EPEC粘附素IntiminC300抗血清,通过体外细胞实验观察该抗血清能否影响EPEC对Hep-2细胞的粘附,以确定IntiminC300是否可作为EPEC感染的保护性抗原。方法:用酶切和PCR方法鉴定IntiminC300表达质粒pET32a-intiminC300,把质粒转化表达宿主菌BL21,构建IntiminC300原核表达系统;用IPTG诱导His标签-IntiminC300融合蛋白的表达,SDS-PAGE分析其表达形式;用NI柱亲和层析方法纯化融合蛋白,用该纯化蛋白作为抗原辅以佐剂免疫家兔,4次免疫后,心脏采血收集抗血清;对流免疫电泳检测抗血清的效价,并用该抗血清作为一抗,用Westen Blotting方法检测了EPEC粘附素IntiminC300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:1.成功构建了BL21/pET32a-intiminC300原核表达系统,并用SDS-PAGE检测到His-IntiminC300融合蛋白既有可溶性表达形式又有包涵体表达形式,但以包涵体表达形式为主;2.通过His标签蛋白纯化试剂盒(包涵体蛋白),得到纯化蛋白。3.成功制得高效价抗血清(对流免疫电泳检测效价达1:32)。4.体外中和实验观察到该抗血清在稀释倍数16以内皆能有效抑制EPEC对Hep-2细胞的粘附。结论:EPEC粘附素IntiminC300不仅具有良好的免疫原性,且其抗体能有效阻止细菌对上皮细胞的粘附,可以预防EPEC的感染,因此IntiminC300是EPEC重要保护性抗原,可作为EPEC疫苗研究的理想备选抗原。
[Abstract]:Aim: to prepare IntiminC300 antiserum of EPEC adhesion hormone, and to observe whether the antiserum can affect the adhesion of EPEC to Hep-2 cells by cell experiment in vitro. Methods: the IntiminC300 expression plasmid pET32a-intiminC300 was identified by enzyme digestion and PCR. The plasmid was transformed into the host strain BL21 to construct the IntiminC300 prokaryotic expression system. IPTG induced expression of His tagged -Intimin C300 fusion protein was analyzed by SDS-PAGE, and the fusion protein was purified by NI column affinity chromatography. The purified protein was used as antigen and adjuvant to immunize rabbits for 4 times. The titer of antiserum was detected by convection immunoelectrophoresis, and the expression of EPEC adhesin IntiminC300 was detected by Westen Blotting method. The adhesion of bacteria to Hep-2 cells after neutralization was observed under microscope with different dilution antiserum neutralization. Results: 1. The prokaryotic expression system of BL21/pET32a-intiminC300 was successfully constructed. The His-IntiminC300 fusion protein was detected by SDS-PAGE in both soluble expression and inclusion body expression. The purified protein was obtained by His tag protein purification kit. 3. The high titer antiserum was prepared successfully (the titer of Convective immunoelectrophoresis was 1: 32. 4). The neutralization experiment in vitro showed that. The antiserum can effectively inhibit the adhesion of EPEC to Hep-2 cells within the dilution multiple of 16. Conclusion the antiserum IntiminC300 has good immunogenicity. Its antibody can effectively prevent bacterial adhesion to epithelial cells and prevent the infection of EPEC. Therefore, IntiminC300 is an important protective antigen for EPEC and can be used as an ideal alternative antigen for the study of EPEC vaccine.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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