EPEC粘附素IntiminC280抗血清的制备及其中和能力测定  

Preparation of antiserum against IntiminC280(an EPEC antigen responsible for adhesion)and examination of its neutralization ability

作　　者：康迁[1] 黄荣 冯亚岚 皮光环[1] 杨健 KANG Qian;HUANG Rong;FENG Ya-lan;PI Guang-huan;YANG Jian(Pediatrics, North Sichuan Medical College Hospital, Nanchong, Sichuan 637000, China;Experimental Teaching Center for Pathogens and Immunology, School of Basic Medical Sciences, North Sichuan Medical College)

机构地区：[1]川北医学院附属医院儿科,四川南充637000 [2]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

出　　处：《中国病原生物学杂志》2017年第10期934-938,共5页Journal of Pathogen Biology

基　　金：四川省教育厅项目(No.15ZA0205)

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。Objectives To prepare antiserum against IntiminC280（an EPEC antigen responsible for its adhesion）and to observe whether the antiserum could affect the adhesion of EPEC to Hep-2 cells. Methods The IntiminC280 expression plasmid pET32（α）-intiminC280 was constructed using gene cloning and transformed into the host strain BL21.Expression of the His-IntiminC280 fusion protein was induced with IPTG and that expression was analyzed with SDSPAGE.The fusion protein was purified with Ni column affinity chromatography and the purified protein was used to immunize rabbits with adjuvants.After 4 immunizations,serum was collected and the antiserum titer was measured using a convective immunoassay.The antiserum was used to recognize the expression of EPEC Intimin in Western blotting.The adhesion of EPEC to Hep-2 cells was observed after EPEC was neutralized with different dilutions of antiserum. Results A BL21/pET32（α）-intiminC280 prokaryotic expression system was constructed,the molecular weight of the expressed protein is 50 ku,which is consistent with expectations.A high-titer antiserum against IntiminC280 was obtained and the adhesion of EPEC to Hep-2 cells was inhibited by the antiserum up to a dilution of 16. Conclusion IntiminC280 antiserum inhibits the adhesion of EPEC to Hep-2 cells and IntiminC280 can serve as an ideal antigen for EPEC vaccine research.

关 键 词：肠致病性大肠埃希菌 紧密素 抗血清 粘附 

分 类 号：R378.21[医药卫生—病原生物学]