致病性大肠埃希菌EspA抗血清制备及其对细菌粘附的影响  

Preparation of an antiserum against the EspA protein of EPEC and examination of its ability to neutralize adherence by EPEC

作　　者：郭永明[1] 黄荣[1] 王朝莉[2] 杨晓红[1] 杨健[1] 

机构地区：[1]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,四川南充637000 [2]川北医学院分子生物学与免疫学研究所

出　　处：《中国病原生物学杂志》2016年第11期995-998,1003,共5页Journal of Pathogen Biology

基　　金：四川省科技厅项目(No.2009SZ0259;2009SZ0260)

摘　　要：目的制备致病性大肠埃希菌(EPEC)分泌蛋白A(EspA)抗血清,并检测其对EPEC粘附的中和作用。方法以EPEC标准株E2348/69为模板,采用PCR方法获得EspA基因,将目的基因连接载体pET32a,构建重组表达质粒pET32a-EspA,转入表达菌株BL21后用IPTG诱导表达并采用Ni-柱亲和层析法纯化EspA;以纯化EspA辅以佐剂免疫家兔,多次免疫获得抗血清,采用中和试验检测抗血清中和EPEC粘附Hep-2细胞的能力。结果成功构建EspA原核表达质粒,表达蛋白分子质量单位为37ku,与预期值相符。用纯化的表达蛋白EspA免疫家兔,对流免疫电泳检测抗血清的效价为1∶2,但稀释度≤1∶16时仍能效抑制EPEC粘附Hep-2细胞。结论 EspA抗血清具有抑制EPEC粘附Hep-2细胞作用,有望为疫苗研发的候选抗原。Objectives To prepare an antiserum against EspA and to detect its ability to inhibit enteropathogenic Escherichia coli（EPEC）adherence to Hep-2cells. Methods The EspA gene was amplified from E2348/69 as a template and cloned into pET32 ato yield pET32a-EspA.The recombinant plasmid was transformed into BL21 to construct a recombinant strain.Expression of the EspA protein was induced with IPTG and the protein was purified with Ni-column affinity chromatography.Rabbits were immunized with the purified protein as an antigen to prepare antiserum.The antiserum was harvested from immunized rabbits and the ability of antiserum to inhibit EPEC adherence to Hep-2cells was detected. Results The recombinant plasmid pET32a-EspA was constructed.Expression of an EspA fusion protein（37ku）was induced with IPTG and the protein was successfully purified with Ni column affinity chromatography.Antiserum was harvested,and counter immunoelectrophoresis indicated a titer of 1：2.However,the antiserum efficiently inhibited EPEC adherence to Hep-2cells at a dilution of 1：16. Conclusion EspA antiserum inhibited the adherence of EPEC to Hep-2cells.EspA is an antigen that can protect against EPEC infection and the protein is a candidate for vaccine development.

关 键 词：致病性大肠埃希菌 EspA 抗血清 粘附 

分 类 号：R378.21[医药卫生—病原生物学]