论文部分内容阅读
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。
Objective To prepare Escherichia coli (EspA) antiserum against pathogenic Escherichia coli (EPEC) and determine its neutralization on EPEC adhesion. Methods The recombinant plasmid pET32a-EspA was obtained by PCR using the EECEC standard strain E2348 / 69 as a template. The recombinant plasmid pET32a-EspA was inserted into the vector pET32a. The recombinant plasmid pET32a-EspA was transformed into E.coli BL21 and induced by IPTG. EspA was purified by chromatography. Rabbit was immunized with purified EspA and adjuvant. The antiserum was obtained by multiple immunizations. Neutralization assay was used to detect the ability of antiserum to neutralize EPEC adhesion to Hep-2 cells. Results The prokaryotic expression plasmid of EspA was successfully constructed. The molecular mass of expressed protein was 37ku, which was consistent with the expected value. Rabbit immunized with the purified expression protein EspA, the antiserum titer was 1: 2 by flow cytometry, but it still inhibited the adhesion of EPEC to Hep-2 cells when the dilution was less than 1:16. Conclusion EspA antiserum can inhibit the adhesion of Hep-2 cells to EPEC and is expected to be a candidate antigen for vaccine development.