抗大肠杆菌curli菌毛CsgA蛋白特异性抗体的制备及鉴定

Preparation and identification of antisera specific against curli major subunit CsgA in Escheriachia coli

目的为进一步研究curli菌毛主要蛋白CsgA的功能,利用MBP-CsgA融合蛋白免疫兔子制备抗体。方法用PCR方法扩增大肠杆菌MHC4100株的csgA基因,在大肠杆菌中表达MBP-CsgA融合蛋白,免疫家兔以制备抗血清,用Westernblotting和免疫电镜鉴定抗MBP-CsgA融合蛋白抗体的特异性。结果PCR扩增出约476 bp的csgA基因,在大肠杆菌XL1-Blue中成功地表达了Mr约为58 000的MBP-CsgAⅢ融合蛋白,Western blotting和免疫电镜检测证明,针对MBP-CsgA融合蛋白的抗血清,不仅可特异性地识别MBP-CsgA,也可识别源于大肠杆菌的csgA蛋白,抗血清的最高滴度达1∶50 000。结论亚克隆csgA基因,成功表达MBP-CsgA融合蛋白,制备并获得其特异性抗curli菌毛抗体。

Objective To study the biological function of the CsgA of bacterial fimbriae curli in E. coli. Methods The major subunit gene csgA was amplified from E. coli MC4100. The MBP-CsgAⅢ fusion protein was over-expressed in E. coli XLl-blue. The fusion protein was used to immunize rabbit for preparing polyclonal antiserum. The specificity of antiserum was identified by Western blotting and electron microscopy. Results A unique band about 476 bp （csgA gene） was amplified by PCR.The SDS-PAGE analysis showed that the fusion protein MBP-CsgA Ⅲ was about Mr 58 000. The purified polyclonal antiserum against MBP-Csg, A Ⅲ fusion protein could recognize both MBP- CsgA fusion protein and CsgA protein specifically. The highest titer of the antisermn was about 1：50 000, confirmed by Western blotting and FJJSA. Conclusion The gene csgA has been sub-cloned and high titer polyclonal antiserum specific against curli major subunit CsgA is obtained.