Pg FimA Ⅱ型特异性单克隆抗体制备及鉴定

Preparation and identification of specific monoclonal antibody against Pg FimA TypeⅡ

[目的]诱导表达、纯化牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)FimAⅡ蛋白,将其作为免疫原制备抗Pg FimAⅡ单克隆抗体(mAbs),对抗体特性进行初步研究。[方法]将BL21-pET28a-FimAⅡ菌株诱导表达,镍柱亲和层析纯化重组蛋白并作为抗原免疫BALB/c小鼠,经PEG融合法和腹水法获得FimAⅡ单克隆抗体;分别采用SDS-PAGE法、酶联免疫吸附实验检测抗体纯度、效价和亚型,Western Blot和Biacore检测抗体的特异性和亲和力。[结果]FimAⅡ重组蛋白为可溶性表达,最适诱导条件为1 mmol/L IPTG诱导12 h;纯化后的重组蛋白纯度为90%以上,该免疫原制备的FimAⅡ单克隆抗体为IgG1亚类,kappa亚型,效价为1:8×104以上,特异性良好,亲和力可达到9nM。[结论]成功诱导表达、纯化FimAⅡ蛋白并制备抗Pg FimAⅡ特异性单克隆抗体,为后续开发快速检测Pg诊断试纸条奠定基础。

[Objective]Porphyromonas gingivalis(Pg)FimAⅡprotein was induced,purified and used as immunogen to prepare anti-PG FimAⅡmonoclonal antibody(mAbs),and the antibody characteristics were preliminarily studied.[Method]BL21-pET28a-FimAⅡstrain was induced to express the recombinant protein.The recombinant protein was purified by nickel column affinity chromatography and immunized BALB/c mice as antigen.FimAⅡmonoclonal antibody was obtained by PEG fusion and ascites method.The purity,titer and subtype of the antibody were detected by SDS-PAGE and enzyme-linked immunosorbent assay,and the specificity and affinity of the antibody were detected by Western Blot and Biacore.[Result]FimAⅡrecombinant protein was soluble,and the optimal induction condition was 12 hours induced by 1 mmol/L IPTG.The purity of the purified recombinant protein was more than 90%.The monoclonal antibody of FimAⅡprepared by the immunogen was IgG1 subclass and kappa subtype.The titer was more than 1:8×10~4,with good specificity and affinity up to 9nM.[Conclusion]The expression of FimAⅡprotein was successfully induced and purified,and the specific monoclonal antibody against Pg FimAⅡwas prepared,which laid the foundation for the subsequent development of the rapid detection of Pg diagnostic strip.