单增李斯特菌InternalinA的表达、纯化及多克隆抗体的制备

【目的】克隆表达单增李斯特菌膜表面蛋白InternalinA(InlA),经免疫家兔获得多克隆抗体,为建立其免疫磁珠富集快速检测方法奠定基础。【方法】利用生物软件设计单增李斯特菌inlA基因的引物,通过PCR扩增出inlA基因,并将其克隆至pET28a()原核表达载体,转化大肠杆菌BL21进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,ELISA分析其免疫原性。免疫家兔,制备其多克隆抗体。间接ELISA检测多抗的效价及交叉性,免疫荧光分析多抗与单增李斯特菌菌体结合的特异性。【结果】成功表达了InlA蛋白,融合表达产物分子量约为92 kD,质谱鉴定其为InlA蛋白;免疫家兔获得的抗血清效价为1:100 000,除与金黄色葡萄球菌约20%的交叉外,与副溶血弧菌等其它病源菌均无交叉;免疫荧光证实该多抗特异性结合于单增李斯特菌膜表面,与同种属的威尔斯李斯特菌不结合。【结论】成功制备了单增李斯特菌特异性的兔多克隆抗体,为单增李斯特菌免疫磁珠富集快速检测方法的建立奠定了基础。

Expression and purification of InternalinA from Listeria monocytogenes and preparation rabbit polyclonal antibody against InternalinA

Objective] To clone and express protein InternalinA (InlA) from Listeria monocytogenes (Lm) and prepare the polyclonal antibody against InlA. To provide basis for preparing the immunomagnetic beads which separated Lm efficiently and specifically. Methods] Use biological software to design the primers of inlA gene, amplify the inlA gene from Lm by PCR and construct the gene into prokaryotic expression vector PET28a(+), then transform the gene into Escherichia coli BL21 and express optimally; purify the recombinant product InlA by nickel affinity chromatography, identify the recombinant protein by mass spectrometry analysis, and test the immunogenicity by ELISA. The purified protein is used as antigen for immunization of rabbit to prepare polyclonal antibody and the binding affinity between polyclonal antibody and Lm is determined. Results] The recombinant InlA protein with relatively molecular mass of 92 kD was over-expressed in E.coli BL21. The InlA antiserum were obtained with a titer as high as 1:100 000. The antibodies had no cross reaction with other pathogenic microorganisms except Staphylococcus aureus. Immunofluorescent staining showed that it only binds to the cell surface of Lm but not L. welshimeri. Conclusion] The polyclonal antibodies which binds to the cell surface of Lm specifically are prepared successfully. These results would provide basis for preparing the immunomagnetic beads which separated Lm efficiently and specifically.