生殖支原体粘附素蛋白MgPa的表达及其多克隆抗体的制备与纯化

Expression of the adhesion protein of Mycoplasma genitalium and preparation and purification of it's polyclonal antibodies

目的:制备并纯化生殖支原体(Mg)粘附素蛋白(MgPa)的多克隆抗体,为进一步探讨MgPa的生物学功能及其在Mg的临床诊断与预防方面的应用提供实验基础。方法:构建原核表达载体,在大肠杆菌中诱导表达生殖支原体粘附素蛋白(rMgPa′),重组蛋白经Western blot鉴定后用Ni-NAT亲和层析柱纯化,然后免疫新西兰兔以制备兔抗rMgPa′血清。用饱和硫酸铵法和溴化氰活化的琼脂糖4B亲和层析柱纯化多克隆抗体,并经Western blot鉴定免疫血清的特异性,用间接ELISA法检测免疫血清的效价。结果:在大肠杆菌中成功表达一分子量约为37kD的6×His-rMgPa′融合蛋白,免疫新西兰兔后获得了相应的多克隆抗体,利用饱和硫酸铵法和亲和层析法纯化后得到具有较高纯度的多克隆抗体,SDS-PAGE分析的结果表明所制备的多克隆抗体分子量约为150kD,Western blot结果表明制备的抗体具有较高的特异性,ELISA结果显示抗体的效价为1∶25600。结论:成功制备了高效价、高特异性的兔抗rMgPa′的多克隆抗体,为进一步研究MgPa的生物学功能、Mg的临床诊断与预防提供了重要的实验基础。

Objective：To prepare and purify rabbit anti-Mycoplasma genitalium adhesion protein（MgPa）polyclonal antibody for providing a tool for the study of the biological function of MgPa and the clinical diagnosis and prevention of M.genitalium.Methods：The recombinant prokaryotic expression plasmid was transformed into E.coli RosettaTM2（DE3）to express the adhesion protein of M.genitalium.The recombinant protein（rMgPa）was identified by Western blot,and then purified by Ni-NAT immunoaffinity chromatography.New Zealand whites rabbits were immunized with the purified protein to generate polyclonal antibody.The immune sera were initially purified by saturated ammonium sulfate,and then by affinity chromatography with CNBr-activated Sepharose 4B coupled with rMgPa.Purified polyclonal antibody was characterized by ELISA and Western blot analysis.Results：The adhesion protein of M.genitalium was successfully expressed in E.coli RosettaTM2（DE3）.SDS-PAGE analysis proved that the antibody had high purity and it′s molecular weight was about 150 kD.The results showed that the polyclonal antibody had high specificity and it′s titer was 1∶25 600.Conclusion：The polyclonal antibody against rMgPa is successfully generated,and therefore this study can provide a tool for the study of the biological function of MgPa and the clinical diagnosis and prevention of M.genitalium.