鸡髓样分化因子88的原核表达及单克隆抗体制备

Prokaryotic Expression of Chicken MyD88 Fragment and Preparation of Mouse Anti-Chicken MyD88 Monoclonal Antibody

目的:克隆、表达、纯化鸡髓样分化因子88(MyD88),制备其单克隆抗体。方法:从脾脏cDNA中扩增857bp的MyD88基因片段,插入pMAL-c5X表达载体,转化大肠杆菌BL21(DE3)获得表达菌株,IPTG诱导表达,用SDS-PAGE分析MBP(麦芽糖结合蛋白)-MyD88重组融合蛋白的表达,切胶纯化目的蛋白;免疫BALB/c小鼠,制备针对MyD88的单克隆抗体,Western印迹检测抗体特异性,制备腹水并进行抗体亚型鉴定和效价测定。结果:构建了鸡MyD88原核表达载体pMAL-MyD88,并在大肠杆菌中获得高表达,目的蛋白以可溶性和包涵体两种形式存在;建立了3株抗鸡MyD88单克隆抗体细胞株,制备了腹水,亚型分别为IgG1、IgG1和IgG2a,轻链均为κ,腹水抗体的效价均为1∶2×105。结论:在原核表达系统中表达、纯化了重组鸡MyD88,制备了针对鸡MyD88的单克隆抗体,为后续的MyD88定量和功能研究奠定了基础。

Objective： To prokaryotic express,purify the chicken myeloid differentiation primary response protein,（MyD88） and prepare monoclonal antibody（mAb） against MyD88.Methods： A 857 bp gene fragment of MyD88 was amplified by PCR from spleen cDNA and sub-cloned into prokaryotic expression vector pMAL-c5X.The MyD88 expression vector was then transformed into E.coli BL21（DE3）.The expression of recombinant MBP-MyD88 was induced by adding IPTG and identified by SDS-PAGE.The unique band of recombinant MBP-MyD88 separated by SDS-PAGE was cut off and purified.BALB/c mouse was immunized and mAb against MyD88 was prepared.The specificity of mAb was identified by Western blot analysis while the immunoglobulin subtype and the titer of that were measured by ELISA analysis,respectively.Results： MyD88 expressing vector pMAL-c5X was constructed,and the recombinant MBP-MyD88 was expressed in high concentration of endosome and soluble protein.Three Cell lines secreting anti-MyD88 monoclonal antibody was developed by cellular fusion.Antibodies in ascites fluid,with the same light chain κ and titer（1∶2×105）,showed positive reaction with MyD88 antigen when identified by Western blot and the subclass specificity was IgG1,IgG1 and IgG2,respectively.Conclusion： Recombinant MBP-MyD88 was successfully expressed and purified,anti-MyD88 monoclonal antibody was prepared.This research provided materials and foundation for further study on the function and quantification of MyD88.