鸭疫里默氏杆菌OmpA/motb蛋白的表达纯化及免疫学特性分析

摘要

本研究对血清1型鸭疫里默氏杆菌(Riemerella anatipestifer,RA)CH3株的ompA/motb基因核苷酸序列及其蛋白进行生物学信息分析,用PCR技术扩增获得去除信号肽编码区段的ompA/motb基因,并构建重组表达质粒pET30a-OmpA/mtob,成功表达分子量约为55 kDa的重组蛋白rOmpA/motb.Western blot结果显示,rOmpA/motb能与血清1、2、10和15型RA阳性血清发生特异性反应,表明rOmpA/motb具有交叉免疫原性.以纯化的rOmpA/motb免疫新西兰大白兔,经3次免疫后兔血清中抗OmpA/motb蛋白的抗体效价为1:256000;Western blot结果显示OmpA/motb兔多抗可与血清1型、2型、10型和15型RA菌株发生特异性结合反应,与其他菌株无反应性.本研究制备的RA OmpA/motb多克隆抗体具有良好的反应性及特异性,为进一步研究OmpA/motb蛋白的免疫学功能及建立鸭疫里默氏杆菌病的诊断方法奠定了基础.%The OmpA/motb domain protein(OmpA/motb)has been identified as an immunogenic antigen of Riemerella anatipestifer by using immunoproteomics analysis.In this study,OmpA/motb of R.anatipestifer was expressed and recombinant protein was evaluated for its immunogenicity.The ompA/motb gene was amplified from serotype 1 strain CH3 of R.anatipestifer by polymerase chain reaction (PCR)and cloned into expression vector pET-30a(+).The pET30a-OmpA/motb plasmid was constructed and transformed into E.coli strain BL21(DE3).The recombinant OmpA/motb(rOmpA/motb)was expressied in E.coli with induction of IPTG and confirmed to be a recombinant protein of 55 kDa in SDS-PAGE with Coomassie blue staining. The recombinant protein rOmpA/motb reacted with duck antisera against R.anatipestifer CH3,NJ3,HXb2 and LAG1 in Western blot,suggesting it was a cross-immunogenic protein among R.anatipestifer strains of different serotypes.Subsequently,rabbit anti-OmpA/motb antibodies were generated by immunization of rabbits with purified rOmpA/motb. The titer of antiserum was assessed to be over 1:256 000 in ELISA. The rabbit anti-OmpA/motb serum reacted with R.anatipestifer CH3,NJ3,HXb2 and LAG1 in Western blotting but did not react with avian pathogenic E.coli,Salmonella enterica and Pasteurella multocida strains.The results showed that rabbit anti-OmpA/motb serum was specific to and cross-reactive with R. anatipestifer strains of serotypes 1,2,10 and 15,suggesting it might be used for developing diagnostic kits in the further study.