鸭疫里默氏杆菌OmpA/motb蛋白的表达纯化及免疫学特性分析  

PROKARYOTIC EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF RECOMBINANT RIEMERELLA ANATIPESTIFER OMPA/MOTB PROTEIN

作　　者：张雪梅[1,2] 王小兰 任晓梅[2] 丁铲 窦亚峰[2] 李涛 王桂军[1] 于圣青[2] 

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2018年第1期37-42,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划项目(2016YFD0500800)

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1:256 000;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 identi？ed 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 ampli？ed 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 con？rmed 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 speci？c 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.

关 键 词：鸭疫里默氏杆菌 rOmpA/motb蛋白 多克隆抗体 免疫学特性 

分 类 号：S852.615[农业科学—基础兽医学]