摘要
旨在筛选和鉴定与鸭补体调节因子Vitronectin(Vn)结合的鸭疫里默杆菌(Riemerella anatipestifer,R.anatipestifer)外膜蛋白。在酵母表达鸭Vn及间接免疫荧光和免疫斑点杂交试验检测鸭疫里默杆菌结合Vn的基础上,以重组Vn为诱饵蛋白进行His pull-down及LC-MS/MS质谱鉴定,筛选外膜蛋白并进行生物信息学分析;原核表达候选外膜蛋白及截短片段,制备多克隆抗体,利用Far-Western blot验证与鸭Vn结合,并用间接ELISA测定肝素和氯化钠对结合的影响;测定Vn及抗外膜蛋白抗体对血清杀菌活性的影响。结果显示,鸭疫里默杆菌能在体外结合鸭Vn,经His-pull down、质谱鉴定及蛋白质结构分析,筛选出的外膜蛋白Omp45具有β桶状结构及7个暴露于细胞膜外的多肽环;成功原核表达Omp45及截短片段,制备的抗Omp45多克隆抗体间接ELISA效价超过1∶12800,Western blot检测多克隆抗体可以与重组蛋白发生特异性反应;Far-Western blot及间接ELISA结果显示,Omp45、包含2个及以上细胞膜外多肽环的截短片段同时以肝素和氯化钠敏感方式结合Vn;血清杀菌试验结果显示,鸭疫里默杆菌对缺乏Vn的健康鸭血清的抵抗力降低,在抑制补体经典激活途径条件下抗Omp45抗体也能极显著降低细菌在鸭血清中的存活率(P<0.01)。结果表明,成功筛选并鉴定了与鸭Vn结合的鸭疫里默杆菌外膜蛋白Omp45,为进一步阐明鸭疫里默杆菌致病机制奠定了基础。
To screen and identify the outer membrane proteins of Riemerella anatipestifer(R.anatipestifer)which bind to complement regulator vitronectin(Vn).Based on the expression of duck Vn in yeast and detection of R.anatipestifer binding to Vn by indirect immunofluorescence assay and immuno-dot blot hybridization assay,the recombinant Vn was used as bait protein for His pull-down assay.The outer membrane proteins were screened based on LC-MS/MS identification and bioinformatics analysis.The candidate proteins and truncated fragments were expressed in E.coli to prepare polyclonal antibodies.Far-Western blot was conducted to verify the interaction between outer membrane proteins of R.anatipestifer and duck Vn.The effects of heparin and sodium chloride on the binding were determined by indirect ELISA.The effects of Vn and outer membrane protein antibody on the survival of bacteria were determined by serum bactericidal assay.The results showed that R.anatipestifer could bind duck Vn protein in vitro.The outer membrane protein Omp45 was identified by His-pull down,mass spectrometry and protein structure analysis.Omp45 was found to have a β-barrel structure and 7 polypeptide rings exposed outside the cell membrane.The recombinant protein Omp45(rOmp45)and its truncated fragments were successfully expressed in prokaryotic cells.The anti-rOmp45 polyclonal antibody could react specifically with the recombinant protein,and its titer of indirect ELISA was more than 1:12800.The rOmp45 and its truncated fragments containing two or more polypeptide rings combined duck Vn in a heparin-and sodium chloride-sensitive manner.Serum bactericidal assay showed that the resistance of R.anatipestifer to ΔVn NDS was decreased,the anti-Omp45 antibody could also significantly reduce the survival rate of the bacteria in normal duck serum(NDS)under the condition of inhibiting the classical pathway of complement(P<0.01).This study successfully screened out and identified one outer membrane protein(Omp45)of R.anatipestifer binding to the duck Vn,which provide a basis to further study the pathogenic mechanism.
作者
何忆箫
秦豆豆
彭兰青
程中林
高继业
李继祥
HE Yixiao;QIN Doudou;PENG Lanqing;CHENG Zhonglin;GAO Jiye;LI Jixiang(College of erinary Medicine,Southwest University,Chongqing 402460,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2023年第12期2472-2479,共8页
Chinese Journal of Veterinary Science
