何忆箫,秦豆豆,彭兰青,程中林,高继业,李继祥

• 1:西南大学动物医学院

摘要(Abstract)：

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∶12 800,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。

关键词(KeyWords)： 鸭疫里默杆菌;补体调节因子;Vitronectin;外膜蛋白

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 何忆箫,秦豆豆,彭兰青,程中林,高继业,李继祥

DOI: 10.16303/j.cnki.1005-4545.2023.12.11

参考文献(References)：

• [1] 包涛涛，鲜思美，顾庆林，等.鸭疫里默氏杆菌Porin蛋白的原核表达及其间接ELISA检测方法的建立与应用[J].中国兽医学报，2022,42(9):1830-1837.
• [2] XIHUI Z,YANLAN L,ZHIWEI W,et al.Antibiotic resistance of Riemerella anatipestifer and comparative analysis of antibiotic-resistance gene detection methods[J].Poult Sci,2023,102(3):102405.
• [3] LEAVITT S,AYROUD M.Riemerella anatipestifer infection of domestic ducklings[J].Can Vet J,1997,38(2):113.
• [4] FAN M,CHEN S,ZHANG L,et al.Riemerella anatipestifer extracellular protease S blocks complement activation via the classical and lectin pathways[J].Avian Pathol,2017,46(4):426-433.
• [5] 李德龙，谭理娟，谷九龙，等.鸭C4BPα的克隆、表达及其与鸭疫里默氏菌的互作[J].生物工程学报，2020,36(4):693-699.
• [6] LI D,WANG X,XU X,et al.Duckcomplement ractor H binds to outer membrane protein Omp24 of Riemerella anatipestifer[J].Avian Dis,2021,65(2):261-268.
• [7] MERLE N,CHURCH S,FREMEAUX-BACCHI V,et al.Complementsystem part i-molecular mechanisms of activation and regulation[J].Front Immunol,2015(6):262.
• [8] SHEEHAN M,MORRIS C,PUSSELL B,et al.Complement inhibition by human vitronectin involves non-heparinbinding domains[J].Clin Exp Immunol,1995,101(1):136-141.
• [9] TSCHOPP J,MASSON D,SCHAEFER S,et al.The heparin binding domain of S-protein/vitronectin binds to complement components C7,C8,and C9 and perforin from cytolytic T-cells and inhibits their lytic activities[J].Biochemistry,1988,27(11):4103-4109.
• [10] SATO K,KUMAGAI Y,SEKIZUKA T,et al.Vitronectin binding protein,BOM1093,confers serum resistance on Borrelia miyamotoi[J].Sci Rep,2021,11(1):1-13.
• [11] HALLSTROM T,BLOM A,ZIPFEL P,et al.Nontypeable Haemophilus influenzae protein E binds vitronectin and is important for serum resistance[J].J Immunol,2009,183(4):2593-2601.
• [12] HALLSTR?M T,UHDE M,SINGH B,et al.Pseudomonas aeruginosa uses dihydrolipoamide dehydrogenase (Lpd) to bind to the human terminal pathway regulators vitronectin and clusterin to inhibit terminal pathway complement attack[J].PLoS One,2015,10(9):e0137630.
• [13] AW R,POLIZZI K.Liquid PTVA:A faster and cheaper alternative for generating multi-copy clones in Pichia pastoris[J].Microb Cell Fact,2016,15(1):1-11.
• [14] TAN Y,GAMAGE A,GAN Y.Complement-activated vitronectin enhances the invasion of nonphagocytic cells by bacterial pathogens Burkholderia and Klebsiella[J].Cell Microbiol,2017,19(8):e12732.
• [15] SIEGEL C,HALLSTR?M T,SKERKA C,et al.Complement factor H-related proteins CFHR2 and CFHR5 represent novel ligands for the infection-associated CRASP proteins of Borrelia burgdorferi[J].PloS One,2010,5(10):e13519.
• [16] KELLEY L,MEZULIS S,YATES C,et al.The Phyre2 web portal for protein modeling,prediction andanalysis[J].Nat Protoc,2015,10(6):845-858.
• [17] HALLSTR?M T,SINGH B,KRAICZY P,et al.Conserved patterns of microbial immune escape:Pathogenic microbes of diverse origin target the human terminal complement inhibitor vitronectin via a single common motif[J].PLoS One,2016,11(1):e0147709.
• [18] HOVINGH E,VAN DEN BROEK B,JONGERIUS I.Hijacking complement regulatory proteins for bacterial immune evasion[J].Front Microbiol,2016(7):2004.
• [19] HALLSTR?M T,ZIPFEL P,BLOM A,et al.Haemophilus influenzae interacts with the human complement inhibitor factor H[J].J Immunol,2008,181(1):537-545.
• [20] THOFTE O,BETTONI S,SU Y,et al.Nontypeable Haemophilus influenzae P5 binds human C4b-binding protein,promoting serum resistance[J].J Immunol,2021,207(6):1566-1577.
• [21] RILEY S,PATTERSON J,NAVA S,et al.Pathogenic Rickettsia species acquire vitronectin from human serum to promote resistance to complement-mediated killing[J].Cell Microbiol,2014,16(6):849-861.
• [22] FISH A,RILEY S,SINGH B,et al.The Rickettsia conorii Adr1 interacts with the C-terminus of human vitronectin in a salt-sensitive manner[J].Front Cell Infect Microbiol,2017(7):61.
• [23] BARTRA S,DING Y,FUJIMOTO L,et al.Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin[J].Microbiology,2015,161(11):2174-2183.