Evaluation of immunogenicity of Streptococcus equi ssp. zooepidemicus surface protein FIPP
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摘要: 【目的】评价马链球菌兽疫亚种(Streptococcus equi ssp. zooepidemicus,SEZ)表面蛋白FIPP(纤维蛋白原和Ig结合蛋白前体)的免疫效力,为预防SEZ感染提供新的候选疫苗抗原。【方法】根据NCBI数据库公布的FIPP基因序列设计引物进行PCR扩增,将扩增片段克隆到原核表达载体pColdⅠ,并转化至大肠杆菌BL21(DE3)感受态细胞,FIPP蛋白通过IPTG诱导在大肠杆菌中表达及纯化,以SDS-PAGE进行鉴定。纯化后的重组蛋白FIPP(rFIPP)对BALB/c小鼠进行免疫,二免14 d后对小鼠进行免疫保护试验并采集血清;解剖感染SEZ小鼠的肝脏、脾脏和肺脏进行病理组织学观察。以酶联免疫吸附试验(ELISA)方法检测小鼠血清中的特异性抗体IgG水平,以蛋白质印迹(Western blotting)方法检测重组蛋白的反应原性,通过全血杀菌试验检测血清的杀菌活性。【结果】通过SDS-PAGE电泳可观察到约96 k D的蛋白片段,与FIPP蛋白的理论分子量相符。重组蛋白rFIPP免疫小鼠后对SEZ攻击的保护率达70.0%。ELISA方法检测结果表明,重组蛋白rFIPP免疫小鼠后可诱导产生特异性抗体免疫球蛋白G(IgG),且极显著高于阴性对照(P<0.01,下同),通过IgG亚型分析,发现IgG1的抗体水平显著高于IgG2a(P<0.05),说明重组蛋白rFIPP免疫后产生的抗体亚型以IgG1为主。全血杀菌试验结果表明,重组蛋白rFIPP超免血清对SEZ具有极显著杀灭作用。组织病理学观察结果显示,重组蛋白rFIPP免疫后能有效减弱小鼠各类器官的病理损伤。【结论】经表达纯化的重组蛋白rFIPP在小鼠感染SEZ模型中具有良好的免疫保护效力,可用作制备SEZ亚单位疫苗的有效候选抗原。Abstract: 【Objective】This study aimed to investigate the immune efficacy of the surface protein FIPP(fibrinogenand Ig-binding protein precursor) of Streptococcus equi ssp. zooepidemicus(SEZ) and to provide a new candidate vaccine antigen for SEZ. 【Method】According to the FIPP gene sequence published in the NCBI database, the primer was designed for PCR amplification, and the amplified fragment was cloned into the prokaryotic expression vector pColdⅠ, and then transformed into Escherichia coli BL21(DE3) receptor cells. The FIPP protein was expressed and purified in E. coli through IPTG induction. Identification by SDS-PAGE. BALB/c mice were immunized with purified recombinant FIPP(rFIPP). 14 d later, the mice were immunologically protected and serum was collected. The liver, spleen and lungs of the mice infected with SEZ were dissected for histopathological observation. Enzyme-linked immunosorbent assay(ELISA) was used to detect specific antibody IgG levels in serum of mice, the reactivity of recombinant protein was detected by Western blotting analysis, and the bactericidal activity of serum was detected by whole blood bactericidal test. 【Result】The protein fragments of about 96 kD were observed by SDS-PAGE electrophoresis, which was consistent with the theoretical molecular weight of FIPP protein. The protection rate of rFIPP against SEZ attack was 70.0% in mice immunized with RFIPP. ELISA assay results showed that rFIPP immunized mice with high titer specific antibody immunoglobulin G(IgG), which was extremely significantly higher than that of negative control(P<0.01, the same below). By IgG subtype analysis, the antibody level of IgG1 was significantly higher than that of IgG2a(P<0.05). These results indicated that IgG1 was the main antibody subtype produced after rFIPP immunization. The results of whole blood bactericidal test showed that rFIPP superimmune serum had an extremely significant killing effect on SEZ. The histopathological observation showed that rFIPP immunization could effectively reduce the pathological damage of various organs in mice. 【Conclusion】In this study,recombinant protein rFIPP obtained by expression purification has good immunoprotective efficacy in a mouse infecting SEZ model and can be used as a valid candidate antigen for SEZ subunit vaccine.
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Keywords:
- Streptococcus equi ssp. zooepidemicus /
- FIPP protein /
- immune protection /
- evaluation
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耿建新,陆承平,范红结.2007.马链球菌兽疫亚种10株国内猪源分离株对小鼠免疫效力的评价[J].中国人兽共患病学报,23(2):201-204.[Geng J X,Lu C P,Fan H J.2007.Evaluation on the immunization potency of 10 strains of Streptococcus equi subsp.zooepidemicus of pig-orgin iso-lated in China[J].Chinese Journal of Zoonoses,23(2):201-204.] 尚天甜,彭婕,鲁强生,刘旭明,曾巧英.2022.内肽酶O对马链球菌兽疫亚种突破血脑屏障的影响[J].中国兽医学报,42(10):2022-2030.[Shang T T,Peng J,Lu Q S,Liu X M,Zeng Q Y.2022.Role of endopeptidase O on the breakthrough of the blood-brain barrier of Strep-tococcus equi ssp.zooepidemicus[J].Chinese Journal of Veterinary Science,42(10):2022-2030.]doi: 10.16303/j.cnki.1005-4545.2022.10.15. 邵俊高,姜慧娇,常建新,张宝江,李善春,苏艳.2016.马链球菌马亚种Ig G结合蛋白的原核表达和免疫原性[J].生物工程学报,32(5):577-583.[Shao J G,Jiang H J,Chang JX,Zhang B J,Li S C,Su Y.2016.Prokaryotic expression and immunogenicity of Ig G-binding protein of Streptococ-cus equi subspecies equi[J].Chinese Journal of Biotech-nology,32(5):577-583.]doi: 10.13345/j.cjb.150405. 孙琼飞,张家瑞,屈勇刚,梁晏.2023.1株豚鼠源马链球菌兽疫亚种的分离鉴定与耐药性分析[J].中国兽医学报,43(5):993-997.[Sun Q F,Zhang J R,Qu Y G,Liang Y.2023.Isolation and identification of a Streptococus equi subspecies of guinea pig origin and analysis of drug resis-tance[J].Chinese Journal of Veterinary Science,43(5):993-997.]doi: 10.16303/j.cnki.1005-4545.2023.05.21. 汪梦竹,蒲飞洋,赵泽阳,冯茜莉,李易聪,王慧慧,马忠仁,李倬.2022.新型疫苗的研究进展[J].畜牧与兽医,54(9):141-147.[Wang M Z,Pu F Y,Zhao Z Y,Feng X L,Li YC,Wang H H,Ma Z R,Li Z.2022.Advances in develop-ment of novel types of vaccines[J].Animal Husbandry&Veterinary Medicine,54(9):141-147.] 徐全圆,石明,杨夷平,李娜,刘建华,加尔肯,冉多良.2015.马链球菌马亚种FNEB蛋白的截短表达及其免疫保护性研究[J].中国预防兽医学报,37(2):132-135.[Xu Q Y,Shi M,Yang Y P,Li N,Liu J H,Jia E K,Ran D L.2015.Expression and immunogenicity identification on trun-cated FNEB protein of Streptococcus equi subsp.equi[J].Chinese Journal of Preventive Veterinary Medicine,37(2):132-135.]doi: 10.3969/j.issn.1008-0589.2015.02.12. 徐宇萌,郭政,李亚娟,邓静飞,何颍欣,黄云飞,付强.2023.马链球菌兽疫亚种Scl E蛋白原核表达及其免疫效力评价[J].南方农业学报,54(8):2465-2473.[Xu Y M,Guo Z,Li Y J,Deng J F,He Y X,Huang Y F,Fu Q.2023.Pro-karyotic expression of Scl E protein from Streptococcus equi ssp.zooepidemicus and evaluation on its immune effi-cacy[J].Journal of Southern Agriculture,54(8):2465-2473.]doi: 10.3969/j.issn.2095-1191.2023.08.028. 张泽华,张欢,汪丽,古丽米热·对山巴依,吕芬芬,蒲小峰,张宝江,苏艳.2021.马源马链球菌兽疫亚种3株新疆分离株基因型的鉴定及MLST分析[J].微生物学通报,48(12):4742-4755.[Zhang Z H,Zhang H,Wang L,Guli-mire D,LüF F,Pu X F,Zhang B J,Su Y.2021.Identifica-tion and MLST analysis of three novel genotypes of Strep-tococcus equi subsp.zooepidemicus Xinjiang strains[J].Microbiology China,48(12):4742-4755.]doi: 10.13344/j.microbiol.china.210337. Agarwal V,Sroka M,Fulde M,Bergmann S,Riesbeck K,Blom A M.2014.Binding of Streptococcus pneumoniae endo-peptidase O(Pep O) to complement component C1q modu-lates the complement attack and promotes host cell adherence[J].The Journal of Biological Chemistry,289(22):15833-15844.doi: 10.1074/jbc.M113.530212.
Ahmed E A,Penfound T A,Brewer S C,Tennant P A,Chiang EY,Dale J B.2010.Streptococcal protective antigens (Spa):A new family of type-specific proteins of group A strepto-cocci[J].European Journal of Clinical Microbiology&Infectious Diseases,29(1):51-57.doi: 10.1007/s10096-009-0819-0.
Baracco G J.2019.Infections caused by group C and G strepto-coccus(Streptococcus dysgalactiae subsp.equisimilis and Others):Epidemiological and clinical aspects[J].Microbiology Spectrum,7(2):10.1128.doi: 10.1128/microbiol-spec.GPP3-0016-2018.
Boyle A G,Timoney J F,Newton J R,Hines M T,Waller A S,Buchanan B R.2018.Streptococcus equi infections in horses:Guidelines for treatment,control,and prevention of strangles-revised consensus statement[J].Journal of Veterinary Internal Medicine,32(2):633-647.doi: 10.1111/jvim.15043.
Chen X,Zhao Y,Su L,Wang L,Ma X,Zhang B,Su Y.2021.Enhanced immune effects and protection conferred by simultaneously targeting GAPDH,Se M,and EAG of S.equi via TLR4[J].Research in Veterinary Science,138:100-108.doi: 10.1016/j.rvsc.2021.06.001.
Godehardt A W,Hammerschmidt S,Frank R,Chhatwal G S.2004.Binding of α2-macroglobulin to GRAB(protein G-related α2-macroglobulin-binding protein),an important virulence factor of group A streptococci,is mediated by two charged motifs in the Delta A region[J].The Biochemi-cal Journal,381:877-885.
Gruszynski K,Young A,Levine S J,Garvin J P,Brown S,Turner L,Fritzinger A,Gertz R E,Murphy J M,Vogt M,Beall B.2015.Streptococcus equi subsp.zooepidemicus infections associated with Guinea pigs[J].Emerging Infec-tious Diseases,21(1):156-158.doi: 10.3201/eid2101.140640.
Guss B,Flock M,Frykberg L,Waller A S,Robinson C,Smith K C,Flock J I.2009.Getting to grips with strangles:An effective multi-component recombinant vaccine for the protection of horses from Streptococcus equi infection[J].PLo S Pathogens,5(9):e1000584.doi: 10.1371/journal.ppat.1000584.
Hau S J,Buckley A,Brockmeier S L.2022.Bacterin vaccina-tion provides insufficient protection against Streptococcus equi subspecies zooepidemicus infection in pigs[J].Fron-tiers in Veterinary Science,9:827082.doi: 10.3389/fvets.2022.827082.
Hulting G,Flock M,Frykberg L,Lannergard J,Flock J I,Guss B.2009.Two novel Ig G endopeptidases of Streptococcus equi[J].FEMS Microbiology Letters,298(1):44-50.doi: 10.1111/j.1574-6968.2009.01698.x.
Keane F M,Loughman A,Valtulina V,Brennan M,Speziale P,Foster T J.2007.Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A,an MSCRAMM of Staphylococcus aureus[J].Molecu-lar Microbiology,63(3):711-723.doi: 10.1111/j.1365-2958.2006.05552.x.
Kerdsin A,Chopjitt P,Hatrongjit R,Boueroy P,Gottschalk M.2022.Zoonotic infection and clonal dissemination of Strep-tococcus equi subspecies zooepidemicus sequence type194 isolated from humans in Thailand[J].Transboundary and Emerging Diseases,69(4):e554-e565.doi: 10.1111/tbed.14331.
Lannergard J,Flock M,Johansson S,Flock J I,Guss B.2005.Studies of fibronectin-binding proteins of Streptococcus equi[J].Infect Immun,73(11):7243-7251.doi: 10.1128/IAI.73.11.7243-7251.2005.
Lee H,Yun S H,Hyon J Y,Lee S,Yi Y,Choi C,Jun S,Park EC,Kim S I.2021.Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infection[J].Veterinary Microbiology,259:109165.doi: 10.1016/j.vetmic.2021.109165.
Li Z,Ma Z,Li Y,Gao S,Xiao S.2020.Porcine epidemic diar-rhea virus:Molecular mechanisms of attenuation and vac-cines[J].Microbial pathogenesis,149:104553.doi: 10.1016/j.micpath.2020.104553.
Liang H,Tang B,Zhao P,Deng M,Yan L L,Zhai P,Wei Z G.2018.Identification and characterization of a novel protec-tive antigen,Sec_205 of Streptococcus equi ssp.Zooepi-demicus[J].Vaccine,36(6):788-793.doi: 10.1016/j.vac-cine.2017.12.072.
Lin H X,Huang D Y,Wang Y,Lu C,Fan H.2011.A novel vac-cine against Streptococcus equi ssp.zooepidemicus infec-tions:The recombinant swinepox virus expressing M-like protein[J].Vaccine,29(40):7027-7034.doi: 10.1016/j.vaccine.2011.07.074.
Lin Z H,Xia Y,Guo J F,Xu G,Liu Y,Yang Y,Xie H,Huang Y,Fu Q.2022.Caspase-1 deficiency impairs neutrophils recruitment and bacterial clearance in Streptococcus equi ssp.zooepidemicus infected mice[J].Veterinary Microbiol-ogy,268:109411.doi: 10.1016/j.vetmic.2022.109411.
Lord J,Carter C,Smith J,Locke S,Phillips E,Odoi A.2022.Antimicrobial resistance among Streptococcus equi subspe-cies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky,USA[J].Peerj,10:e13682.doi: 10.7717/peerj.13682.
Madžar D,Hagge M,Möller S,Regensburger M,Lee D,Schwab S,Jantsch J.2015.Endogenous endophthalmitis complicating Streptococcus equi subspecies zooepidemicus meningitis:A case report[J].BMC Research Notes,8(1):184.doi: 10.1186/s13104-015-1133-9.
Mitchell A M,Mitchell T J.2010.Streptococcus pneumoniae:Virulence factors and variation[J].Clinical Microbiology and Infection,16(5):411-418.doi: 10.1111/j.1469-0691.2010.03183.x.
Tang F,Li D Z,Wang H J,Ma Z,Lu C,Dai J.2015.Prophage lysin ply30 protects mice from Streptococcus suis and Streptococcus equi subsp.zooepidemicus infections[J].Applied and Environmental Microbiology,81(21):7377-7384.doi: 10.1128/AEM.02300-15.
Timoney J F,Qin A,Muthupalani S,Artiushin S.2007.Vaccine potential of novel surface exposed and secreted proteins of Streptococcus equi[J].Vaccine,25(30):5583-5590.doi: 10.1016/j.vaccine.2007.02.040.
Valentin W P,Traore M Y,Blobel H,Chhatwal G S.1990.Role of alpha 2-macroglobulin in phagocytosis of group Aand C streptococci[J].Fems Microbiology Letter,58(3):321-324.doi: 10.1111/j.1574-6968.1990.tb13997.x.
Voyich J M,Vuong C,De Wald M,Nygaard T K,Kocianova S,Griffith S,Jones J,Iverson C,Sturdevant D E,Braughton K R,Whitney A R,Otto M,De Leo F R.2009.The Sae R/Sgene regulatory system is essential for innate immune eva-sion by Staphylococcus aureus[J].The Journal of Infec-tious Diseases,199(11):1698-1706.doi: 10.1086/598967.
Wei Z G,Fu Q,Chen Y S,Li M,Cong P,Mo D,Liu X.2013.Streptococcus equi ssp.zooepidemicus C5a peptidase,a putative invasin,induces protective immune response in mice[J].Research in Veterinary Science,95(2):444-450.doi: 10.1016/j.rvsc.2013.03.026.
Xie H L,Wei Z G,Ma C Q,Li S,Liu X,Fu Q.2018.Character-ization of Sese C_01411 as a surface protective antigen of Streptococcus equi ssp.zooepidemicus[J].Research in Veterinary Science,118:517-521.doi: 10.1016/j.rvsc.2018.05.007.
Xu G,Guo Z,Liu Y,Yang Y,Lin Y,Li C,Huang Y,Fu Q.2022.Gasdermin D protects against Streptococcus equi subsp.zooepidemicus infection through macrophage pyrop-tosis[J].Frontiers in Immunology,13:1005925.doi: 10.3389/fimmu.2022.1005925.
Yi L,Wang Y,Ma Z,Lin H X,Xu B,Grenier D,Fan H J,Lu CP.2016.Identification and characterization of a Streptococ-cus equi ssp.zooepidemicus immunogenic Gro EL protein involved in biofilm formation[J].Veterinary Research,47(1):1-9.doi: 10.1186/s13567-016-0334-0.
Yi L,Wang Y,Ma Z,Zhang H,Li Y,Zheng J X,Yang Y C,Lu C P,Fan H J.2013.Contribution of fibronectin-binding protein to pathogenesis of Streptococcus equi ssp.zooepi-demicus[J].Pathogens and Disease,67(3):174-183.doi: 10.1111/2049-632X.12029.
Zhou Y,Yan K,Sun C F,Liu F,Peng W,Chen H C,Yuan F Y,Bei W C,Li J Q.2021.Binding of plasminogen to Strepto-coccus suis protein endopeptidase O facilitates evasion of innate immunity in Streptococcus suis[J].Frontiers in Microbiology,12:694103.doi: 10.3389/fmicb.2021.694103.
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