支原体(Mycoplasma)属于原核生物界柔膜体纲支原体科,普遍存在于自然界,有200多个种,其中动物支原体多达几十种[1]。兽医上最重要的支原体有丝状支原体丝状亚种(牛传染性胸膜肺炎-牛肺疫病原)、猪肺炎支原体(猪地方性肺炎病原)、山羊支原体山羊肺炎亚种(山羊传染性胸膜肺炎病原)、鸡毒支原体等[2],此外,牛支原体、滑液支原体等对养殖业的危害日益严重,越来越受到重视。动物支原体感染主要表现为慢性、持续性,难以根治,已成为威胁养殖业发展的重要病原。
目前,动物支原体病的防控主要依赖疫苗和抗菌药物,抗菌药使用存在菌株耐药性增加[3-7]、药物残留、病原很难被彻底清除等问题,临床上应用效果很不理想。疫苗是防控动物支原体病的重要手段,目前,主要为灭活疫苗和弱毒疫苗[8-10]。亚单位疫苗具有安全高效、成本低廉等优点,是支原体病疫苗研发的一个重要发展方向。目前,已对多种支原体病亚单位疫苗进行研究,病原包括丝状支原体丝状亚种(Mycoplasma mycoides subsp. Mycoides,Mmm)、猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)、牛支原体(Mycoplasma bovis,M. bovis)、鸡毒支原体(Mycoplasma gallisepticum,MG)和山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp. capripneumoniae,Mccp)等,但迄今未有商业化的动物支原体病亚单位疫苗。笔者对这几种重要动物支原体病相关蛋白的免疫原性进行综述,以期为动物支原体的免疫和疫苗研究提供参考。
1 丝状支原体丝状亚种Mmm是牛传染性胸膜肺炎病原,是最重要的动物支原体之一。Mmm免疫蛋白研究较为深入,部分免疫蛋白已试制亚单位疫苗,并进行了牛体免疫保护效果评价。应用蛋白组学方法鉴定了Mmm一些免疫蛋白和毒力相关蛋白[11-12],如H2O2产生和荚膜合成相关蛋白。L-α-甘油磷酸盐氧化酶(GlpO)是支原体H2O2产生的重要酶,是Mmm重要的毒力因子和免疫蛋白,GlpO重组蛋白混合弗氏完全佐剂免疫牛,可诱导产生抗体,却不能保护Mmm感染[13]。跨膜脂蛋白LppQ被认为是Mmm的主要抗原和毒力因子,Mulongo等[14]用纯化重组蛋白LppQ-N混合弗氏佐剂免疫牛,疫苗组表现出血清转化强阳性,但与对照组相比,临床症状和大体病变评分无显著差异,且在攻毒后肾小球肾炎增强,提示LppQ-N不适合作为亚单位疫苗抗原。对Mmm 4种脂蛋白LppA、LppB、LppC和LppQ的研究发现,虽然它们均能诱导体液免疫,但是体内试验显示仅LppA能被淋巴细胞识别,诱导产生IFN-γ,表明LppA是CD4+ T细胞抗原,可诱导细胞免疫,可作为潜在疫苗抗原[15]。荚膜多糖(CPS)是Mmm一种重要的毒力因子,CPS偶联卵清蛋白免疫牛能诱导牛产生特异性免疫反应,能够减轻病变,具有一定的免疫保护作用[16]。Perez-Casal等[17]通过反向疫苗学和基因组分析方法系统鉴定了66个潜在亚单位疫苗候选抗原,将这些蛋白表达后按其被牛传染性胸膜肺炎阳性牛血清识别的能力强弱进行打分排序和分组,每组4~5个重组蛋白,配以CpG2007 ODN和30% EmulsigenTM佐剂,制成疫苗免疫小牛,检测发现在第35天各组针对每种蛋白的IgG1效价比免疫前显著升高,且高于对照组,每种蛋白的IgG1滴度彼此之间无显著差异;大多数蛋白可诱导IgG2和IgG1反应,无免疫干扰;通过PBMC增殖试验来检测细胞免疫应答,发现所有蛋白的抗原刺激指数与对照组之间无显著差异,提示制备亚单位疫苗不能很好诱导细胞免疫。攻毒保护试验发现A组(MSC-0136+MSC-0957+MSC-0499+ MSC-0431+ MSC-0776,假定脂蛋白组)和C组(假定蛋白YP 004400559.1+假定蛋白YP 004399807.1 + Vsp+TE-Tu+ 脂蛋白MSC-0775)亚单位疫苗能够提供保护,其中疫苗C组肺分离不到病原,N组(YP004400127.1+YP004399790.1+YP004400580.1+YP004400610.1) 疫苗组可减轻病变,且肺病原分离阴性;其他重组蛋白疫苗组反而加重了病变;A组、C组和N组疫苗保护率分别为79.2%、83.0%和73.3%,结果表明,重组蛋白多组分亚单位疫苗可成功用于预防牛传染性胸膜肺炎。研究提示,抗体介导的体液免疫在牛传染性胸膜肺炎亚单位疫苗免疫保护中发挥主要作用[18]。
2 牛支原体国内外研究者做了大量的工作,鉴定出多个牛支原体免疫蛋白,如GAPDH、PdhA、Tuf、P48、P81等。最近郭爱珍教授团队通过基因组预测分析结合分泌蛋白组学方法,发现牛支原体HB0801株有60个分泌蛋白,分泌蛋白MbovP0581是一种ABC转运蛋白,具有强的免疫原性,能与牛支原体感染血清反应,有作为疫苗抗原的潜力[19]。一些牛支原体蛋白的免疫应答反应和免疫保护作用已被评估。GAPDH在牛支原体菌株中非常保守,参与糖酵解过程,被认为是牛支原体的一种毒力因子和免疫保护抗原。Prysliak等[20]将牛支原体GAPDH与宿主防御肽组成嵌合蛋白Gap-I、嵌合蛋白Gap-I和牛支原体提取物作为疫苗抗原,与CpG2007佐剂混合制成亚单位疫苗,结果显示亚单位疫苗可引起强烈的体液免疫,而细胞免疫较弱,不具有免疫保护作用。Mulongo等[21]将两株牛支原体总蛋白和/或膜蛋白结合CpG ODN2007佐剂制备成疫苗,发现总蛋白和膜蛋白混合组能显著引起IgG1血清学反应,总蛋白组诱导明显的IgG2反应,但是均不能引起淋巴细胞记忆性应答;攻毒后免疫组与对照组在临床症状和病原分布无显著差异,表明研制疫苗不具有免疫保护作用。Prysliak等[22]应用SDS-PAGE、2D结合免疫印迹方法,鉴定出PdhA、Tuf、PepA、P48、P81、DeoB、OppA、LppB、O256、PepQ,它们可作为潜在疫苗候选蛋白,将这些重组蛋白、膜蛋白、总蛋白配合佐剂TriAdj后免疫犊牛,重组蛋白可产生IgG1或IgG2反应,但没有诱导T细胞免疫应答,不具有免疫保护作用。Th17细胞免疫应答在多种支原体的抗感染中发挥重要作用,如肺支原体(M. pulmonis)[23]、肺炎支原体(M. pneumoniae)[24]等。研究者使用MontanideTM ISA 61VG和Th17应答诱导剂凝胶多糖为佐剂,牛支原体膜组分、总蛋白和9个重组蛋白(PdhA、Tuf、PepA、LppB、O256、OppA、DeoB、P81、PepQ)组成混合抗原,制成疫苗免疫犊牛,发现除膜组分外,其余抗原刺激PBMC均能引起Th17细胞反应;该疫苗能够诱导体液免疫反应,血清中IgG1、IgG2和IgA水平较对照组显著上升;但疫苗组仅能轻微减轻牛支原体引起的肺部病变和体重下降,不具有免疫保护作用,表明该疫苗诱导的Th17细胞免疫应答不能对牛支原体感染提供有效保护[25]。牛支原体具有膜表面抗原高频率变异、规避吞噬细胞吞噬作用、入侵宿主细胞、形成生物被膜以及免疫调节作用等方面逃避宿主天然和适应性免疫[9, 26-28],使得亚单位疫苗的研制面临诸多挑战。本实验室近期研究发现牛支原体生物被膜与浮游细胞共有的免疫原性蛋白,如endoglucanase、thiol peroxidase、MilA,有可能作为潜在疫苗靶标预防牛支原体急性感染和生物被膜感染[29]。最近发现,针对膜蛋白P81和UgpB的多抗能够抑制牛支原体的生长,提示膜蛋白P81和UgpB可作为潜在疫苗候选抗原[30]。到目前为止,一些免疫原性蛋白被鉴定并进行了牛体验证,可引起强烈的以抗体为介导的体液免疫,但都不具有免疫保护作用。细胞免疫在牛支原体免疫中发挥着重要作用,Th17细胞免疫不具有免疫保护作用。因此,鉴定有效新抗原或抗原组合,平衡Th1/Th2细胞免疫,对于牛支原体病亚单位疫苗研发非常重要。
3 猪肺炎支原体目前,已鉴定了一些猪肺炎支原体免疫蛋白,如P36、P42、P46、P95、P97、P102、P116、Nrdf等,可作为疫苗候选抗原。刘茂军等[31]原核表达了猪肺炎支原体P65基因,发现P65重组蛋白具有良好的免疫原性。Galli等[32]发现猪肺炎支原体p42和p95能够在BALB/c小鼠诱导细胞和体液免疫反应,对猪肺炎支原体7个重组蛋白在小鼠的免疫反应发现,能诱导IgG1和IgG2a抗体反应。马丰英等[33]重组表达了猪肺炎支原体的主要免疫蛋白p36、p46、p65和p97R1-Nrdf,重组蛋白组合制备亚单位疫苗,检测发现疫苗组(p36、p46、p65和p97R1-Nrdf)能够诱导小鼠产生很强的体液免疫反应,血清中抗体水平高于常规疫苗,同时,也激活了细胞免疫,IFN-γ水平显著高于常规疫苗组。对猪肺炎支原体的P1C、P116 N、P30评价,串联嵌合这3个蛋白的MP559能够引起较高水平的体液免疫反应[34]。巴西学者构建了猪肺炎支原体rP97R1P46P95P42嵌合抗原亚单位疫苗,发现在小鼠试验中具有良好的免疫原性,可作为一种潜在的疫苗[35]。猪肺炎支原体表面黏附素P97蛋白的C端重复区域R1和R1R2,与大肠杆菌不耐热肠毒素B亚基融合形成嵌合蛋白rLTBR1和rLTBR1R2,与IMS 1113佐剂混合乳化后免疫小鼠,每个重组蛋白均能诱导抗R1特异性的体液抗体(IgG)、黏膜抗体(IgG和IgA)和IFN-γ的产生,嵌合蛋白rLTBR1R2在重组蛋白中引起最快的体液抗体应答,有作为亚单位疫苗的前景[36]。很多猪肺炎支原体亚单位疫苗研究在小鼠中进行免疫保护效果评价,并未进行猪体试验。小鼠并不能代表猪体免疫反应,一些在小鼠上具有潜力的亚单位疫苗需要进行猪体试验验证。对P97/P102类似物亚单位疫苗猪体免疫发现,尽管可以诱导很强的体液免疫,保护纤毛免受损伤,肺组织病变却加重,不具有免疫保护作用[37]。热休克蛋白P42重组蛋白在猪体能诱导细胞免疫和体液免疫反应,有作为亚单位疫苗候选蛋白的潜力[38]。目前,一些免疫蛋白或片段具有免疫原性,可作为亚单位疫苗的候选抗原,但免疫保护效果都不理想。鉴定新的有效疫苗候选蛋白或多抗原蛋白组合(或嵌合)是猪支原体肺炎亚单位疫苗研究的一个重要发展方向[39]。
4 鸡毒支原体鸡毒支原体的一些疫苗候选免疫蛋白已被鉴定,如GroEL、EF-Tu、greA、PDHC、DnaK、P67 (pMGA)、P52[40]等;通过免疫蛋白组学发现,EF-Tu、greA、PDHC、DnaK、GroEL等具有良好的免疫原性。对GroEL蛋白进一步研究发现,其具有ATPase活性,参与MG PrpC蛋白的重折叠。补体依赖杀菌试验表明,MG rGroEL的兔抗血清具有良好的杀菌效果,与灭活疫苗诱导的抗血清相似,提示MG GroEL是一种保护性抗原,可作为MG亚单位疫苗候选抗原[41]。MGC1和MGC2是MG重要的黏附因子,MGC1和MGC2重组蛋白混合油佐剂制成亚单位疫苗,不同浓度重组蛋白免疫家禽,ELISA结果显示,两种蛋白制剂均引起较强的免疫应答,且不同剂量反应水平接近,证明MGC1和MGC2重组蛋白具有良好的免疫原性,能诱导产生抗体,但却不具有免疫保护作用[42]。
5 山羊支原体山羊肺炎亚种随着Mccp基因组解析[43-45]和免疫蛋白组学等技术的发展,一些潜在的疫苗抗原被发现,如PDHC、HSP70、转酮醇酶、延长因子G、LDH、NAD家族蛋白、NADPH、Ef-Tu等[46-47],有作为亚单位疫苗候选抗原的潜力。GlpO是Mccp的一个毒力因子,具有作为疫苗抗原和治疗靶标的潜力[48]。然而最新研究发现GlpO存在于包括Mccp和Mmm SC等在内的所有丝状支原体簇成员的细胞质,GlpO并不是丝状支原体簇理想的保护性应答的候选抗原[49]。PDHA、PDHB、PDHC是Mccp主要的免疫原性蛋白,预测并筛选其相关的B细胞表位和T细胞表位,构建多重抗原肽(MAP),原核表达后免疫小鼠,结果显示3种抗原均能和小鼠免疫血清发生很好的反应,免疫血清具有体外代谢抑制作用;淋巴细胞增殖试验显示,当用单个T细胞表位及Mccp超声破碎抗原分别刺激免疫小鼠的淋巴细胞时均产生明显的增殖现象,免疫小鼠的IFN-γ、TNF-α、IL-1和IL-10产量明显升高,而IL-12产量明显下降。研究表明,构建的MAP能够引起小鼠的体液和细胞免疫反应[50]。
6 动物支原体病亚单位疫苗研发的主要难题动物支原体病亚单位疫苗研发尚处于探索初期,国内外尚未见商业化亚单位疫苗问世。以下是阻碍亚单位疫苗研发的3个主要难题:
1) 抗原设计:支原体病亚单位疫苗研发,首先需要确定支原体的保护性抗原基因。支原体保护性免疫成分主要包括参与病原黏附与致病的成分。细胞免疫和黏膜免疫在支原体保护性免疫应答中往往起重要作用。因此,在亚单位疫苗抗原选择时应充分考虑到细胞免疫与黏附免疫相关抗原和表位。一些保护性抗原中可能存在参与免疫逃避、免疫病理损伤、诱导免疫副反应或自身免疫应答的表位,因此在设计时需要对相应表位进行修饰或删除;在抗原中添加靶向修饰序列,如内质网腔靶向转运的KDAEL基序,可增强抗原的免疫效果[51]。由于支原体无细胞壁结构,膜蛋白是其主要免疫原之一,膜抗原包括糖蛋白、脂蛋白、热休克蛋白等,是支原体主要的免疫抗原[52]。
2) 抗原的高效递送系统:在基因工程亚单位疫苗的研制中,需要根据表达抗原的特点选择合适的表达系统;目前,基因工程亚单位疫苗的表达系统有原核生物、酵母、昆虫细胞、哺乳动物细胞、植物细胞表达系统等;在选择表达系统时需注意所表达抗原是否有糖基化等翻译后修饰。由于原核表达系统缺乏相关的翻译后修饰,会影响蛋白抗原的生物学活性和免疫原性。此外,需要根据重组蛋白的免疫原形式确定表达系统,如目的蛋白是单体形式还是多聚体形式。另外,在设计和构建重组表达载体时,可以将佐剂与免疫原融合表达,提高细胞免疫和体液免疫[51]。
3) 细胞免疫、黏膜免疫佐剂:重组蛋白抗原往往需要配以合适的佐剂以增强免疫原性。细胞免疫和黏附免疫在支原体保护性免疫应答中发挥重要作用,细胞免疫和黏附免疫佐剂(如IMS 1113、LTB亚基)可增强猪肺炎支原体的免疫效果[36]。
7 展望运用反向疫苗学的方法,在筛选鉴定丝状支原体丝状亚种、猪肺炎支原体等重要支原体保护性抗原方面取得进展,MSC-0136、MSC-0957、MSC-0499、MSC-0431、MSC-0776等为丝状支原体丝状亚种保护性抗原,能诱导牛体产生保护性免疫,可用于预防牛传染性胸膜肺炎[18]。P97、P42、NrdF等为猪肺炎支原体的保护性抗原[53]。现有研究提示,单一支原体免疫蛋白往往难以诱导理想的免疫反应和产生良好的免疫保护,多组分抗原(组合或嵌合表达)可克服这一缺陷,诱导较全面的免疫反应,可能会产生较好的免疫保护效果。理想的支原体亚单位疫苗应该有效激活机体体液免疫和细胞免疫,甚至黏膜免疫,抵御或清除病原的感染。以猪肺炎支原体为例,发现和鉴定更加有效的免疫保护抗原或抗原组合,根据抗原特点筛选高效的抗原递送系统,如减毒鼠伤寒沙门菌[54]或腺病毒等[55]递送系统,相应抗原配合更有效的佐剂,如IMS 1113[36]、LTB亚基[56]、氯化锂[57]、介孔二氧化硅纳米颗粒[58]等,增强疫苗免疫保护效果[59]。因此,深入研究支原体蛋白的免疫反应,筛选保护性抗原、抗原表位,抗原设计,选择高效的抗原递送系统,配合合适的佐剂可提高支原体亚单位疫苗免疫保护效果,为新型疫苗研制提供科学依据。
[1] |
吴移谋, 叶元康.
支原体学[M]. 2版. 北京: 人民卫生出版社, 2008.
WU Y M, YE Y K. Mycoplasma[M]. 2nd ed. Beijing: People's Medical Publishing Press, 2008. (in Chinese) |
[2] | World Organization for Animal Health. Chapter 3.7.4 Contagious caprine pleuropneumonia[EB/OL]//Manual of diagnostic tests and vaccines for terrestrial animals 2018. [2020-06-05]. https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.07.04_CCPP.pdf. |
[3] | KLEIN U, DE JONG A, YOUALA M, et al. New antimicrobial susceptibility data from monitoring of Mycoplasma bovis isolated in Europe[J]. Vet Microbiol, 2019, 238: 108432. DOI: 10.1016/j.vetmic.2019.108432 |
[4] | CAI H Y, MCDOWALL R, PARKER L, et al. Changes in antimicrobial susceptibility profiles of Mycoplasma bovis over time[J]. Can J Vet Res, 2019, 83(1): 34–41. |
[5] | ABD EL-HAMID M I, AWAD N F S, HASHEM Y M, et al. In vitro evaluation of various antimicrobials against field Mycoplasma gallisepticum and Mycoplasma synoviae isolates in Egypt[J]. Poult Sci, 2019, 98(12): 6281–6288. DOI: 10.3382/ps/pez576 |
[6] | GAUTIER-BOUCHARDON A V. Antimicrobial resistance in Mycoplasma spp.[J]. Microbiol Spectr, 2018, 6(4). DOI: 10.1128/microbiolspec.ARBA-0030-2018 |
[7] | KLEIN U, DE JONG A, MOYAERT H, et al. Antimicrobial susceptibility monitoring of Mycoplasma hyopneumoniae and Mycoplasma bovis isolated in Europe[J]. Vet Microbiol, 2017, 204: 188–193. DOI: 10.1016/j.vetmic.2017.04.012 |
[8] | TAO Y, SHU J H, CHEN J, et al. A concise review of vaccines against Mycoplasma hyopneumoniae[J]. Res Vet Sci, 2019, 123: 144–152. DOI: 10.1016/j.rvsc.2019.01.007 |
[9] | CALCUTT M J, LYSNYANSKY I, SACHSE K, et al. Gap analysis of Mycoplasma bovis disease, diagnosis and control: an aid to identify future development requirements[J]. Transbound Emerg Dis, 2018, 65(S1): 91–109. |
[10] | MAES D, SIBILA M, KUHNERT P, et al. Update on Mycoplasma hyopneumoniae infections in pigs: knowledge gaps for improved disease control[J]. Transbound Emerg Dis, 2018, 65(S1): 110–124. |
[11] | WELDEAREGAY Y B, PICH A, SCHIECK E, et al. Proteomic characterization of pleural effusion, a specific host niche of Mycoplasma mycoides subsp.mycoides from cattle with contagious bovine pleuropneumonia (CBPP)[J]. J Proteomics, 2016, 131: 93–103. DOI: 10.1016/j.jprot.2015.10.016 |
[12] | KRASTEVA I, LILJANDER A, FISCHER A, et al. Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp.mycoides, the causative agent of contagious bovine pleuropneumonia[J]. Vet Microbiol, 2014, 168(1): 116–123. DOI: 10.1016/j.vetmic.2013.10.025 |
[13] | MULONGO M M, FREY J, SMITH K, et al. Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs.mycoides fail to generate neutralizing antibodies and succumb to disease on challenge[J]. Vaccine, 2013, 31(44): 5020–5025. DOI: 10.1016/j.vaccine.2013.08.100 |
[14] | MULONGO M, FREY J, SMITH K, et al. Vaccination of cattle with the N terminus of LppQ of Mycoplasma mycoides subsp.mycoides results in type Ⅲ immune complex disease upon experimental infection[J]. Infect Immun, 2015, 83(5): 1992–2000. DOI: 10.1128/IAI.00003-15 |
[15] | DEDIEU L, TOTTE P, RODRIGUES V, et al. Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp.mycoides small colony identifies LppA as a major T-cell antigen[J]. Comp Immunol Microbiol Infect Dis, 2010, 33(4): 279–290. DOI: 10.1016/j.cimid.2008.08.011 |
[16] | MWIRIGI M, NKANDO I, OLUM M, et al. Capsular polysaccharide from Mycoplasma mycoides subsp.mycoides shows potential for protection against contagious bovine pleuropneumonia[J]. Vet Immunol Immunopathol, 2016, 178: 64–69. DOI: 10.1016/j.vetimm.2016.07.002 |
[17] | PEREZ-CASAL J, PRYSLIAK T, MAINA T, et al. Analysis of immune responses to recombinant proteins from strains of Mycoplasma mycoides subsp.mycoides, the causative agent of contagious bovine pleuropneumonia[J]. Vet Immunol Immunopathol, 2015, 168(1-2): 103–110. DOI: 10.1016/j.vetimm.2015.08.013 |
[18] | NKANDO I, PEREZ-CASAL J, MWIRIGI M, et al. Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia[J]. Vet Immunol Immunopathol, 2016, 171: 103–114. DOI: 10.1016/j.vetimm.2016.02.010 |
[19] | ZUBAIR M, MUHAMED S A, KHAN F A, et al. Identification of 60 secreted proteins for Mycoplasma bovis with secretome assay[J]. Microb Pathog, 2020, 143: 104135. DOI: 10.1016/j.micpath.2020.104135 |
[20] | PRYSLIAK T, VAN DER MERWE J, PEREZ-CASAL J. Vaccination with recombinant Mycoplasma bovis GAPDH results in a strong humoral immune response but does not protect feedlot cattle from an experimental challenge with M.bovis[J]. Microb Pathog, 2013, 55: 1–8. DOI: 10.1016/j.micpath.2012.12.001 |
[21] | MULONGO M, PRYSLIAK T, PEREZ-CASAL J. Vaccination of feedlot cattle with extracts and membrane fractions from two Mycoplasma bovis isolates results in strong humoral immune responses but does not protect against an experimental challenge[J]. Vaccine, 2013, 31(10): 1406–1412. DOI: 10.1016/j.vaccine.2012.12.055 |
[22] | PRYSLIAK T, PEREZ-CASAL J. Immune responses to Mycoplasma bovis proteins formulated with different adjuvants[J]. Can J Microbiol, 2016, 62(6): 492–504. DOI: 10.1139/cjm-2015-0762 |
[23] | SUN X L, JONES H P, HODGE L M, et al. Cytokine and chemokine transcription profile during Mycoplasma pulmonis infection in susceptible and resistant strains of mice: macrophage inflammatory protein 1β (CCL4) and monocyte chemoattractant protein 2(CCL8) and accumulation of CCR5+ Th cells[J]. Infect Immun, 2006, 74(10): 5943–5954. DOI: 10.1128/IAI.00082-06 |
[24] | WU Q, MARTIN R J, RINO J G, et al. IL-23-dependent IL-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory Mycoplasma pneumoniae infection[J]. Microbes Infect, 2007, 9(1): 78–86. DOI: 10.1016/j.micinf.2006.10.012 |
[25] | PRYSLIAK T, MAINA T, PEREZ-CASAL J. Th-17 cell mediated immune responses to Mycoplasma bovis proteins formulated with Montanide ISA61 VG and curdlan are not sufficient for protection against an experimental challenge with Mycoplasma bovis[J]. Vet Immunol Immunopathol, 2018, 197: 7–14. DOI: 10.1016/j.vetimm.2018.01.004 |
[26] |
季文恒, 储岳峰, 赵萍, 等. 牛支原体逃避宿主免疫的研究进展[J]. 畜牧兽医学报, 2017, 48(3): 393–402.
JI W H, CHU Y F, ZHAO P, et al. Advances in the research of immune evasion by Mycoplasma bovis[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(3): 393–402. (in Chinese) |
[27] | PEREZ-CASAL J. Pathogenesis and virulence of Mycoplasma bovis[J]. Vet Clin North Am Food Anim Pract, 2020, 36(2): 269–278. DOI: 10.1016/j.cvfa.2020.02.002 |
[28] |
康浩然, 刘重阳, 于勇, 等. 2017-2018年我国部分地区牛支原体的分离鉴定及多位点序列分型[J]. 畜牧兽医学报, 2019, 50(9): 1857–1863.
KANG H R, LIU C Y, YU Y, et al. Isolation, identification and multilocus sequence typing analysis of Mycoplasma bovis strains in some regions of China during 2017 to 2018[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(9): 1857–1863. (in Chinese) |
[29] | CHEN S L, HAO H F, ZHAO P, et al. Differential immunoreactivity to bovine convalescent serum between Mycoplasma bovis biofilms and planktonic cells revealed by comparative immunoproteomic analysis[J]. Front Microbiol, 2018, 9: 379. DOI: 10.3389/fmicb.2018.00379 |
[30] | ZHANG Y K, LI X, ZHAO H R, et al. Antibodies specific to membrane proteins are effective in complement-mediated killing of Mycoplasma bovis[J]. Infect Immun, 2019, 87(12): e00740–19. |
[31] |
刘茂军, 张悦, 杜改梅, 等. 猪肺炎支原体P65基因原核表达及免疫原性分析[J]. 畜牧兽医学报, 2014, 45(2): 262–267.
LIU M J, ZHANG Y, DU G M, et al. Prokaryotic expression and immunogenicity identification of P65 from Mycoplasma hyopneumoniae[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(2): 262–267. (in Chinese) |
[32] | GALLI V, SIMIONATTO S, MARCHIORO S B, et al. Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines[J]. Vaccine, 2012, 31(1): 135–140. DOI: 10.1016/j.vaccine.2012.10.088 |
[33] |
马丰英, 姚睿玉, 邹浩勇, 等. 猪支原体肺炎亚单位疫苗的免疫应答[J]. 中国兽医学报, 2012, 32(4): 529–533.
MA F Y, YAO R Y, ZOU H Y, et al. Development of the subunit vaccine against Mycoplasmal pneumonia of swine[J]. Chinese Journal of Veterinary Science, 2012, 32(4): 529–533. (in Chinese) |
[34] | CHEN C, YONG Q, JUN G, et al. Designing, expression and immunological characterization of a chimeric protein of Mycoplasma pneumoniae[J]. Protein Peptide Lett, 2016, 23(7): 592–596. DOI: 10.2174/0929866523666160502155414 |
[35] | DE OLIVEIRA N R, JORGE S, GOMES C K, et al. A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice[J]. Vet Microbiol, 2017, 201: 146–153. DOI: 10.1016/j.vetmic.2017.01.023 |
[36] | BARATE A K, CHO Y, TRUONG Q L T, et al. Immunogenicity of IMS 1113 plus soluble subunit and chimeric proteins containing Mycoplasma hyopneumoniae P97 C-terminal repeat regions[J]. FEMS Microbiol Lett, 2014, 352(2): 213–220. DOI: 10.1111/1574-6968.12389 |
[37] | WOOLLEY L K, FELL S A, GONSALVES J R, et al. Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs[J]. Vaccine, 2014, 32(34): 4333–4341. DOI: 10.1016/j.vaccine.2014.06.008 |
[38] | JORGE S, DE OLIVEIRA N R, MARCHIORO S B, et al. The Mycoplasma hyopneumoniae recombinant heat shock protein P42 induces an immune response in pigs under field conditions[J]. Comp Immunol Microbiol Infect Dis, 2014, 37(4): 229–236. DOI: 10.1016/j.cimid.2014.07.001 |
[39] |
付启欢, 丁红雷, 吴建云. 猪支原体肺炎疫苗研究进展[J]. 中国兽医学报, 2016, 36(7): 1251–1255.
FU Q H, DING H L, WU J Y. Advance in research of Mycoplasma Hyopneumoniae vaccine[J]. Chinese Journal of Veterinary Science, 2016, 36(7): 1251–1255. (in Chinese) |
[40] | JAN G, LE HéNAFF M, FONTENELLE C, et al. Biochemical and antigenic characterisation of Mycoplasma gallisepticum membrane proteins P52 and P67(pMGA)[J]. Arch Microbiol, 2001, 177(1): 81–90. DOI: 10.1007/s00203-001-0364-4 |
[41] | TAN L, HU M R, YU S Q, et al. Characterization of the chaperonin GroEL in Mycoplasma gallisepticum[J]. Arch Microbiol, 2015, 197(2): 235–244. DOI: 10.1007/s00203-014-1047-2 |
[42] | MOURA L, DOHMS J, ALMEIDA J M, et al. Development and evaluation of a novel subunit vaccine for Mycoplasma gallisepticum[J]. Arq Bras Med Vet Zootec, 2012, 64(6): 1569–1576. DOI: 10.1590/S0102-09352012000600024 |
[43] | CHEN S L, HAO H F, ZHAO P, et al. Genome-wide analysis of the first sequenced Mycoplasma capricolum subsp.capripneumoniae strain M1601[J]. G3(Bethesda), 2017, 7(9): 2899–2906. |
[44] | HAO H F, CHEN S L, LI Y G, et al. Complete genome sequence of Mycoplasma capricolum subsp.capripneumoniae Strain zly1309F, isolated from endangered Tibetan Antelope[J]. Genome Announc, 2017, 5(29): e00496–17. |
[45] | LI Y, WANG R, SUN W J, et al. Comparative genomics analysis of Mycoplasma capricolum subsp.capripneumoniae 87001[J]. Genomics, 2020, 112(1): 615–620. DOI: 10.1016/j.ygeno.2019.04.013 |
[46] | ZHAO P, HE Y, CHU Y F, et al. Identification of novel immunogenic proteins in Mycoplasma capricolum subsp.capripneumoniae strain M1601[J]. J Vet Med Sci, 2012, 74(9): 1109–1115. DOI: 10.1292/jvms.12-0095 |
[47] | YATOO M I, PARRAY O R, MUHEET, et al. Novel candidates for vaccine development against Mycoplasma capricolum subspecies Capripneumoniae (Mccp)-current knowledge and future prospects[J]. Vaccines, 2019, 7(3): 71. DOI: 10.3390/vaccines7030071 |
[48] | LILJANDER A, SACCHINI F, STOFFEL M H, et al. Reproduction of contagious caprine pleuropneumonia reveals the ability of convalescent sera to reduce hydrogen peroxide production in vitro[J]. Vet Res, 2019, 50(1): 10. DOI: 10.1186/s13567-019-0628-0 |
[49] | SCHUMACHER M, NICHOLSON P, STOFFEL M H, et al. Evidence for the cytoplasmic localization of the L-α-Glycerophosphate oxidase in members of the "Mycoplasma mycoides Cluster"[J]. Front Microbiol, 2019, 10: 1344. DOI: 10.3389/fmicb.2019.01344 |
[50] |
赵萍, 陈胜利, 郝华芳, 等. 一种山羊支原体山羊肺炎亚种多重抗原肽的小鼠免疫试验[J]. 畜牧兽医学报, 2016, 47(12): 2476–2482.
ZHAO P, CHEN S L, HAO H F, et al. Immune responses in mice immunized with a recombinant multiple antigenic peptide from Mycoplasma capricolum subsp.capripneumoniae[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(12): 2476–2482. DOI: 10.11843/j.issn.0366-6964.2016.12.018 (in Chinese) |
[51] |
夏业才, 陈光华, 丁家波.
兽医生物制品学[M]. 2版. 北京: 中国农业出版社, 2018.
XIA Y C, CHEN G H, DING J B. Science of veterinary biologicals[M]. 2nd ed. Beijing: China Agriculture Press, 2018. (in Chinese) |
[52] |
张悦, 刘茂军, 邵国青. 猪肺炎支原体主要抗原蛋白的研究进展[J]. 中国农学通报, 2013, 29(2): 16–22.
ZHANG Y, LIU M J, SHAO G Q. Advancement of major immunogenicity proteins on Mycoplasma hyopneumoniae[J]. Chinese Agricultural Science Bulletin, 2013, 29(2): 16–22. DOI: 10.3969/j.issn.1000-6850.2013.02.004 (in Chinese) |
[53] |
王文秀, 王宝琴, 徐晴晴, 等. 猪肺炎支原体主要保护性抗原的研究进展[J]. 家畜生态学报, 2018, 39(6): 79–85.
WANG W X, WANG B Q, XU Q Q, et al. Research progress on major immunogenicity proteins on Mycoplasma hyopneumoniae[J]. Acta Ecologiae Animalis Domastici, 2018, 39(6): 79–85. DOI: 10.3969/j.issn.1673-1182.2018.06.016 (in Chinese) |
[54] | FAGAN P K, DJORDJEVIC S P, CHIN J, et al. Oral immunization of mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma hyopneumoniae antigen (NrdF)[J]. Infect Immun, 1997, 65(6): 2502–2507. DOI: 10.1128/IAI.65.6.2502-2507.1997 |
[55] | OKAMBA F R, ARELLA M, MUSIC N, et al. Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine[J]. Vaccine, 2010, 28(30): 4802–4809. DOI: 10.1016/j.vaccine.2010.04.089 |
[56] |
卢会英, 沈青春, 宁宜宝. 猪肺炎支原体p97 R1区基因和大肠杆菌LTB基因的重组和表达[J]. 中国兽医杂志, 2010, 46(4): 3–6.
LU H Y, SHEN Q C, NING Y B. The recombination and expression of R1 region of Mycoplasma hyopneumoniae p97 adhesin with Escherichia coli heat-labile enterotoxin B subunit[J]. Chinese Journal of Veterinary Medicine, 2010, 46(4): 3–6. DOI: 10.3969/j.issn.0529-6005.2010.04.001 (in Chinese) |
[57] | ISHAG H Z A, WU Y Z, LIU M J, et al. In vitro protective efficacy of Lithium chloride against Mycoplasma hyopneumoniae infection[J]. Res Vet Sci, 2016, 106: 93–96. DOI: 10.1016/j.rvsc.2016.03.013 |
[58] | VIRGINIO V G, BANDEIRA N C, DOS ANJOS LEAL F M, et al. Assessment of the adjuvant activity of mesoporous silica nanoparticles in recombinant Mycoplasma hyopneumoniae antigen vaccines[J]. Heliyon, 2017, 3(1): e00225. DOI: 10.1016/j.heliyon.2016.e00225 |
[59] |
陶宇, 李高建, 舒建洪, 等. 猪支原体肺炎基因工程疫苗的研究进展[J]. 中国生物工程杂志, 2018, 38(2): 95–101.
TAO Y, LI G J, SHU J H, et al. Advances in the research of genetically engineering vaccine of Mycoplasmal pneumoniae[J]. China Biotechnology, 2018, 38(2): 95–101. (in Chinese) |