1. Inner Mongolia Agriculture University, Hohhot 010018, Inner Mongolia, China;
2. Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Progress in research of immune related antigen of Brucella
WANG Na1,2, Huhebateer1, CUI Bu-yun2
Abstract
Brucellosis is a zoonotic infectious disease in the world. A lot of researches have been conducted on this disease and following problems are realized: first, it is difficult to determine whether the animals are vaccinated or natural infected in quarantine. Second, no effective vaccine is available for human use, and initial symptoms are not typical, so the treatment might be delayed. This paper summarizes the progress in research of some related diagnostic antigens for brucellosis, vaccine candidate antigens and some other important antigens.
布鲁氏菌免疫相关抗原研究进展
王娜1,2, 呼和巴特尔1, 崔步云2
1. 内蒙古农业大学,呼和浩特 010018;
2. 中国疾病预防控制中心传染病预防控制所,北京 102206
2. 中国疾病预防控制中心传染病预防控制所,北京 102206
收稿日期：2013-11-13
基金项目：国家“863”课题（No.2007AA02Z410）；国家“十二五”科技重大专项课题（No.2013ZX10004221，No.2012ZX10004215）
通讯作者：呼和巴特尔，Email：Hhbte@163.com；崔步云，Email：cuibuyun@icdc.cn
摘要
布鲁氏菌病是世界范围内存在的一种人畜共患传染病，目前许多学者对此展开研究并总结一些瓶颈问题：其一，在动物检疫方面，无法确定被检动物是疫苗接种还是自然感染，这对畜牧业经济造成了很大影响；其二，在疫苗接种方面，人类预防布鲁氏菌感染没有理想的疫苗，使得感染率增加，再加之短时间内临床症状不典型，使得病情极易被延误。针对这两个问题本研究主要介绍了一些布鲁氏菌病诊断相关抗原、疫苗候选抗原和一些热点关注抗原。
内容大纲
1 布病诊断性抗原
关于布病的诊断抗原的研究，一方面要选出布病检出率较高的抗原，利于布病的筛查，进一步还需寻找区分自然感染和疫苗接种的抗原，进而精确筛查结果。尚德秋等[5]用两性离子去污剂从牛羊猪3种布氏菌不同生物型的光滑型菌株中提取外膜蛋白并进行了SDS-PAGE图谱的比较，结果发现三者在>97.4×103、28～31×103以及<28×103的3个相对分子质量区段内，谱带一致；此外，羊种和猪种布氏菌的外膜蛋白在42.7×103附近有共同的谱带，而牛种布氏菌的外膜蛋白没有，可见种间蛋白存在差异。张洪丽[6]为了寻找羊种布氏菌Ⅰ型的强毒株16M和疫苗株M5的差异，于2006年通过双相电泳(2-DE) 对两者蛋白进行了分离，用Image Master TM 2D Platinum 软件对其结果进行分析，发现强毒株16M的蛋白点为797±22个，疫苗株M5蛋白点为831±29个，两者匹配上的蛋白点共有580个，而且在表达模式上也非常相似。另外选取了其中的53个差异蛋白点，经过酶切、肽提取，最终采用最新的电子飞行时间质谱进行鉴定(MALDI-TOF-MS)，其中4个无信号，在剩余的49个蛋白中，22个蛋白在疫苗株M5中高度表达，27个在强毒株16M中高度表达，共代表了35个不同的开放性阅读框。可见两者在蛋白种类和表达水平等方面均存在差异，为鉴别诊断奠定了基础。
1.1 Omp28蛋白
布氏菌外膜蛋白Omp28存在于菌体表面和细胞外周浆质中，是一种可由细胞内向外释放的可溶性蛋白，相对于固定的外膜蛋白来说，它具有易于检测的优点[7]，而且Omp28包含250个氨基酸，在所有已知的布氏菌种中均可以检测到Omp28抗原。大量的间接酶联免疫吸附试验(iELISA)和免疫印迹(WB)都证明了该蛋白敏感性强、特异性好，适用于血清学诊断。Thavaselvam等[8]用Omp28作为抗原，通过iELISA对布氏菌细菌培养阳性、临床诊断阳性和健康人3组人群进行了评价，结果发现Omp28只与细菌培养阳性的血清发生特异性反应。侯慧玉[9]用Omp28做抗原检测河南100份牛血清(RBPT和SAT检测结果有56份为布病阳性，44份为阴性)，其判定为阳性的血清共45份，相比较灵敏度为75%，特异度为93.2%，假阴性率为25%，假阳性率为6.8%，在动物布病诊断方面也证明了Omp28是一种重要的布氏菌诊断抗原。
国外曾有报道omp28基因在部分弱毒苗中会出现缺失和弱化现象。Debbarh等[10]和Cloeckaert等 [11]在研究中发现，用减毒活疫苗和致病强毒株免疫动物血清，前者Omp28所引发的免疫反应要远弱于后者，omp28确实在一些目前所使用的疫苗中存在这一现象，这也许会成为区分自然感染和疫苗免疫的可行性方法。
1.2 Omp31蛋白
布氏菌omp31基因由723个核苷酸序列组成，Omp31蛋白由240个氨基酸所组成。通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和Southern blot杂交技术对其多态性进行了研究，结果显示Omp31存在于除牛种之外所有的布氏菌中，羊种和猪种之间有大约9个核苷酸的差异，同时Omp31也是猪种和犬种的种属特异性标志 。Omp31能诱导产生保护性免疫反应 ，而且保护性作用很强，免疫动物以后发现血清检测和皮试反应结果均为阳性，可以说明布氏菌的Omp31不仅可以引起体液免疫，同时也可以诱导针对布氏菌感染的细胞免疫反应[18]，而且其单抗的保护性作用不受干扰。国外学者也做了大量相关的实验，都验证了Omp31具有良好的免疫识别效果，可用于血清学诊断[19]，而且发展空间很大。所以可以将Omp31运用于血清学诊断和分子分型中。
国外研究已证实，在目前所出现的致弱菌苗中omp31基因缺失株均不改变亲本株的免疫保护作用[20]，omp31被认为是目前最适合的基因缺失标记疫苗之一，若将Omp31与其他免疫原性蛋白联合用于血清学诊断中，可以区分疫苗接种和自然感染[21]。
杨星雅[22]对Omp31进行了克隆表达并且进行了WB验证，其结果为13份非牛种菌的免疫血清中有11份与Omp31蛋白产生了免疫性条带，而在10份自然感染的牛血清中，均没有发生特异性抗原抗体反应。可见，牛种菌感染不会产生该蛋白的抗体，而非牛种菌感染血清中均存在其抗体，证明Omp31可作为种间差异的鉴别诊断蛋白，可以鉴别牛种菌和非牛种菌。
1.3 BCSP31外膜蛋白
该外膜蛋白相对分子质量约为31×103，经证实牛、羊、猪布氏菌BCSP31基因的种间同源性很高可达100%[23]。不同研究者的免疫蛋白质组学分析也均显示，该蛋白既有良好的免疫原性，又有较高的特异性 。但也有学者利用蛋白质芯片对布氏菌蛋白进行免疫学检测时发现该抗体只存在于感染的山羊血清中，而在被确定的62份已感染的患者血清中并未发现其抗体[26]，也许说明该外膜蛋白可作为区分人和山羊的鉴别诊断抗原。
1.4 P17 蛋白
P17是由158个氨基酸编码的相对分子质量为17.3×103蛋白质。Fabienne等[27]首先克隆表达了P17，并分别用自然感染的羊血清和牛血清对其抗原性进行了验证，采用WB方法，检出率分别为51%和39%；而用竞争酶联免疫吸附试验(c-ELISA)方法，检测结果分别为70%和61%。所以认为P17在布病的血清学诊断方面可能有应用潜质。
1.5 其他
Omp10是一种具有免疫原性的抗原，但也有文献报道，该蛋白与自然感染的牛不发生血清学反应，但可与自然感染的羊发生血清学反应[28]，由此Omp10可能可以作为牛种布氏菌疫苗株与野毒株的鉴别诊断蛋白。
此外，还有文章报道，Omp19蛋白与布氏菌的毒力有关，也可作为布氏菌的血清学诊断的候选抗原之一[29]，有研究者对一定数量的牛血清样品进行分析后发现，只有已感染的牛才能产生抗Omp19的抗体。也有国外研究者提到在收集的马耳他型布氏菌自然感染的羊血清中，大部分能与Omp19抗原发生抗原抗体特异性结合反应，而流产布氏菌自然感染的牛血清中几乎不发生血清学反应 。
2 疫苗候选抗原
2.1 Omp10蛋白 Omp10 蛋白是布氏菌表面的一种脂蛋白，能编码126个氨基酸。omp10基因由381 bp的核苷酸序列组成。Omp10存在于已知所有的种型中，有人证实S2和M5中omp10的同源性为100%，与牛种544A菌株的同源性为99.7% ，且该序列在不同种属间非常保守。
Omp10具有良好的抗原性与免疫保护性，不仅可刺激体液免疫而且对细胞免疫也有刺激作用[5]。Cassataro等[34]的研究表明，以羊种布氏菌为模板对omp10进行克隆表达，首先设计引物，将omp10基因大量扩增，通过测序在确保扩增片段与 GenBank中omp10基因序列完全一致后，将含相应抗性的表达载体各自进行酶切后，与目的片段两者进行连接、原核表达、诱导，将纯化后的蛋白进行iELISA检测，其结果表明，所表达的蛋白能被布氏菌免疫小鼠血清所识别。免疫动物后，发现其保护力与完整的细胞表面蛋白极其相似，可见产生了保护性免疫反应。
2.2 Omp16蛋白
Omp16存在于布氏菌所有6个种的34种生物型中，能够编码166个氨基酸，2010年的一项研究表明[35]，由于其自身的脂质部分有佐剂的活性，在无外部佐剂的条件下可诱导小鼠特异性CD4(+)和CD8(+)T细胞产生γ干扰素，能够达到与减毒活疫苗S19相似的保护水平[29]，而且未脂化Omp16的蛋白质部分在口服途径或全身免疫也能诱导Th1细胞发生特异性免疫。Omp16用于口服疫苗将会有很大的应用前景。
2.3 Omp25外膜蛋白
Omp25是布氏菌的重要外膜蛋白之一，牛种、羊种等5种布氏菌都具有高度的同源性，羊种布氏菌缺乏EcoR V位点。在GenBank中查询可知，omp25基因序列全长642 bp，序列比较后发现只有绵羊附睾布氏菌从第561个序列开始有36 bp的缺失序列。而有报道显示，omp25基因在布氏菌不同种型中高度保守，利用该成分制备的新型疫苗有望能够在不同的布氏菌中形成交叉保护作用 。omp25作为BvrR/BvrS双组份系统的靶基因，通过调节能抑制TNF-a的产生和分泌，对布氏菌的胞内生存有利。Jubier等[39]的研究也发现Omp25 对巨噬细胞分泌INF-a有抑制作用。如果采用omp25基因敲除疫苗，就可通过TNF-a的释放来刺激吞噬细胞，起到吞噬和杀菌的作用，而且能减弱毒力。有报道显示，羊种和绵羊附睾种布氏菌的omp25的基因突变株与疫苗株Rev.1具有相似或者更强的免疫保护性。如果omp25基因敲除疫苗能广泛应用于市场，也能达到区分布氏菌疫苗接种和自然感染的效果。
2.4 核蛋白L7/L12
L7/L12是一种核糖体蛋白，能刺激T细胞、是一种T细胞免疫优势抗原。在布氏菌的核糖体亚单位中以4个拷贝的形式存在。有研究发现，从布氏菌中提取的L7/L12蛋白能特异性地刺激感染动物的单核细胞，激活T细胞，而且对INF-γ的转录和表达有上调作用，从而起到保护作用[40]。
1997年Kurar等[41]利用pcDNA3真核表达载体构建了编码布氏菌核蛋白L7/L12的DNA疫苗，对小鼠肌内注射后能够诱导细胞免疫，并产生特异性抗体，与对照组相比产生较高水平的免疫保护力，是目前公认的保护性抗原分子。对M5、S2、S19和104M 4株菌的保护性基因进行测序，发现L7/L12序列在核苷酸的第77、197、200、268位碱基发生改变，为新型疫苗的研发提供理论支持。2004年曾政等[42]分别将L7/L12构建至pET-32a和pCDNA3.1载体中，重组质粒经转化表达纯化后，对小鼠进行肌注免疫，最后通过iELISA和WB检测到了免疫小鼠体内有特异性抗体产生，说明L7/L12可作为潜在的布氏菌新型疫苗抗原。
2.5 胞质结合蛋白P39
胞质结合蛋白P39是T细胞免疫显著性抗原，既能使布氏菌致敏的豚鼠发生明显的迟发型超敏反应，也能刺激牛外周血单核细胞IFN-γ的产生，能够产生明显的细胞免疫反应和体液免疫反应，P39作为保护性抗原使人们对它产生了很大的研究兴趣[43]。用重组P39蛋白肌内注射免疫小鼠后，发现小鼠体内产生的特异性抗体滴度较高，以IgG1和IgG2a为主，而后用牛种布氏菌544A野毒株攻毒后，产生了长时间的、强烈的免疫记忆反应[44]。
3 部分关注较多的蛋白
3.1 热休克蛋白(GroEL) GroEL基因是热休克蛋白家族成员之一，存在于各种病原微生物之中，是体液和细胞免疫的首选目标[45]。它存在于胞质内，在受到外界刺激时可分泌到细胞外，并在菌体表面进行表达，这可能与Ⅳ分泌系统有很大关系，有可能是Ⅳ型分泌系统的效应子蛋白[46]。 2002年Leclerq等[47]采用拷贝牛种布氏菌的GroEL DNA苗，对小鼠分别进行肌内注射和基因枪两种免疫方式，得出两种方式都没有产生明显的保护作用。
3.2 Ⅳ型分泌系统
Ⅳ型分泌系统(Type Ⅳ secretion system， TFSS)是布氏菌的主要毒力因子，是由12个基因(表1)组成的操纵子VirB编码的多蛋白复合物家族，Wang等 通过比较蛋白质组学的方法发现：virB操纵子可通过影响一些相关蛋白的功能来调节布氏菌的胞内寄生，如压力蛋白基因(dnaK)、耐高温基因(htrA)、omp25等，并且TFSS影响外膜蛋白的特性，可能与布氏菌适应胞内胞外的生存环境紧密相关。缺失了virB基因的所有突变株都将失去在细胞内繁殖的能力。
表1 Ⅳ型分泌系统的12个基因及其各自的作用
Table 1 The 12 genes of type Ⅳ secretion system and their respective function
| 名称 | 作用 |
| VirB1 | 溶解糖基转移酶位点，利于Ⅳ型系统装配和集合； 影响其他VirB蛋白[50] |
| VirB2 | 毒力因子，利于布氏菌在吞噬细胞内复制 |
| VirB6 | 毒力因子，利于布氏菌在吞噬细胞内复制 |
| VirB3 | 细菌跨膜信号传送蛋白 |
| VirB7 | 细菌跨膜信号传送蛋白 |
| VirB10 | 细菌跨膜信号传送蛋白 |
| VirB5 | 调节吞噬作用和细胞内转运[51] |
| VirB8 | 毒力因子 |
| VirB12 | 致病性，血清学检测标记抗原[52] |
3.3 糖基转移酶(WboA)
布氏菌的wboA基因编码的糖基转移酶，与布氏菌脂多糖的O链合成相关，布氏菌16M和猪型4号株的wboA基因进行断裂突变后，所得到的突变株毒性低于它们的原始株，不产生O链抗体，但能诱导产生细胞免疫反应。Carmen等[53]证实去除wboA基因的牛种布氏菌变异株，其毒力要比原代菌株S2308株明显减弱，且在体内其产生抗体抵抗布氏菌的能力比RB51弱菌苗强。可见wboA基因的缺失或突变将导致光滑型布氏菌的表现型或毒力的改变。
3.4 Omp89蛋白
国内有关Omp89的研究相对较少。1992年，Axel等[54]从布氏菌中提取了Omp89，并对其抗原性进行验证，发现该蛋白有抗原性，但进行c-ELISA实验后，结果表明Omp89蛋白对布氏菌病诊断没有特异性。Limet等[55]从羊种布氏菌中提取并纯化了Omp89，并将其作为抗原进行包被，分别用免疫接种的不同时段的牛血清和自然感染牛血清作为一抗进行ELISA检测，并与用S-LPS做为抗原的iELISA检测进行了比较，结果发现Omp89的免疫应答反应弱于S-LPS，可能不适用于布氏菌的诊断。
4 结论
国内外许多学者利用一系列技术从基因、氨基酸等水平对布氏菌菌体蛋白进行了研究，本研究总结了部分与诊断和疫苗相关蛋白，可以看出某些蛋白在疫苗研制方面有显著的作用，某些更适用于布病的诊断、甚至鉴别诊断。Omp31可以作为种间差异蛋白，牛种自然感染株不产生此抗体，其他菌株和疫苗株均产生抗体。在诊断方面，我们可以考虑将这些有价值的抗原进行条件优化，用于ELISA等试验中，进而提高检测的灵敏度。融合蛋白的构建也将会是一种趋势，将免疫原性较高的一种蛋白和鉴别诊断蛋白进行克隆表达，最终用血清学诊断进行评估并对其结果进行分析，查看这种蛋白是否既能起到提高诊断效率，又能区分自然感染与疫苗接种的作用。布氏菌的种型鉴定方面基质辅助激光解吸电离飞行时间质谱也会成为一种新趋势，通过已创建的布氏菌质谱图数据库，将采集的布氏菌菌株与之进行比对，进而区分自然感染和疫苗接种，简单快捷，有利于布病疫情的净化与控制。
参考文献
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[8] Thavaselvam D,Kumar A,Tiwari S,et al.Cloning and expression of the immunoreactive Brucella melitensis 28 kDa outer-membrane protein (Omp28) encoding gene and evaluation of the potential of Omp28 for clinical diagnosis of brucellosis[J].Med Microbiol,2010,59(4):421-428.
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[13] Vizcaìno N,Kittelberger R,Cloeckaert A,et al.Minor nucleotide substitutions in the OMP31 gene of Brucella ovis result in antigenic differences in the major outer membrane protein that it encodes compared to those of the other Brucella species[J].Infect Immun,2001,69(11):7020-7028.
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[17] Cassataro J, Estein SM, Pasquevich KA, et al.Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD+4 T helper 1 response that protects against Brucella melitensis infection[J]. Infect Immun,2005,73(12):8079-8088.
[18] Vizcaino N, Caro-Hernandez P, Cloeckaert A, et al. DNA polymorphism in the OMP25 /OMP 31 family of Brucella spp:identification of a 1.7-kb inversion in Brucella cetaceae and of a15.1-kb genomic island,absentfrom Brucella ovis,related to the synthesis of smooth lipopolysaccharide[J].MicrobesInfect,2004,6(9):821-834.
[19] Cassataro J, Velikovsky CA, Bruno L, et al. Improved immunogenicity of a vaccination regimen combining a DNA vaccine encoding Brucella melitensis outer membrane protein 31 (Omp31) and recombinant Omp31 boosting[J]. Clin Vaccine Immunol,2007,14(7):869-874.
[20] Jacques I,Verger JM,Laroucau K,et al.Immunological responses and protective efficacy against Brucella melitensis induced by bp26 and omp31 B.melitensis Rev.1 deletion mutants in sheep[J].Vaccine,2007,25(5):794-805.
[21] Wu JB,Qiu JL,Wang WJ,et al.Cloning,expression and antigenicity of recombinant protein OMP31 of Brucella melitensis[J].Chin ese Journary of Public Health,2013,29(4):596-598.(in Chinese)吴静波,丘金浪,王文敬,等.羊布鲁菌OMP蛋白基因表达及抗原性分析[J].中国公共卫生,2013,29(4):596-598.
[22] Yang XY.The study on key proteins for the identification of Brucella Species[D].Beijing:China CDC.(in Chinese)杨星雅.布鲁氏菌种间差异标识蛋白研究[D].北京:中国疾病预防控制中心,2012.
[23] Li P,Luo DY,Gao YH,et al. The building of expression plasmid BCSP31/PBAX1 and the reseach in immune protection of brucella[J]. Chinese Journal of Zoonoses,2006,22 (6): 493-497. (in Chinese)李鹏,罗德炎,高永辉,等.布氏杆菌BCSP31 /pVAX1重组表达质粒的构建及其免疫保护效果的研究[J].中国人兽共患病学报,2006,22(6):493-497.
[24] Connolly JP, Comerci D, Alefantis TG,et al. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins forvaccine development[J].Proteomics,2006,6(13):3767-3680.
[25] Dahouk S, Nckler K, Scholz HC, et al.Immunoproteomic characterization of Brucella abortus 1119-3 pre-parations usedforthe serodiagnosis of Brucella infections[J].Immun Methods,2006,309(1/2):34-47.
[26] Liang L, Leng D, Burk C,et al.Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensisantigens[J]. PLoS Negl Trop Dis,2010,4(5):673.
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