下载文档原格式
简述免疫球蛋白的主要生物学功能。
免疫球蛋白是一种重要的免疫分子，在机体的免疫防御过程中起到关键作用。
其主要生物学功能如下：
1. 负责识别抗原：免疫球蛋白的变异区域可以识别和结合不同的抗原，从而激活机体的免疫反应。
2. 参与抗原递呈：免疫球蛋白可以与抗原结合并形成免疫复合物，提高其在抗原递呈细胞表面的稳定性和特异性，从而促进抗原递呈和T细胞的激活。
3. 中和病原微生物：免疫球蛋白可以结合病原微生物表面的抗原，从而中和其毒性和侵袭性，防止其感染机体。
4. 参与细胞毒作用：部分免疫球蛋白可以结合特定的癌细胞或病原微生物，活化和引导细胞毒性T细胞对其进行杀伤。
5. 调控免疫反应：免疫球蛋白可以调节机体的免疫反应，包括提高抗体亲和力、调节炎症反应和参与免疫抑制等功能。
6. 传递免疫记忆：免疫球蛋白在机体抗原暴露后会留下一定量的记忆B细胞和记忆T细胞，从而为下一次抗原感染提供更快速、更有效的免疫应答。
免疫球蛋白检验方法
免疫球蛋白检验方法是一种常用的血液学检验方法，用于检测患者体内免疫球蛋白的水平。
这是一种重要的检测方法，可以为医生提供有用的信息，以帮助诊断和治疗患者。
免疫球蛋白是一种由B细胞产生的蛋白质，它们在身体中的免疫反应中起着关键作用。
免疫球蛋白可以识别和结合各种病原体，如细菌、病毒和真菌等，从而启动免疫反应，以保护身体免受感染。
在免疫球蛋白检验过程中，医生会采集患者的血液样本，并将其送往实验室进行分析。
实验室技术人员会使用特定的试剂来测量患者血液中的免疫球蛋白水平。
这些试剂通常是特定抗体，它们能与免疫球蛋白结合，从而形成复合物。
通过测量这些复合物的形成，实验室技术人员可以确定患者体内免疫球蛋白的水平。
免疫球蛋白检验方法通常用于检测多种疾病。
例如，它可以用于检测感染性疾病，如流感和肝炎，以及自身免疫性疾病，如类风湿性关节炎和系统性红斑狼疮等。
此外，免疫球蛋白检验方法还可用于监测治疗过程中的疾病进展和治疗效果。
虽然免疫球蛋白检验方法是一种常用的检测方法，但它也存在一些局限性。
例如，它不能确定特定免疫球蛋白的功能状态，也不能确定免疫球蛋白是否与某种病原体结合。
此外，免疫球蛋白检验方法还可能会出现假阳性或假阴性的结果，这可能会影响诊断和治疗的
准确性。
免疫球蛋白检验方法是一种重要的血液学检验方法，可用于检测患者体内免疫球蛋白的水平。
它可以为医生提供有用的信息，以帮助诊断和治疗患者。
然而，它也存在一些局限性，需要医生综合考虑其他相关因素，才能做出准确的诊断和治疗方案。
免疫球蛋白（immunoglobulin，Ig）具有抗体活性的血清蛋白称为免疫球蛋白，又称为抗体。
是由机体的B淋巴细胞在抗原的刺激下分化、分裂而成的一组特殊球蛋白。
在人体至少有五种免疫球蛋白，即IgG、IgA、IgM、IgD、IgE。
免疫球蛋白是机体受抗原（如病原体）刺激后产生的，其主要作用与抗原起免疫反应，生成抗原-抗体复合物，从而阻断病原体对机体的危害，使病原体失去致作用。
免疫球蛋白是一组具有抗体活性的蛋白质，主要存在于生物体血液、组织液和外分泌液中，是检查机体体液免疫功能的一项重要指标。
IgD和IgE含量很低，常规所测定的Ig主要为lgG、lgA、IgM三项。
血清中免疫球蛋白异常，主要可分为三类：1.几种不同的Ig水平增加：主要见于感染、肿瘤、自身免疫病、慢性活动性肝炎、肝硬化及淋巴瘤等。
自身免疫病中，如系统性红斑狼疮(SLE)以lgG、lgA、IgM升高多见，类风湿性关节炎以IgG、IgM升高多见。
2.单一的Ig水平增加：又称为“M”蛋白病，主要见于：多发性骨髓瘤(MM)，表现为仅有某一种Ig异常增高，而其他种明显降低或维持正常。
其中以IgG型MM最常见，血清中lgG含量可高达70g／L，IgA型次之，IgD型较少见，IgE 型最为罕见。
巨球蛋白血症，是产生IgM的浆细胞恶性增生，血清中IgM可高达20g／L以上。
3.一种或多种Ig水平减少：分为原发性或继发性的，前者属于遗传性，如瑞士丙种球蛋白缺乏症，选择性IgA、IgM缺乏症等。
继发性缺损见于网状淋巴系统的恶性疾病、慢性淋巴细胞性白血病、肾病综合征、大面积烧伤烫伤患者、长期大剂量使用免疫抑制剂或放射线照射所致。
lgD升高主要见于lgD型MM。
流行性出血热、过敏性哮喘、特应性皮炎患者IgD升高。
妊娠末期、吸烟者中IgD也可出现生理性升高。
IgE升高常见于超敏反应性疾病，如过敏性鼻炎、外源性哮喘、枯草热、慢性荨麻疹，以及寄生虫感染、急慢性肝炎、药物所致的间质性肺炎、支气管肺曲菌病、类风湿性关节炎和IgE型MM等。
简述免疫球蛋白的生物学效应免疫球蛋白（Immunoglobulin，简称Ig）是一类特殊的蛋白质，也被称为抗体。
它在人体免疫系统中起着重要的生物学效应。
免疫球蛋白的生物学效应主要体现在以下几个方面：1. 识别和结合抗原：免疫球蛋白具有高度的特异性，可以识别和结合各种不同的抗原。
抗原是指能够诱导机体免疫应答的物质，如细菌、病毒、肿瘤细胞等。
免疫球蛋白的结构中有一对可变区域，使其能够与特定的抗原结合，形成免疫复合物。
2. 中和毒素和病原体：免疫球蛋白可以中和毒素和病原体。
当免疫球蛋白与毒素或病原体结合时，可以阻断其对宿主细胞的侵袭和损害，从而发挥保护作用。
例如，IgG类免疫球蛋白可以中和病毒，阻止其进入宿主细胞；而IgE类免疫球蛋白则可以结合过敏原，中和过敏反应。
3. 激活补体系统：免疫球蛋白可以激活补体系统。
补体系统是机体先天免疫系统的一部分，具有溶解病原体、促进炎症反应等功能。
当免疫球蛋白与抗原结合时，可以激活补体系统的级联反应，引发一系列的炎症反应和免疫细胞的吞噬作用，起到清除病原体的作用。
4. 介导细胞毒性作用：一些免疫球蛋白可以介导细胞毒性作用。
例如，某些IgG亚类的免疫球蛋白可以结合到病原体表面，然后与免疫细胞的Fc受体结合，激活免疫细胞，如巨噬细胞和自然杀伤细胞，释放毒性物质并杀死病原体。
5. 调节免疫应答：免疫球蛋白可以调节免疫应答。
IgG亚类的免疫球蛋白可以通过与抗原结合，影响免疫细胞的活化和功能，调节免疫应答的强度和方向。
此外，一些免疫球蛋白还可以与调节性T细胞相互作用，参与免疫耐受的建立和维持。
免疫球蛋白在机体的免疫系统中具有多种生物学效应，包括识别和结合抗原、中和毒素和病原体、激活补体系统、介导细胞毒性作用以及调节免疫应答。
这些效应共同作用，形成机体的免疫防御机制，保护机体免受各种病原体和疾病的侵袭。
免疫球蛋白的研究对于理解免疫系统的功能和疾病的发生发展具有重要意义，并为开发新的免疫疗法和疫苗提供了理论基础。
免疫球蛋白的分类和特点免疫球蛋白的奇妙世界嘿，你知道吗？人体的免疫系统就像是一个超级英雄团队，而免疫球蛋白就是这个团队中的关键角色。
它们就像是超级英雄的盾牌和武器，保护我们免受细菌和病毒的侵害。
今天，我们就来聊聊这些超级英雄——免疫球蛋白，看看它们有哪些神奇的特点吧！咱们来说说免疫球蛋白的种类。
你知道有A、B、C三个大类吗？没错，它们就像是三兄弟，各有各的特点和用处。
比如，IgG就像是个大块头，它可是抗体大军的统帅，负责对付那些入侵者。
而IgM呢，就像是个小前锋，专门在前线冲锋陷阵，把敌人打得落花流水。
还有IgA，它就像是个小护士，默默地在体内巡逻，确保我们的健康不受影响。
接下来说说免疫球蛋白的特点。
它们可不是一般的蛋白质，而是拥有超能力的存在！比如说，IgG家族的成员们，它们可以记住上次遇到的敌人是谁，下次再见面就能一击必杀，让敌人无处可逃！还有IgM家族的成员们，它们的速度简直快得跟闪电似的，一秒钟就能赶到战场，把敌人打得落花流水！再来说说免疫球蛋白的作用。
它们就像是超级英雄们的战袍，让我们的身体变得更加强大。
有了它们，我们就能轻松应对各种细菌和病毒的攻击，保持身体健康。
而且，它们还能帮助我们预防疾病，减少生病的几率。
想象一下，如果我们没有免疫球蛋白的保护，那岂不是随时都可能被疾病侵袭，变得虚弱不堪？除了这些，免疫球蛋白还有一些有趣的故事呢。
比如，有一种叫做IgD的蛋白质，它的存在就像是给免疫系统做标记的神秘符号。
有了它，我们就能更好地识别和记忆那些曾经遇到过的敌人。
还有IgE家族的成员们，它们就像是过敏反应的小精灵，一旦接触到过敏原，就会引发一系列的反应，让我们感到不舒服。
总的来说，免疫球蛋白就像是我们身体中的超级英雄团队，它们各有各的职责和技能，共同守护着我们的健康。
虽然它们看起来不起眼，但如果没有它们，我们可能会面临更多的危险和挑战。
所以，我们要感谢这些可爱的超级英雄，是它们让我们的生活变得更加美好和安全！好了，关于免疫球蛋白的介绍就到这里啦。
免疫球蛋白的作用是什么
免疫球蛋白，也称为抗体，是一种在人体免疫系统中起关键作用的蛋白质分子。
它们由免疫系统的特定细胞称为B细胞产生。
免疫球蛋白的主要作用是识别并结合到入侵人体的病原体，如细菌、病毒或其他病原微生物。
通过结合到病原体表面的特定结构，免疫球蛋白能够中和病原体，阻止其进一步感染或侵入人体细胞。
抗体还可以激活免疫系统中的其他细胞来参与清除病原体，例如通过与巨噬细胞结合促进吞噬作用，或通过与自然杀伤细胞结合促进细胞毒性作用。
此外，抗体还可以激活补体系统，引发一系列的免疫反应来破坏病原体。
除了对病原体的直接作用，免疫球蛋白还具有其他重要的功能。
例如，它们能够与病毒感染的细胞结合，标记这些细胞以被免疫系统清除。
免疫球蛋白还参与调节免疫应答，调控免疫细胞的活动，并参与到炎症反应和修复过程中。
总的来说，免疫球蛋白的作用是保护机体免受病原体的感染和损害，维持机体的免疫平衡和健康状态。
免疫球蛋白的主要生物学功能免疫球蛋白，也被称为抗体，是免疫系统中一类重要的蛋白质分子。
它们在人体内发挥着多种生物学功能，对维持机体免疫稳态至关重要。
免疫球蛋白可以识别和结合病原体。
在机体遭遇病原体侵袭时，免疫球蛋白能够识别和结合病原体表面的抗原，形成抗原-抗体复合物。
这个过程起到了阻断病原体侵入的作用，从而保护机体免受感染。
免疫球蛋白通过其特异性的结合能力，可以识别和结合各种不同的病原体，如细菌、病毒、真菌等，起到了广泛的防御作用。
免疫球蛋白可以激活免疫系统。
当抗原与免疫球蛋白结合后，免疫球蛋白可以激活免疫系统中的其他细胞和分子，促进免疫应答的发生。
例如，在免疫球蛋白与病原体结合后，会激活补体系统，进而引发炎症反应和吞噬细胞的活化，以消灭病原体。
此外，免疫球蛋白还可以与其他免疫细胞表面的Fc受体结合，从而激活这些细胞，增强免疫应答的效果。
除了直接参与免疫应答，免疫球蛋白还具有调节作用。
它们可以调节免疫系统中不同免疫细胞的活性和功能。
例如，某些类型的免疫球蛋白可以促进细胞毒性T细胞杀伤肿瘤细胞，从而发挥抗肿瘤作用。
另一些免疫球蛋白则可以抑制免疫细胞的活性，以避免过度免疫反应引发的损伤。
通过这种调节作用，免疫球蛋白能够平衡机体的免疫应答，维持免疫稳态。
免疫球蛋白还具有传递母体免疫到胎儿的功能。
在胎儿发育过程中，母体的免疫球蛋白可以通过胎盘传递到胎儿体内，为胎儿提供一定的免疫保护。
这种传递的免疫球蛋白被称为胎儿免疫球蛋白，可以在胎儿出生后的早期提供免疫防御，直到胎儿自身免疫系统的发育成熟。
总结起来，免疫球蛋白的主要生物学功能包括识别和结合病原体、激活免疫系统、调节免疫应答以及传递母体免疫到胎儿。
这些功能使得免疫球蛋白成为机体免疫系统中不可或缺的组成部分。
通过免疫球蛋白的作用，机体能够有效应对各种病原体的侵袭，维持免疫稳态，保护人体健康。
希望本文对您有所帮助，如有需要，请随时与我联系。
免疫蛋白知识点总结免疫蛋白的种类主要包括IgG、IgM、IgA、IgD和IgE等。
1. IgGIgG是最常见的免疫蛋白，在全血抗体总量的70~75%约为IgG。
它是唯一可以通过胎盘转移给胎儿的免疫蛋白，也是唯一能在组织间穿行的免疫球蛋白。
IgG参与调节细胞介导的免疫反应，能够中和病原微生物，促进吞噬细胞清除细菌，保护机体免受细菌、病毒的感染。
2. IgMIgM是人体最早出现的一种免疫球蛋白，也是生物体内含有多聚体结构的免疫球蛋白，它的分子量最大，以400KD的形式存在。
一般来说IgM是通过对外界抗原的初次反应产生的，是机体对感染的早期反应。
IgM对细菌及毒素有强大的保护能力。
它是一种功能活性最早成熟的免疫球蛋白，其可以作为胎儿体内的初次免疫系统防护作用，对新生儿免疫系统的帮助很大。
3. IgAIgA是人体分泌液体和黏膜上最多的抗体，对保护呼吸道黏膜、肠道黏膜和各种进入体内的细菌、病毒和有害物质具有特殊的保护作用。
IgA异体含有两种亚型分别是IgA1和IgA2。
它是保护婴儿不受肠道接触感染。
成年后IgA帮助保护呼吸道和消化道黏膜。
4. IgDIgD是人体内数量最少的抗体种类，分别占全血抗体总量的0.2%。
IgD是一种传递体型抗原特异性阳电荷抗体，也是免疫球蛋白的一类蛋白质。
正常人的细胞表面有IgD分子存在。
5. IgEIgE是机体内一种含有两条重链，轻链结构的生物大分子，在血清中占全血抗体的0.002%。
是免疫球蛋白中浓度最低的一种，其生物学效应与抗体的其他类型截然不同，主要是通过经皮抗原刺激后抗原特异性T细胞的表达上升而刺激黏膜的分泌性免疫反应，有很强的致敏性，有助于机体抗细菌、寄生虫、病毒感染或其他形式的外源性抗原。
免疫蛋白的结构和功能免疫蛋白的结构具有两个重要的特点，一是抗原结合部位，也称为抗原决定簇（antigen-binding site），是与抗原结合的部位，决定了抗体的特异性和识别抗原的能力。
简述免疫球蛋白生物学功能
免疫球蛋白是一类蛋白质，也被称为抗体。
它们是机体对抗感染和外来物质的主要武器。
免疫球蛋白的生物学功能包括：
1. 识别病原体和其他外来物质：免疫球蛋白可以识别并结合多种病原体和其他外来物质，使它们易于被机体的免疫系统识别和清除。
2. 中和毒素和病原菌：一旦免疫球蛋白与毒素或病原菌结合，可以中和它们的活性，从而避免它们对机体造成伤害。
3. 激活免疫反应：免疫球蛋白可以激活机体的免疫反应，包括促进炎症反应和增强细胞毒性T细胞的杀伤活性。
4. 保护胎儿：在怀孕期间，免疫球蛋白可以穿过胎盘传递给胎儿，保护它们免受母体免疫系统的攻击。
5. 诱导免疫记忆：一旦机体产生免疫球蛋白应对某种病原体，它们可以诱导机体产生免疫记忆，从而使机体在下一次遭遇相同病原体时能更快地产生更多的免疫球蛋白。
免疫球蛋白的分型依据主要基于抗体结构和功能的不同。
免疫球蛋白是存在于人体血液中的蛋白质，它们可以识别和结合病毒或细菌等外来物质，从而保护人体的健康。
免疫球蛋白主要有IgG、IgM、IgA、IgD和IgE这五种类型，这些类型之间的区别主要基于其基因表达、结构以及功能的不同。
免疫球蛋白的主要分型有以下三种：
1. IgG型免疫球蛋白是最常见的类型，具有很强的抗病毒和细菌能力，几乎在所有体液中都有分布，是机体抗感染、抵御病毒和细菌入侵的重要武器之一。
2. IgM型免疫球蛋白是仅次于IgG的抗体，也是人体最主要的抗体之一，其识别和结合病毒或细菌的速度非常快，有助于快速保护机体不受感染。
3. IgA型免疫球蛋白主要存在于人体的黏膜表面，如胃肠道、呼吸道等部位，具有保护黏膜和皮肤免受感染和外界刺激的作用。
此外，免疫球蛋白还有IgD和IgE这两种特殊类型。
IgD型免疫球蛋白在某些情况下可以作为“哨兵”来检测潜在的感染或过敏原，有助于机体提前做出反应。
IgE型免疫球蛋白则主要用于机体对过敏原的特异性免疫应答，具有高度亲和性和专一性，有助于机体抵御特定的感染。
总的来说，免疫球蛋白的分型依据主要基于其结构和功能的差异。
不同类型免疫球蛋白在人体内发挥着不同的作用，它们共同协作，帮助人体抵御各种感染，维护身体健康。
FULL PAPER BacteriologyIdentification of Novel Immunogenic Proteins in Mycoplasma capricolum subsp. Capripneumoniae Strain M1601Ping ZHAO1), Ying HE1), Yue-feng CHU1), Peng-cheng GAO1), Xuan ZHANG1), Nian-zhang ZHANG1),Hai-yan ZHAO1), Ke-shan ZHANG1) and Zhong-xin LU1)*1)State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Epizootic Diseases of Grazing Animals of Ministry of Agriculture, Key Laboratory of Veterinary Public Health of Ministry of Agriculture, Engineering Research Center of Biological Detection of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China(Received 1 March 2012/Accepted 17 April 2012/Published online in J-STAGE 14 May 2012)ABSTRACT.The purpose of this study was to obtain immunogenic proteins and potential proteins of interest that were isolated from Myco-plasma capricolum subsp. capripneumoniae (Mccp) by MALDI-TOF mass spectrometry. One-dimensional SDS-PAGE and two-dimen-sional gel electrophoresis of whole cell preparation were conducted, and membrane proteome maps were prepared by immunoblotting. One-dimensional SDS-PAGE identified three immunogenic proteins with molecular masses in the range 29–97.2 kDa, two of which were in the membrane protein fraction. After two-dimensional gel electrophoresis, 20 highly immunogenic proteins were identified in the whole cell protein preparation while 9 immunogenic proteins were identified in the membrane protein fraction. This indicated that membrane proteins were the principle immunogenic proteins in Mccp. These proteins may have potential for the development of improved diagnostic tests and possible vaccines.KEY WORDS:homology, identification, immunoblotting, immunogenic proteins, Mycoplasma capricolum subsp. Capripneumonia.doi: 10.1292/jvms.12-0095; J. Vet. Med. Sci. 74(9): 1109–1115, 2012Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the causative agent of contagious capripleuropneumonia in goats [13], which is a disease characterized by coughing, dyspnea, lagging behind the herd, lying down, fever (40.5–41.5°C) and mouth breathing in the terminal stages [21]. In chronic cases, animals present sporadic coughing, emacia-tion, and diarrhea. Animals that recover from this form of the disease do not show any signs or lesions after recovery. Although there are no sequelae in recovered animals, the survivor goats may remain carriers of the infection.The genome sequence of Mccp has been completed but not annotated by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. There have been few efforts to identify and characterize Mccp antigens, which might potentially be useful for immunodiagnosis and/or vaccination. These antigens could also be virulence factors and their characterization may help to elucidate the mechanisms of pathogenicity. There is an obvious need for better antigens to improve the serological methods that are currently used for goat herd surveillance, which is an important step before the implementation of preventive or therapeutic measures.Proteome analysis is a useful complementary method for studying bacteria, which supports genome annotation and protein identification [9, 22], and it could be used to charac-terize differentially expressed or post-translationally modi-fied antigenic proteins [11]. This approach is particularly attractive in mycoplasmae, because the high A + T content and the use of TGA as a tryptophan codon rather than as a stop codon prevent a genetic approach using Escherichia coli expression libraries. Two-dimensional gel electropho-resis (2DE) mapping of Mccp proteins will improve ge-nome annotation, and it will help to characterize the extent of post-translational modifications, as well as identifying potential virulence factors.In this study, we isolated three dominant immunogenic proteins in Mccp with SDS-PAGE and immunoblotting, 2 of which were membrane proteins. To further elucidate the identity of these proteins, 2DE maps of the whole cell protein preparation and the membrane protein fraction were produced, followed by immunoblotting. We identified 20 novel immunogenic proteins in the whole cell protein preparation and 9 immunogenic proteins in the membrane protein fraction. These important results have established the immunogenic protein profile of Mccp for the 1st time, which will be valuable for studying its pathogenicity and developing vaccines and serodiagnostic markers in the future.MATERIALS AND METHODSBacterial strains and cultivation: The Mccp strain was isolated from an infected goat in GanSu, China. The strain*C orrespondenCe to: L u, Z., State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Epizootic Diseases of Grazing Animals of Ministry of Agriculture, Key Laboratory of Veterinary Public Health of Ministry of Agriculture, Engineer-ing Research Center of Biological Detection of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.e-mail: luzhongxin@©2012 The Japanese Society of Veterinary ScienceP. ZHAO ET AL. 1110was cultivated at 37°C in KM2 broth containing 0.5% w/v Minimum Essential Medium, 0.5% w/v lactalbumin hydro-lysate, 0.5% w/v yeast extract, 15% v/v horse serum, 0.08% w/v phenol sulfonphthalein, 0.01% w/v thallium acetate, and 2,000 IU/l–1 penicillin. The pH was maintained at 7.4–7.6. Preparation of the whole cell protein samples: Cells were harvested at a density of 108 CCU m l–1 by centrifugation at 12,000 × g for 20 min and resuspended in 1 m l 25 mM Tris–HCl, pH 7.2. Cell suspensions were then lysed by sonication (25 Hz in a VC601 Sonics and Materials Inc. sonicator, Syd-ney, Australia) in an ice bath using five 30 sec cycles with a 1 min interval between pulses. Proteins were quantified us-ing the Bradford method (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA, U.S.A.).Preparation of the membrane protein fraction: Membrane proteins were prepared essentially as previously described, with slight modifications [16, 25]. Briefly, the harvested cells were washed twice with pre-cooled 50 mM Tris-HCl buffer, pH 7.2, and resuspended in the same buffer. Cell lysis was performed by sonication, and undisrupted cells were removed by centrifugation at 12,000 × g for 20 min. Membrane proteins were then collected by centrifugation at 100,000 × g for 30 min and washed 3 times with distilled water. The pellet was resuspended in distilled water. Convalescent sera: Five Mccp-free goats were inoculated tracheally with Mccp at a dose of 2.5 × 1010 CCU goat–1. Seven weeks after the 1st injection, blood was collected, and serum samples were evaluated by ELISA [26]. Specific titers were obtained from 1:5,000 to 1:10,000, and the serum with the highest titer was used in the immunoblotting ex-periments. The pre-infection serum was used as a negative control. The Institutional Animal Care and Use Committee at Lanzhou Veterinary Research Institute approved the study. One-dimensional SDS-P AGE and immunoblotting: The whole cell protein preparation and membrane protein frac-tion of Mccp were separated by 1-dimensional SDS-PAGE using a PROTEAN1 II xi 2D Cell (Bio-Rad). Proteins were electroblotted onto PVDF membranes (Amersham Biosciences, Waukesha, WI, U.S.A.). Blotted membranes were blocked with 3% w/v albumin bovine V (Roche) in phosphate buffered saline (PBS) (10 mM Na2HPO4, 1.7 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl) and then incubated with goat anti-Mccp serum diluted 1:50 in block-ing solution. Membranes were washed 3 times in PBS for 5 min, incubated with a secondary antibody (anti-goat IgG HRP, Sigma; 1:3,000), washed and developed with 4-chloro-1-naphthol (Sigma) according to the manufacturer’s instruc-tions. Controls were carried out using the corresponding pre-infection serum.Two-dimensional gel electrophoresis and immunoblotting: Protein samples were solubilized in an isoelectric focusing buffer (IEF buffer) containing 7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% w/v dithiothreitol (DTT), and 0.2% v/v ampholytes, pH 3–10 (Bio-Rad). We passively rehydrated 17 cm immobilized pH gradient (IPG) strips (pH 3–10, Bio-Rad) for 16 hr with 300 µl cell extract samples containing 1–4 mg of protein. Isoelectric focusing was performed in a Protean IEF cell system (Bio-Rad) with up to 50,000 VH at a maximum voltage of 10,000 V. Strips were equilibrated for15 min in equilibration buffer I (30% v/v glycerol, 6 M urea, 1% DTT, and a trace of bromophenol blue) and for 15 min in equilibration buffer II (equilibration solution I where DTT was replaced with 4% iodoacetamide). In the 2nd dimen-sion, IPG strips were run vertically onto 12% SDS-PAGE gels using a PROTEAN II xi 2D Cell (Bio-Rad). Gels were subsequently stained with Coomassie Brilliant Blue R250 (Bio-Rad) or with silver. The stained gels were scanned with an Image Scanner (Amersham Biosciences) and analyzed using the PDQuest Basic 8.0 program (Bio-Rad), followed by additional visual analysis. Immunoblotting was carried out as described above.Protein identification and homology analysis: Protein spots were manually excised from Coomassie-stained gels and digested in-gel with trypsin. Gel plugs received three washing steps with 180 µl of 50% acetonitrile and 50 mM ammonium bicarbonate for 15 min, followed by one wash-ing step with 180 µl of acetonitrile. After the washing pro-cedures, gel plugs were dried by vacuum centrifugation and digested for 18–24 hr at 37°C using 12 µl of 10 µg m l–1 modified porcine trypsin (sequencing grade modified tryp-sin, Promega Corporation, Madison, WI, U.S.A.) diluted to 25 mM in NH4HCO3. After tryptic digestion, peptides were extracted in two washing steps with 50 m l 50% acetonitrile and trifluoroacetic acid (TFA) for 1 hr. Extracted peptides were dried and resuspended in 10 µl of Milli-Q water. A sample of 1 µl of crude digest was mixed with 1.0 µl of α-cyano-4-hydroxycinnamic acid (10 mg m l–1 in 0.1% TFA in 1:1 acetonitrile/methanol), and an aliquot of 0.5 µl was delivered to the target plate, and then dried at room tem-perature. MS was conducted using an AB 4700 TOF/TOF Proteomics Analyzer (Applied Biosystems), operated in the reflector mode for MALDITOF peptide mass fingerprint-ing (PMF) or collision-induced dissociation-MALDI-MS/ MS (MS/MS). Each spectrum was based on the accumulat-ing data from 2,000 consecutive laser shots. All samples that were analyzed by PMF were further analyzed by MS/ MS, where up to 5 PMF precursor ions per sample were selected and submitted to MS/MS analysis. Peptides were identified using the MASCOT search engine (http://www. ) by searching against the NCBI database (release 7442,25/3/2010, Mycoplasma). Maximum mass er-rors of 100 and 200 ppm were allowed for valid PMF and MS/MS protein identifications, respectively. No functions of Mccp proteins have been previously described, so a BLAST search was performed for the identified proteins using the sequences obtained.RESULTSOne-dimensional SDS-P AGE and immunoblotting: A western blot was generated using convalescent serum from goats. We repeated this assay 3 times, with a very high de-gree of reproducibility (data not shown). Three dominant im-munogenic proteins were detected with molecular masses in the range 29–97.2 kDa, two of which were in the membrane protein fraction (Fig. 1). Controls were carried out using theIDENTIFICATION OF NOVEL IMMUNOGENIC PROTEINS 1111corresponding pre-infection serum, and no immunoreactive protein was detected .This showed that membrane proteins were the principle immunogenic proteins.Two-dimensional gel electrophoresis : To identify these immunogenic proteins, we performed 2DE of the whole cell protein preparation and the membrane protein fraction. As technical controls, we repeated all the protein prepara-tion and subsequent 2DE experiments 3 times, with a very high degree of reproducibility (data not shown). About 300 prominent protein spots were resolved in Coomassie-stained 2DE gels with pH 3–10 IPG strips (Fig. 2a), while 150 pro -tein spots were detected in the membrane protein fraction (Fig. 3a).Identification of immunogenic proteins by mass spec -trometry and homology analysis : Immunoblot assays of the 2DE gels were performed using the serum of an infected goat to identify novel immunogenic proteins (Fig. 2b and Fig. 3b). Controls were carried out using the corresponding pre-infection serum, and no immunoreactive protein spots were detected (data not shown). We repeated this assay 3 times, with a very high degree of reproducibility . The con-valescent serum produced IgG recognition patterns contain-ing 40 spots in the whole cell protein preparation with 15 spots in the membrane protein fraction. These spots were identified and they corresponded to 20 different proteins and 9 different proteins with post-translational modifica -tions. Nine immunogenic proteins (heat shock protein 70; cytosol aminopeptidase family, catalytic domain protein; L-lactate dehydrogenase; four members of the pyruvate dehydrogenase complex; NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; elongation factor Tu) thatwere identified in the membrane protein fraction were also identified in the whole cell protein preparation. No Mccp proteins have been previously annotated, so we performed a BLAST search of the identified proteins using the sequences obtained. The results showed that the homology to proteins from related species was all>92.5%. In particular, 9 pro -teins had homologies>99%, as follows: 4 members of the pyruvate dehydrogenase complex, heat shock protein 70, transketolase, elongation factor G, phosphoenolpyruvate-protein phosphotransferase, and glutamyl-tRNA (Gln) ami-dotransferase subunit A. Others had homologies>98%, such as L-lactate dehydrogenase, cytosol aminopeptidase family catalytic domain protein, aldehyde dehydrogenase (NAD) family protein, thioredoxin reductase (NADPH), elongation factor Tu, and peptidase M24 family. The homologies of the remaining proteins were between 92.5% and 96.5% (Table 1 and Table 2).DISCUSSIONWe have described the results of an immunoproteome-based analysis of Mccp pathogenic strain M1601. One-dimensional SDS-PAGE and immunoblot analysis identified 3 principal immunogenic proteins, with molecular masses in the range 29–97.2 kDa, 2 of which were also present in the membrane protein fraction. The whole cell protein 2DE facilitated the resolution of over 300 protein spots in the pH range 3–10, with 150 protein spots in the membrane protein fraction. However, multiple spots may correspond to a single gene, if post-translational modification occurs, e.g., chemi -cal modification or proteolytic cleavage [4, 15, 25].ThisFig. 1. Western-blot of antiserum of Mycoplasma capricolum subsp. Capripneumoniae (the detected immunogenic proteins were marked with arrows) Mr: Protein molecular weight marker;1, 2:M1601 whole cell Protein sample;3, 4:M1601 membrane protein fraction; 5, M1601 whole cell Protein sample against pre-infection serum; 6, M1601 membrane protein fraction against pre-infection serum.P. ZHAO ET AL.1112Fig. 3. a. 2DE gels of the membrane protein fraction from Mycoplasma capricolum subsp. capripneumoniae strain M1601. Proteins were separated by IEF using 17 cm IPG strips, pH 3–10, followed by SDS-PAGE on 12% gels, and stained with 0.1% Coomassie Brilliant Blue R250. The approximate molecular weights are shown on the right of the gel and the acid to alkaline gradient is from left to right. The spot numbers correspond to those identified by MS and are listed in Table 2. b. Identification of immunogenic proteins from the membrane protein fraction of Mycoplasma capricolum subsp. capripneu-moniae strain M1601. Proteins resolved by 2DE as in Fig. 3.a were electroblotted onto PVDF membranes, and probed with an anti-Mccp goat antiserum (1:50 dilution). Anti-goat IgG HRP (1:3,000 dilution) was used to develop antigen-antibodyreactions. The acid to alkaline gradient is from left to right. Groups of immunogenic spots are indicated by numbers.Fig. 2. a. 2DE gels of whole cell protein sample from Mycoplasma capricolum subsp. capripneumoniae strain M1601. Proteins were separated by IEF using 17 cm IPG strips, pH 3–10, followed by SDS-PAGE on 12% gels, and stained with 0.1% Coomassie Brilliant Blue R250. The approximate molecular weights are shown on the right of the gel and the acid to alkaline gradient is from left to right. The spot numbers correspond to those identified by MS and are listed in Table1. b. Identification of immunogenic proteins from the whole cell protein sample from Mycoplasma capricolum subsp. capri-pneumoniae strain M1601. Proteins resolved by 2DE as in Fig. 2.a were electroblotted onto PVDF membranes, and probed with an anti-Mccp goat antiserum (1:50 dilution). Anti-goat IgG HRP (1:3,000 dilution) was used to develop antigen-anti -body reactions. The acid to alkaline gradient is from left to right. Groups of immunogenic spots are indicated by numbers.IDENTIFICATION OF NOVEL IMMUNOGENIC PROTEINS1113result was similar to that reported from proteomic studies of other mycoplasmae [7, 9, 18, 20, 22, 24].We identified 20 novel Mccp antigens in the whole cell protein preparation while 9 novel antigens were identified in the membrane protein fraction. The analysis showed that these proteins had>92.5% homology to proteins from related species. In particular, 9 proteins all had>99% homol-ogy to proteins in the database, as follows: 4 members of the pyruvate dehydrogenase complex, heat shock protein 70, transketolase, elongation factor G, phosphoenolpyruvate-protein phosphotransferase, and glutamyl-tRNA (Gln) ami-dotransferase subunit A. Others had homology>98%, such as L-lactate dehydrogenase, cytosol aminopeptidase family catalytic domain protein, aldehyde dehydrogenase (NAD) family protein, thioredoxin reductase (NADPH), elongation factor Tu, and peptidase M24 family. The homologies of the remaining proteins were between 92.5% and 96.5%. Previ-ous studies have demonstrated the immunogenicity of the pyruvate dehydrogenase complex. Pyruvate dehydrogenase appears to be located on the cell surface where it mediates adhesion in some mycoplasmae and other bacteria [3, 5, 6, 10, 12, 14, 17, 19, 23]. Elongation factor Tu has recently been described as immunogenic, and it is also surface-localized, mediating M. pneumoniae binding to fibronectin [5]. Heat shock protein 70 has been reported to be surface-localized in other mycoplasmae where it mediates host receptor binding [2]. Another vaccine candidate is glyceral-dehyde-3-phosphate dehydrogenase in M. pulmonis, which is surface-localized and capable of binding to mucin [1]. The conserved sequence of this protein makes it a possible vaccine candidate. Interestingly, 4 members of the pyruvate dehydrogenase complex, elongation factor Tu, heat shockTable 1. Proteins identified in the whole cell protein sample by MS that reacted with convalescent sera in the immunoblotting experimentsLocus tag in corresponding genome/ Gene name Protein name/origin Spot no.Mycoplasma capricolumsubsp. capripneumoniaestrain M1601 identity (%)MSC_0532/ldh L-lactate dehydrogenase/Mycoplasma mycoides subsp. mycoides SC str. PG120/2198MSC_0265/pdhA Pyruvate dehydrogenase E1 component alpha subunit/Mycoplasma mycoides subsp. mycoides SC str. PG115/16/1799.1MCAP_0226/pdhB Pyruvate dehydrogenase complex E1component beta subunit/Mycoplasma capricolum subsp. capricolum ATCC 273432299.7MCAP_0227/pdhC Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydro-genase complex/Mycoplasma capricolum subsp. capricolum ATCC 273438/999.2MCAP_0228/pdhD Dihydrolipoamide dehydrogenase/Mycoplasma capricolum subsp. Capricolum ATCC 2734329/30/3199.4MCAP_0369/dnaK Heat shock protein 70/Mycoplasma capricolum subsp. capricolum ATCC 2734313/1499.4MSB_A0207Cytosol aminopeptidase family, catalytic domain protein/Mycoplasma leachii PG501198.2MCAP_0461Aldehyde dehydrogenase (NAD) family protein/Mycoplasma capricolum subsp. capricolum ATCC 273431298.3MCAP_0610/tkt Transketolase/Mycoplasma capricolum subsp. capricolum ATCC 273432599.1MSC_0938/trxB Thioredoxin reductase (NADPH)/Mycoplasma mycoides subsp. mycoides SC. PG12398.3MSC_0263/nox NADH oxidase/Mycoplasma mycoides subsp. mycoides SC str. PG11093.8MSC_0214/tufA Elongation factor Tu /Mycoplasma mycoides subsp. mycoides SC str. PG118/1998MCAP_0153/fusA Elongation factor G /Mycoplasma capricolum subsp. capricolum ATCC 273431/2/399.7MSB_A0355Peptidase, M24 family/Mycoplasma leachii PG502498.9MSB_A0517Putative RNA polymerase sigma factor RpoD/Mycoplasma leachii PG503294.5MCAP_0233/ptsI Phosphoenolpyruvate-protein phosphotransferase/Mycoplasma capricolum subsp. capricolum ATCC2734326/27/2899.0MMCAP1_0508Glutamyl-tRNA (Gln) amidotransferase subunit A/Mycoplasma mycoides subsp. capri str. GM1233 100MMCAP1_0451NADP-dependent glyceraldehyde-3-phosphate dehydrogenase/Mycoplasma mycoides subsp. capri str. GM1238/39/4096.5MMS_A1021DNA topoisomerase/Mycoplasma mycoides subsp. mycoides SC str. Gladysdale4/5/6/796.1MMCAP1_0064 /lysS Lysine-tRNA ligase/Mycoplasma mycoides subsp. capri str. GM1234/35/36/3792.5P. ZHAO ET AL. 1114protein 70, and glyceraldehyde-3-phosphate dehydrogenase were all identified in the whole cell protein preparation and membrane protein fractions in Mccp. This confirms that membrane proteins are the primary immunogenic proteins and these proteins are abundant in the cell.We identified only 9 immunogenic proteins in the mem-brane protein fraction, perhaps because of limiting factors including: (i) the dynamic range of 2DE prevents the de-tection of low abundance proteins; (ii) difficulties with the solubilization of membranes; and (iii) the poor resolution of basic proteins and proteins with a molecular mass<10 kDa or>150 kDa [20]. Thus, different approaches are required to investigate Mccp further, such as enrichment using Triton X-114 fractionation [15], synthesis of potential immuno-genic peptide epitopes [8], or the heterologous expression of potential antigens.In order to elucidate the exact role of these potential colonization factors and their use as vaccine antigens, fu-ture experiments should focus on the dynamics of humoral immune responses during the disease course and the differ-ences between infected animals with acute disease and those that recover.The functions of only a few Mccp proteins have been re-ported. Thus, the immunogenic proteins described here will facilitate comparative studies to identify their localization and their role in colonization by this important disease. ACKNOWLEDGMENTS. We are grateful for funding from the State Key Laboratory of Veterinary Etiological Biol-ogy, The Technology R and D Program of Gansu Province (1011NKCA054), Gansu Province Agricultural Biotech-nology Research and Application Development Project (GNSW-2010-09, The National Scientific-Basic Special Fund (2008FY210200), and China Agriculture Research System (CARS-39), The Natural Science Foundation of Gansu Province (1107RJZA107).REFERENCES1. Alvarez, R. A., Blaylock, M. W. and Baseman, J. B. 2003.Surface localized glyceraldehyde-3-phosphate dehydrogenase of Mycoplasma genitalium binds mucin. Mol. Microbiol.48: 1417–1425. [Medline] [CrossRef]2. Boulanger, J., Faulds, D., Eddy, E. M. and Lingwood, C. A.1995. Members of the 70 kDa heat shock protein family spe-cifically recognize sulfoglycolipids: role in gamete recognition and mycoplasma-related infertility. J. Cell. Physiol.165: 7–17.[Medline] [CrossRef]3. Burnett, T. A., Dinkla, K., Rohde, M., Chhatwal, G. S., Up-hoff, C., Srivastava, M., Cordwell, S. J., Geary, S., Liao, X., Minion, F. C., Walker, M. J. and Djordjevic, S. P. 2006. P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae:defined domains of P159 bind heparin and pro-mote adherence to eukaryote cells. Mol. Microbiol.60: 669–686.[Medline] [CrossRef]4. Cannistraro, V. J. and Kennell, D. 1991. RNase I*, a form ofRNase I, and mRNA degradation in Escherichia coli. J. Bacte-riol.173: 4653–4659. [Medline]5. Dallo, S. F., Kannan, T. R., Blaylock, M. W. and Baseman, J.B. 2002. Elongation factor Tu and E1 beta subunit of pyruvatedehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae. Mol. Microbiol.46: 1041–1051.[Medline][CrossRef]6. De Berardinis, P. and Haigwood, N. L. 2004. New recombinantvaccines based on the use of prokaryotic antigen-display sys-tems. Expert Rev. Vaccines3: 673–679. [Medline][CrossRef]7. Ferrer-Navarro, M., Gomez, A., Yanes, O., Planell, R., Aviles,F. X., Pinol, J., Perez Pons, J. A. and Querol, E. 2006. Proteomeof the bacterium Mycoplasma penetrans. J. Proteome Res.5: 688–694. [Medline][CrossRef]8. Frank, R. 2002. The SPOT-synthesis technique. Synthetic pep-tide arrays on membrane supports–principles and applications. J.Immunol. Methods267: 13–26. [Medline] [CrossRef]Table 2. Proteins identified in the membrane protein fraction by MS that reacted with convalescent sera in the immunoblotting experimentsLocus tag in corresponding genome/ Gene name Protein name/origin Spot no.Mycoplasma capricolumsubsp. capripneumoniaestrain M1601 identity (%)MCAP_0369/dnaK Heat shock protein 70/Mycoplasma capricolum subsp. capricolum ATCC 27343899.4MSB_A0207Cytosol aminopeptidase family, catalytic domain protein/Mycoplasma leachii PG50998.2MSC_0532/ldh L-lactate dehydrogenase/Mycoplasma mycoides subsp. mycoides SC str. PG111/1398MSC_0265/pdhA Pyruvate dehydrogenase E1 component alpha subunit/Mycoplasma mycoides subsp. mycoides SC str. PG1799.1MCAP_0226/pdhB Pyruvate dehydrogenase complex E1component beta subunit/Mycoplasma capricolum subsp. capricolum ATCC 273431299.7MCAP_0227/pdhC Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydroge-nase complex/Mycoplasma capricolum subsp. capricolum ATCC 27343699.2MCAP_0228/pdhD Dihydrolipoamide dehydrogenase/Mycoplasma capricolum subsp. Capricolum ATCC 273431/2/3/4/599.4MMCAP1_0451NADP-dependent glyceraldehyde-3-phosphate dehydrogenase/Mycoplasma mycoides subsp. capri str. GM121096.5MSC_0214/tufA Elongation factor Tu /Mycoplasma mycoides subsp. mycoides SC str. PG114/1598IDENTIFICATION OF NOVEL IMMUNOGENIC PROTEINS11159. Jaffe, J. D., Berg, H. C. and Church, G. M. 2004. Proteogenomicmapping as a complementary method to perform genome an-notation. Proteomics4: 59–77. [Medline] [CrossRef]10. Layh-Schmitt, G., Podtelejnikov, A. and Mann, M. 2000. Pro-teins complexed to the P1 adhesin of Mycoplasma pneumoniae.Microbiology146: 741–747. [Medline]11. Lebeau, I., Lammertyn, E., De Buck, E., Maes, L., Geukens, N.,Van Mellaert, L., Arckens, L., Anne, J. and Clerens, S. 2005.First proteomic analysis of Legionella pneumophila based on its developing genome sequence. Res. Microbiol.156: 119–129.[Medline][CrossRef]12. Lopez, J. E., Siems, W. F., Palmer, G. H., Brayton, K. A., Mc-Guire, T. C., Norimine, J. and Brown, W. C. 2005. Identification of novel antigenic proteins in a complex Anaplasma marginale outer membrane immunogen by mass spectrometry and genomic mapping. Infect. Immun.73: 8109–8118. [Medline][CrossRef] 13. MacOwan, K. J. and Minette, J. E. 1976. A mycoplasma fromacute contagious caprine pleuropneumonia in Kenya. Trop.Anim. Health Prod.8: 91–95. [Medline][CrossRef]14. Marques, M. A., Chitale, S., Brennan, P. J. and Pessolani, M.C. 1998. Mapping and identification of the major cell wall-associated components of Mycobacterium leprae. Infect. Immun.66: 2625–2631. [Medline]15. Meens, J., Selke, M. and Gerlach, G. F. 2006. Identification andimmunological characterization of conserved Mycoplasma hyo-pneumoniae lipoproteins Mhp378 and Mhp651. Vet. Microbiol.116: 85–95. [Medline][CrossRef]16. Molloy, M. P., Herbert, B. R., Slade, M. B., Rabilloud, T.,Nouwens, A. S. and Williams, K. L. 2000. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem.267: 2871–2881. [Medline][CrossRef]17. Olson, L. D., Renshaw, C. A., Shane, S. W. and Barile, M. F.1991. Successive synovial Mycoplasma hominis isolates exhibit apparent antigenic variation. Infect. Immun.59: 3327–3329.[Medline]18. Pinto, P. M., Chemale, G., de Castro, L. A., Costa, A. P., Kich,J. D., Vainstein, M. H., Zaha, A. and Ferreira, H. B. 2007. Pro-teomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins. Vet. Microbiol.121: 83–93.[Medline] [CrossRef]19. Regula, J. T., Boguth, G., Gorg, A., Hegermann, J., Mayer, F.,Frank, R. and Herrmann, R. 2001. Defining the mycoplasma ‘cytoskeleton’: the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry.Microbiology147: 1045–1057. [Medline]20. Regula, J. T., Ueberle, B., Boguth, G., Gorg, A., Schnolzer, M.,Herrmann, R. and Frank, R. 2000. Towards a two-dimensional proteome map of Mycoplasma pneumoniae. Electrophoresis21: 3765–3780. [Medline] [CrossRef]21. Thiaucourt, F., Bolske, G., Leneguersh, B., Smith, D. and We-songa, H. 1996. Diagnosis and Control of Contagious Caprine Pleuropneumonia. Rev. Sci. Tech.15: 1415–1429. [Medline] 22. Ueberle, B., Frank, R. and Herrmann, R. 2002. The proteomeof the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products. Proteomics2: 754–764. [Medline] [CrossRef]23. Wallbrandt, P., Tegman, V., Jonsson, B. H. and Wieslander, A.1992. Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Achole-plasma laidlawii. J. Bacteriol.174: 1388–1396. [Medline] 24. Wasinger, V. C., Pollack, J. D. and Humphery-Smith, I. 2000.The proteome of Mycoplasma genitalium. Chaps-soluble com-ponent. European. Eur. J. Biochem.267: 1571–1582. [Medline] [CrossRef]25. Zhou, M., Zhang, A., Guo, Y., Liao, Y., Chen, H. and Jin, M.2009. A comprehensive proteome map of the Haemophilus para-suis serovar 5. Proteomics9: 2722–2739. [Medline] [CrossRef]26. Zhou, M., Guo, Y., Zhao, J., Hu, Q., Hu, Y., Zhang, A., Chen,H. and Jin, M. 2009. Identification and characterization of novelimmunogenic outer membrane proteins of Haemophilus parasuis serovar 5. Vaccine27: 5271–5277. [Medline] [CrossRef]。
