CN1473166A - 犬埃里希体的同源28kD免疫优势蛋白质基因及其用途 - Google Patents
犬埃里希体的同源28kD免疫优势蛋白质基因及其用途 Download PDFInfo
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Abstract
本发明针对犬埃里希体的一个多形性多基因家族的同源免疫反应性28kDa蛋白质基因,p28-1、-2、-3、-5、-6、-7、-9的克隆、测序和表达。还公开了编码犬埃里希体所有9个同源28kDa蛋白质基因的多基因基因座。重组犬埃里希体28kDa蛋白质与犬埃里希体感染的犬的恢复期抗血清反应,可用于开发对于疾病预防和血清诊断特别有效的疫苗和血清诊断法。
Description
发明背景
发明领域
本发明一般涉及分子生物学领域。本发明更具体涉及犬埃里希体中同源28-kDa蛋白质基因的分子克隆和特征确定,以及编码犬埃里希体该28kDa同源蛋白质的多基因基因座及其用途。
相关领域的描述
犬埃里希体病也称作犬热带全血细胞减少症,是一种犬扁虱携带的立克次氏体疾病,首次于1935年在非洲和1963年在美国发现(Donatien和Lestoquard,1935;Ewing,1963)。该疾病在越战期间,美国军犬中流行性爆发后被更好的认识(Walker等,1970)。
犬埃里希体病的病因是犬埃里希体,一种小革兰氏阴性专性胞内细菌,显示对单核吞噬细胞的嗜性(Nyindo等,1971),并由棕色犬虱(Rhipicephalussanguineus)传播(Groves等,1975)。犬埃里希体病的进展有三个阶段:急性、亚临床和慢性。急性阶段的特征是发热、食欲减退、抑郁、淋巴结病和中度血小板减少(Troy和Forrester,1990)。犬通常从急性期恢复,但是变成在几个月或甚至几年中成为该病菌的持续传染携带者,而没有临床症状(Harrus等,1998)。某些犬进入慢性阶段,特征是血小板减少、高球蛋白血症、食欲减退、消瘦和出血,特别是鼻出血,然后死亡(Troy和Forrester,1990)。
表面抗原性的调节可能是在宿主中建立这种持续感染的重要机制。虽然对该病的发病机制了解得很少,但在相关的埃里希体属、无形体属、考德里体属的成员中所述的多基因家族可能涉及主要表面抗原表达的改变,从而逃脱免疫监视。着边无形体(Anaplasma marginale),一种与犬埃里希体密切相关的生物,显示了主要表面蛋白3(msp-3)基因的改变,导致菌株之间的抗原多态性(Alleman等,1997)。
基于16SrRNA基因的分子分类学分析,确定了犬埃里希体和恰菲埃里希体(人单核细胞埃里希体病(HME)的病因)是密切相关的(Anderson等,1991;Anderson等,1992;Dawson等,1991;Chen等,1994)。报道了犬埃里希体和恰菲埃里希体的64、47、40、30、29和23kDa抗原之间可观的交叉反应性(Chen等,1994;Chen等,1997;Rikihisa等,1994;Rikihisa等,1992)。用人和犬恢复期血清作免疫印迹分析免疫反应性抗原,鉴定出许多犬埃里希体的免疫优势蛋白质,包括30kDa蛋白(Chen等,1997)。另外,已描述犬埃里希体的30kDa蛋白质是免疫应答早期识别的主要免疫优势抗原,它与恰菲埃里希体的30kDa蛋白质是不同的抗原(Rikihisa等,1992;Rikihisa等,1994)。犬埃里希体的其它分子量在20-30kDa之间的免疫优势蛋白质也已被鉴定(Brouqui等,1992;Nyindo等,1991;Chen等,1994;Chen等,1997)。
已在相关生物,包括恰菲埃里希体和反刍类考德里氏体(Cowdria ruminantium)中报道了多基因家族编码的同源28-32kDa免疫优势蛋白质(Sulsona等,1999;Ohashi等,1998a;Reddy等,1998)。近来,描述了恰菲埃里希体中编码23-28kDa蛋白质的21个成员的多基因家族(Yu等,2000)。恰菲埃里希体28kDa外膜蛋白质是表面暴露的,含有三个主要超变区(Oashi等,1998a)。重组恰菲埃里希体P28似乎在小鼠中提供了抵抗同源攻击感染的保护,针对重组蛋白产生的抗血清与犬埃里希体的30kDa蛋白质交叉反应(Ohashi等,1998a)。报道了恰菲埃里希体分离物中p28基因中的多样性(Yu等,1999a),用单克隆抗体的研究进一步证明了表达的P28蛋白质中的多样性(Yu等,1993)。相反,报道了犬埃里希体地理学不同的分离物中p28基因完全保守,提示犬埃里希体在北美洲中可能是保守的(McBride等,1999,2000)。
现有技术的缺陷在于不能克隆和确定犬埃里希体的新的同源性28kDa免疫反应性蛋白质基因,和含有同源28kDa蛋白质基因的单个多基因基因座的特征。另外,现有技术的缺陷在于缺乏犬埃里希体免疫反应性基因的重组蛋白质。本发明填补了本领域这一长期存在的需要。
发明内容
本发明的一些实施例描述了犬埃里希体同源成熟28kDa免疫反应性蛋白质基因(称为p28-1、-2、-3、-5、-6、-7、-9)的分子克隆、测序、特征确定和表达,以及对含有9种犬埃里希体28kDa蛋白质基因(p28-1到-9)的一个基因座的鉴定(10677bp)。p28基因中的8种位于一条DNA链上,一种p28基因在互补链上。9种p28基因成员中的核酸同源性是37-75%,氨基酸同源性是28-72%。
在本发明的一个实施例中,提供了编码犬埃里希体30kDa免疫反应性蛋白质的DNA序列。优选蛋白质具有选自SEQ ID NO:2、4、6、40、42、44、46的氨基酸序列,基因具有选自SEQ ID NO:1、3、5、39、41、43、45的核酸序列,是多态性多基因家族的成员。通常蛋白质具有N-末端信号肽,它在翻译后加工中被切下,产生成熟28-kDa蛋白质。另外,编码28kDa蛋白质的基因优选包含在一个多基因座中,它的大小是10,677bp,编码9种犬埃里希体的同源28kDa蛋白质。
在本发明的另一个实施例中,提供了一种表达载体,它含有编码犬埃里希体28-kDa免疫反应性的基因,并能在引入细胞时表达该基因。
在本发明的另一个实施例中,提供了一种重组蛋白质,它具有选自SEQ IDNO:2、4、6、40、42、44、46的氨基酸序列。优选该氨基酸序列是由选自SEQ ID NO:1、3、5、39、41、43、45的核酸序列编码的。优选该重组蛋白质含有4个可变区,它们可以是暴露于表面、亲水和具有抗原性。重组蛋白质可用作抗原。
在本发明的另一个实施例中,提供了一种产生重组蛋白质的方法,包括步骤:获得一种载体,它含有一个表达区,其中含有与启动子可操纵性连接的、编码选自SEQ ID NO:2、4、6、40、42、44、46的氨基酸序列的序列;将该载体转染入细胞;在有效表达该表达区的条件下培养细胞。
还在一些实施例中描述了本发明,作为在个体中抑制犬埃里希体感染的方法,包括步骤:在接触前或怀疑已接触,或已感染犬埃里希体时鉴定个体;和以抑制犬埃里希体感染有效量施用含有犬埃里希体28kDa抗原的组合物。抑制可通过任何方式,例如刺激个体的体液或细胞免疫应答反应,或通过其它方法,例如抑制28kDa抗原的正常功能,或甚至与抗原竞争与个体体内的一些因子相互作用发生。
可从下文为了公开而提供的本发明优选例的描述中,更加清楚的了解本发明的其它方面、特征和优点。
附图简述
因此,将获得并详细理解本发明上述特征、优点和目的中的内容,以及其它将变得清楚的内容,更具体的、对本发明上面简述的内容将参考某些在附图中说明的实施例。这些图是说明书的一部分。然而要注意的是,附图说明了本发明的优选例,因此不应认为是对其范围的限制。
图1显示了p28-7基因,包括邻近的5’和3’非编码序列的核酸序列(SEQ IDNO:1)和推测的氨基酸序列(SEQ ID NO:2)。ATG起始密码子和TAA终止子用黑体标示,23氨基酸前导信号序列下划线标示。
图2显示了表达的50kDa重组p28-7硫氧还蛋白融合蛋白质(泳道1,箭头)和16kDa硫氧还蛋白对照(泳道2,箭头)的SDS-PAGE,和重组p28-7-硫氧还蛋白融合蛋白用恢复期犬埃里希体犬抗血清的对应免疫印迹(泳道3)。犬埃里希体抗血清未检测到硫氧还蛋白对照(未显示)。
图3显示了p28-7蛋白质(ECa28-1,SEQ ID NO:2)、p28-5蛋白质(ECa28SA2,部分序列,SEQ ID NO:7)、p28-4蛋白质(ECa28SA1,SEQ ID NO:8)、恰菲埃里希体P28(SEQ ID NO:9)、恰菲埃里希体OMP-1家族(SEQ ID NO:10-14)和反刍类考德里氏体(C.ruminantium)MAP-1蛋白质(SEQ ID NO:15)的氨基酸序列的排列对比。p28-7氨基酸序列作为共有序列。未显示的氨基酸与p28-7相同,用点表示。用对应的一字母缩写显示分歧的氨基酸。为了氨基酸序列的最大排列引入的缺口用长划线标出。可变区用下划线标出并命名为(VR1、VR2、VR3和VR4)。箭头表示信号肽的推测的信号肽酶切割位点。
图4显示了用非平衡树构建从推测的氨基酸序列得到的犬埃里希体p28-7(Eca28-1)、p28-5(ECa28SA2,部分序列)、p28-4(ECa28SA1),恰菲埃里希体omp-1多基因家族的成员,和反刍类考德里氏体map-1蛋白质的系统发生学相关性。每对分枝的长度代表配对氨基酸序列之间的距离。比例尺测定序列之间的距离。
图5显示了用6种不同的限制性酶完全消化犬埃里希体基因组DNA,并与p28-7DIG标记的探针杂交的Southern印迹分析(泳道2-7);DIG标记的分子量标记(泳道1和8)。
图6显示了犬埃里希体p28-7的预测的蛋白质特征(ECa28-1,Jake株)和恰菲埃里希体P28(Arkansas株)的比较。用6肽窗口,表面可能性预测了表面残基。表面残基是任何具有2.0nm2以上的水可接触表面积的残基。预测值高于1的六肽被视为表面区。抗原性指数预测可能的抗原决定簇。指数值大于0的区域是可能的抗原决定簇。T-细胞基序用5个氨基酸的基序(残基1-甘氨酸或极性氨基酸,残基2-疏水氨基酸,残基3-疏水氨基酸,残基4-疏水氨基酸或脯氨酸,和残基5-极性或甘氨酸),确定了可能的T-细胞抗原决定簇的位置。比例尺表示氨基酸位置。
图7显示了犬埃里希体28-kDa蛋白质基因p28-5(核苷酸1-849:SEQ ID NO:3;氨基酸序列:SEQ ID NO:4)和p28-6(核苷酸1195-2031:SEQ ID NO:5;氨基酸序列:SEQ ID NO:6),包括基因间非编码序列(NC2,核苷酸850-1194:SEQ ID NO:31)的核酸序列和推测的氨基酸序列。ATG起始密码子和TAA终止子用黑体标示。
图8显示了犬埃里希体28kDa蛋白基因的基因座(5.592Kb,含有5个基因)示意图,表明基因组的朝向和基因间非编码区域(28NC1-4)。基因座1和2(阴影)显示的28kDa蛋白质已被描述过(McBride等,1999;Reddy等,1998;Ohashi等,1998)。p28-5的完整序列和新的28kDa蛋白质基因(称为p28-6)已测定了序列。完成了p28-5、p28-6和p28-7之间非编码基因间区(28NC2-3)与先前未连接的基因座1和2的连接。
图9显示采用非平衡树构建法根据氨基酸序列得到的犬埃里希体p28-4(Eca28SA1)、p28-5(ECa28SA2)、p28-6(ECa28SA3),p28-7(ECa28-1)和p28-8(ECa28-2)的系统发生学相关性。每对分枝的长度代表配对氨基酸序列之间的距离。比例尺测定序列之间的距离。
图10显示了犬埃里希体28kDa蛋白质基因基因间非编码核酸序列(SEQ IDNO:30-33)的排列对比。用点(.)表示的未显示的核酸与非编码区1(28NC1)相同。用对应的一字母缩写显示分歧的氨基酸。为了氨基酸序列的最大排列对比,引入的缺口用长划线(-)标出。推测的转录启动子区(-10和-35)以及核糖体结合位点(RBS)加框标示。
图11显示了9种犬埃里希体基因p28基因座(10,677bp)的示意性图,表明基因组朝向和基因间非编码区域。实施例8中鉴定了p28基因(p28-1、2、3、9)(没有阴影)。先前鉴定了阴影标示的p28基因,并命名如下:p28-4,p30a(Ohashi等,1998b)和ORF1(Reddy等,1998);p28-5和p28-6(McBride等,2000);p28-7、p28(McBride等,1999)和p30(Ohashi等,1998b);和p28-8、p30-1(Ohashi等,1998b)。
图12显示了犬埃里希体P28-1到P28-9根据氨基酸序列的系统发生关系。每对分枝的长度代表配对氨基酸之间的距离。比例尺测定序列之间的分歧百分数。
图13显示了犬埃里希体p28-1基因的核酸序列(SEQ ID NO:39)和推测的氨基酸序列(SEQ ID NO:40)。
图14显示了犬埃里希体p28-2基因的核酸序列(SEQ ID NO:41)和推测的氨基酸序列(SEQ ID NO:42)。
图1 5显示了犬埃里希体p28-3基因的核酸序列(SEQ ID NO:43)和推测的氨基酸序列(SEQ ID NO:44)。
图16显示了犬埃里希体p28-9基因的核酸序列(SEQ ID NO:45)和推测的氨基酸序列(SEQ ID NO:46)。
发明详述
本发明描述了编码犬埃里希体30-kDa蛋白质的同源性基因的克隆、测序和表达。还进行了7种犬埃里希体分离物和恰菲埃里希体omp-1多基因家族中的同源性基因的比较性分子分析。如下鉴定了几种新的28-kDa蛋白质基因:
p28-7(ECa28-1)具有一个834bp的开放阅读框,编码具有30.5kDa的预测分子量的278个氨基酸的蛋白质(SEQ ID NO:2)。鉴定出一个N-末端信号序列,提示蛋白质经过翻译后修饰变成27.7kDa的成熟蛋白质。
P28-6(ECa28SA3)具有840bp的开放阅读框,编码280氨基酸的蛋白质(SEQ IDNO:6)。
用PCR扩增犬埃里希体的28kDa蛋白质基因,得到先前未曾测序的p28-5(Eca28SA2)区域。p28-5的序列分析揭示有一个编码283个氨基酸蛋白质(SEQ IDNO:4)的849bp的开放阅读框。
采用对28kDa蛋白质基因的基因间非编码区特异性的引物作PCR扩增,对连接先前分离的基因座的区域测序,从而鉴定了一个含有5种28kDa蛋白质基因(p28-4、-5、-6、-7和-8)的基因座(5.592-kb)。预测该5种28kDa蛋白质具有信号肽,可得到成熟蛋白质,并且具有51-72%氨基酸同源性。分析了基因间区,揭示各基因的假设启动子区,提示这些基因可独立表达和差异性表达。基因间非编码区(28NC1-4)的大小是299-355bp,具有48-71%的同源性。
另外,对串联排列的p28基因上述5个基因的基因座的DNA上游和下游先前未知区域进行了测序,鉴定到p28-1、-2、-3和-9。因此,本发明鉴定到了横跨10,677bp的9基因犬埃里希体p28基因座。
本发明特别针对犬埃里希体的同源28kDa蛋白质基因p28-1、-2、-3、-6、-7和p28-9,和先前部分测序的p28-5的完整序列。还公开了编码犬埃里希体的9种同源28kDa外膜蛋白的多基因基因座。8种p28基因位于一条DNA链上,在互补链上发现了一个p28基因。9个p28基因成员之间的核酸同源性是37-75%,氨基酸同源性范围是28-72%。
根据本发明,可以使用本领域能力范围内的常规分子生物学、微生物学和重组DNA技术。这些技术在文献中有完整的解释,见例如Maniatis,Fritsch &Sambrook,"Molecular Cloning:A Laboratory Manual(1982);"DNA Cloning:APractical Approach,"卷I和II(D.N.Glover ed.1985);"Oligonucleotide Synthesis"(M.J.Gait ed.1984);"Nucleic Acid Hybridization"[B.D.Hames & S.J.Higgins eds.(1985)];"Transcription and Translation"[B.D.Hames & S.J.Higgins eds.(1984)];"Animal Cell Culture"[R.I.Freshney,ed.(1986)];"Immobilized Cells And Enzymes"[IRL Press,(1986)];B.Perbal,"A Practical Guide To Molecular Cloning"(1984)。
本发明包括基本纯的DNA,它编码犬埃里希体的28kDa免疫反应性蛋白质。本发明的DNA编码的蛋白质可以与SEQ IDNO:2、4、6、40、42、44或46中列出的氨基酸具有至少80%(优选85%,更优选90%,和最优选95%)的序列相同性。更优选的,该DNA包括SEQ IDNO:1、3、5、39、41、43、45的核苷酸编码序列,或这些序列的简并变体。
本领域熟知蛋白质的氨基酸序列是由编码该蛋白质的DNA的核苷酸序列确定的。由于遗传密码的简并性(即对于大多数氨基酸而言,一种氨基酸有一种以上的核苷酸三联体(密码子)),不同的核苷酸序列可以编码特定的氨基酸或多肽。因此,本发明的多核苷酸序列还包含那些编码本发明多肽或其片段或变体的简并序列。
本发明还包括基本纯的DNA,它含有SEQ ID NO:1、3、5、39、41、43或45所列出的核苷酸区域的至少15个连续核苷酸(优选20,更优选30,甚至更优选50和最优选全部)序列。
“基本纯的DNA”指通过将DNA的一些或全部分子从其天然存在的微环境中分离出来,或通过改变本发明DNA的侧接序列,而分离出来(部分或完全纯化)的DNA。因此该术语包括例如掺入载体、自主复制质粒或病毒、或原核细胞或整合细胞的基因组DNA的重组DNA;或独立于其它序列作为单个分子存在的DNA(例如cDNA或基因组或聚合酶链式反应(PCR)或限制性内切酶消化产生的cDNA片段)。还包括重组DNA,它是编码其它多肽序列,例如融合蛋白的杂交基因的一部分。本发明还包括重组DNA,它包括编码犬埃里希体28kDa免疫反应性蛋白质的SEQ ID NO:1、3、5、39、41、43或45中列出的核苷酸的一部分。
DNA应该与SEQ ID NO:1、3、5、39、41、43或45中列出的核苷酸的编码序列具有至少约70%,优选至少75%(例如至少80%),最优选至少90%的序列相同性。两条序列之间的相同性是匹配或相同位置数目的直接函数。当两条序列中都有的亚基位置被同一个单体亚基占据,例如如果两条DNA分子中的给定的位置都被腺嘌呤占据,那么它们在该位置是相同的。例如,如果长10个核苷酸的序列中有7个位置与第二条10个核苷酸的序列中相应的位置相同,呢这么两条序列具有70%的序列相同性。比较序列长度通常至少是50个核苷酸,优选至少60个核苷酸,更优选至少75个核苷酸,最优选100个核苷酸。通常用序列分析软件(例如遗传学计算机小组的序列分析软件包,威斯康星大学生物技术中心,1710University Avenue,Madison,WI 53705)测定序列相同性。
本发明还包括含有编码犬埃里希体28kDa免疫反应性蛋白基因的DNA序列的载体,所述载体能在宿主中复制,含有可操纵性连接的:a)复制起始点;b)启动子;和c)编码所述蛋白质的DNA序列。优选本发明的载体含有SEQ ID NO:1、3、5、39、41、43或45所示的DNA序列的一部分。
“载体”可定义为可复制核酸构建物,例如质粒或病毒核酸。载体可以用于扩增和/或表达编码犬埃里希体28kDa免疫反应性蛋白的核酸。表达载体是一种可复制构建物,其中编码多肽的核酸序列与能影响多肽在细胞中的表达的合适控制序列可操纵性连接。对控制序列的需要可根据所选的细胞和所选的转化方法而变。通常控制序列包括转录启动子和/或增强子、合适的mRNA核糖体结合位点、和控制转录终止和翻译的序列。本领域技术人员熟知的方法可用于构建含有合适转录和翻译控制信号的表达载体。见例如Sambrook等,1989,Molecular Cloning:ALaboratory Manual(2nd Ed.),Cold Spring Harbor Press,N.Y.中描述的技术。如果转录控制序列有效控制基因的转录,基因及其转录控制序列定义为“可操纵性连接”。本发明的载体包括但不限于质粒载体和病毒载体。优选本发明的病毒载体是衍生自反转录病毒、腺病毒、腺伴随病毒、SV40病毒或疱疹病毒。
一般含有促进插入的DNA片段有效转录的启动子序列的表达载体与宿主联用。本文所用的术语“宿主”意味着不仅包括原核细胞,还包括真核细胞,例如酵母、植物和动物细胞。编码犬埃里希体28kDa免疫反应性蛋白质的重组DNA分子或基因可使用任何本领域一般技术人员已知的技术转化宿主。尤其优选的是用含有编码犬埃里希体28kDa免疫反应性蛋白的基因编码序列的载体转化原核细胞。
原核细胞宿主可以包括大肠杆菌(E.coli)、鼠伤寒沙门氏菌(S.tymphimurium)、粘质沙雷氏菌(Serratia marcescens)和枯草杆菌(Bacillus subtilis)。真核宿主包括酵母,例如巴斯德毕赤酵母(Pichia pastoris)、哺乳动物细胞和昆虫细胞。可以用本领域已知的方法发酵并培养转化的宿主,以实现最佳细胞生长。
本文所用的术语“工程改造的”或“重组”的细胞是指一种引入了编码犬埃里希体抗原的重组基因的细胞。因此,工程改造的细胞与不含有重组引入的基因的天然存在的细胞不同。工程改造的细胞是具有人工引入的一种或多种基因的细胞。重组引入的基因可以是cDNA基因、基因组基因拷贝形式,或包含的基因邻接一启动子,但不是天然与该引入基因相连。另外,重组基因可以整合入宿主基因组,或可以包含在载体、或转染入宿主细胞的细菌基因组中。
本发明还针对基本纯的犬埃里希体28-30kDa免疫反应性蛋白,它含有SEQ IDNO:2、4、6、40、42、44或46列出的氨基酸序列。
“基本纯的蛋白质”意味着从与其天然相伴随的至少一部分成分分开的蛋白质。通常当某蛋白质至少60%重量没有其它体内天然相伴的蛋白质和其它天然存在的有机分子时,蛋白质是基本纯的。优选蛋白质制品的纯度至少75%,更优选至少90%,最优选至少99%重量。可通过例如,从天然来源提取;表达编码犬埃里希体的28kDa免疫反应性蛋白的重组核酸;或化学合成该蛋白质来获得犬埃里希体的基本纯的28-kDa免疫反应性蛋白质。可通过任何合适的方法,例如使用对犬埃里希体的28kDa免疫反应性蛋白的特异性抗体进行免疫亲和层析等柱层析,聚丙烯酰胺凝胶电泳,或HPLC分析方法测定纯度。当将蛋白质从其天然状态伴随的至少一些污染物中分离出来时,该蛋白质基本没有天然伴随成分。因此,化学合成的或在不同于天然起源细胞的细胞系统中产生的蛋白质,定义为基本无天然伴随成分。因此,基本纯的蛋白质包括大肠杆菌、其它原核细胞或天然不存在的其它生物中合成的真核蛋白质。
除了基本上全长的蛋白质,本发明还包括犬埃里希体28kDa免疫反应性蛋白的片段(例如抗原片段)(SEQ ID NO:2、4、6、40、42、44或46)。本文所用的术语“片段”用于指一般长度至少10个残基,更通常至少20个残基,优选至少30(例如50)个残基,但比全长完整序列短的多肽。犬埃里希体28kDa免疫反应性蛋白的片段可以用本领域技术人员已知的方法产生,例如通过酶消化天然存在的或重组的犬埃里希体28kDa免疫反应性蛋白,或通过化学合成。可通过本文所述的方法评估候选片段显示犬埃里希体28kDa免疫反应性蛋白的特征的能力(例如,与犬埃里希体28kDa免疫反应性蛋白特异性抗体结合)。
可用纯化的犬埃里希体28kDa免疫反应性蛋白或犬埃里希体28kDa免疫反应性蛋白的抗原性片段产生新的抗体,或通过使用本领域技术人员已知的标准方法测试现有抗体(例如作为诊断试验中的阳性对照)。
如本领域熟知的那样,某给定的多肽其免疫原性可改变。因此常需将免疫原(例如本发明的多肽)与载体偶联。示范性和优选载体是匙孔血蓝蛋白(KLH)和人血清清蛋白。偶联多肽和载体蛋白质的方法是本领域熟知的,包括戊二醛、间马来酰亚胺苯甲酸-N-羟基琥珀酰亚胺酯、碳二亚胺和双固氮二氨基联苯胺法。还知道可通过本领域熟知的遗传工程技术将肽偶联于蛋白质。
本领域还已知可使用免疫应答的非特异性刺激剂(称为佐剂)来增强特定免疫原的免疫原性。示范性和优选的佐剂包括完全BCG、Detox,(RIBI,ImmunochemResearch Inc.)ISCOMS和氢氧化铝佐剂(Superphos,Biosector)。
本发明包括用犬埃里希体28kDa免疫反应性蛋白或犬埃里希体28kDa免疫反应性蛋白的片段作为免疫原,在例如家兔中产生多克隆抗血清。使用本领域技术人员已知的单克隆和多克隆抗体产生的标准方法。可筛选该方法产生的单克隆抗体是否具有鉴定重组犬埃里希体cDNA克隆,和将其与已知cDNA克隆区分开来的能力。
本发明不仅包括完整的单克隆抗体,还包括免疫活性抗体片段,例如Fab或(Fab)2片段;工程改造产生的单链Fv分子;或嵌合分子,例如含有一种抗体(如小鼠抗体)的结合特异性,和另一种抗体(例如人抗体)的剩余部分的嵌合抗体。
在一个实施例中,抗体或其片段可以与毒素或可检测标记,例如放射性标记、非放射性同位素标记、荧光标记、化学发光标记、顺磁标记、酶标记或产色标记连接。本领域一般技术人员应了解这些和其它适用于本发明的标记。这些标记与抗体或其片段的结合可用本领域普通技术人员所知的标准技术来完成。
还考虑了可用本发明的新颖蛋白质制备药物组合物。该药物组合物含有本发明的新颖活性组合物和药物学上可接受的载体。本领域一般技术人员无须过多实验不难确定施用本发明活性成分的合适剂量和给药途径。
词组“药物学上可接受的”指当给予个体时,不会产生过敏或类似不良反应的分子或组合物。含有作为活性成分的蛋白质的水相组合物的制备是本领域熟知的。通常,将这些组合物制备成可注射的溶液或悬液;也可制备成可在注射前溶解或悬浮于液体中的固态形式。还可乳化该制备物。
可将蛋白以中性或盐形式配制在组合物中。药物学上可接受的盐包括酸加成盐(由蛋白质的游离氨基形成),和与无机酸,如盐酸或磷酸,或乙酸、草酸、酒石酸、杏仁酸等有机酸形成的盐。还可与无机碱,如钠、钾、铵、钙或铁的氢氧化物,和有机碱,如异丙基胺、三甲基胺、组氨酸、普鲁卡因等衍生的游离羧基形成盐。
配制后,以与剂量配方兼容的方式,和治疗有效量施用这些溶液。不难以各种剂型,如可注射溶液施用这些制剂。
对于水溶液肠胃道外施药,如需要可适当缓冲该溶液,首先用足够的盐水或葡萄糖将稀释液变成等渗。这些具体水溶液特别适合静脉内、肌肉内、皮下和腹膜内施用。就此而言,本领域技术人员,根据本文公开内容将了解可使用无菌水溶液。例如,可将一剂量溶于1毫升等渗NaCl溶液中,或加到1000毫升作皮下灌注液或注射到预定输液部位(见例如“Remington药物科学”15版,1035-1038页和1570-1580)。根据待治疗个体的情况,须对剂量作某些改动。负责给药的人员在任何情况下都将能确定各个患者的合适剂量。
在本发明的一个实施例中,提供了编码犬埃里希体30kDa免疫反应性蛋白的DNA序列。优选该蛋白质具有选自SEQ ID NO:2、4、6、40、42、44、46的氨基酸序列,基因具有选自SEQ ID NO:1、3、5、39、41、43、45的核酸序列,是多态性多基因家族的成员。更优选的,该蛋白质具有N-末端信号序列,它在翻译后加工后切断,产生成熟的28kDa蛋白质。优选的是编码28kDa蛋白质的DNA包含在一个多基因基因座中,它的大小是10,677bp,编码9个犬埃里希体的同源28kDa蛋白。
在本发明的另一个实施例中,提供了一种表达载体,它含有编码犬埃里希体28kDa免疫反应性蛋白的基因,当引入细胞时该载体能表达该基因。
在本发明的另一个实施例中,提供了一种重组蛋白质,该蛋白质含有选自SEQID NO:2、4、6、40、42、44、46的氨基酸序列。优选氨基酸序列是由选自SEQ IDNO:1、3、5、39、41、3、45的核酸序列所编码。更优选该重组蛋白质含有4个表面暴露的、亲水和抗原性可变区。仍然优选的是该重组蛋白是一种抗原。
本发明的另一个实施例提供了一种产生该重组蛋白的方法,包括以下步骤:获得含有表达区的一载体,该表达区含有选自与启动子可操纵性连接的SEQ IDNO:2、4、6、40、42、44、46的氨基酸序列的编码序列;将载体转染入细胞,并在有效表达该表达区的条件下培养细胞。
在一些实施例中还描述了本发明抑制个体犬埃里希体感染的方法,包括步骤:鉴定怀疑已接触或感染犬埃里希体的个体;和给予能有效抑制犬埃里希体感染剂量的含有犬埃里希体28kDa抗原的组合物。该抑制作用可以通过任何方法,例如刺激个体的体液或细胞免疫应答,或通过其它途径,例如抑制28kDa抗原的正常功能,或甚至同该抗原竞争与个体体内一些的因子相互作用而发生。
给出下列例子,用于说明本发明的各种实施例,而不意味以任何方式限制本发明。
实施例1
ECa28-1(p28-7)基因的未知5’和3’区域的测序
埃里希体和纯化
Dr.Edward Breitschwerdt,(College of Veterinary Medicine,North Carolina StateUniversity,Raleigh,NC)提供了犬埃里希体(Florida株和分离物Demon,DJ,Jake,和Fuzzy)。Dr.Richard E.Corstvet(School of Veterinary Medicine,Louisiana StateUniversity,Baton Rouge,LA)提供了犬埃里希体(Louisana株),Dr.Jacqueline Dawson(Centers for Disease Control and Prevention,Atlanta,GA)提供了犬埃里希体(Oklahoma株)。以补充有10%胎牛血清和2mM L-谷氨酰胺的DMEM培养的DH82细胞中37℃进行埃里希体的增殖。用一般的细胞染色法观察犬埃里希体桑椹体的存在,监测在DH82细胞中的胞内生长。当100%细胞被埃里希体感染时,用细胞刮棒收集DH82细胞。17,000xg离心细胞20分钟。用Braun-Sonic2000超声发生器以40W30秒,在冰上破碎细胞沉淀两次。如前所述纯化埃里希体(Weiss E,1975)。将细胞裂解液加到42%-36%-30%泛影葡胺的不连续梯度上,80,000xg离心60分钟。收集重带和轻带中的埃里希体,并用蔗糖-磷酸-谷氨酰胺缓冲液(SPG,218mM蔗糖、3.8mM KH2PO4、7.2mM K2HPO4、4.9mM谷氨酰胺,pH7.0)洗涤,离心沉淀。
核酸制备
如前所述(McBride等,1996),将泛影葡胺纯化的埃里希体重新悬浮在600微升10mM Tris-HCl缓冲液(pH7.5)和1%十二烷基磺酸钠(SDS,w/v)和100ng/ml蛋白酶K中,制备犬埃里希体基因组DNA。将该混合物在56℃保温1小时,用苯酚/氯仿/异戊醇(24∶24∶1)的混合物抽提核酸两次。用纯乙醇沉淀DNA,用70%乙醇洗涤一次,干燥并重悬浮于10mM Tris(pH7.5)中。用高纯度质粒分离试剂盒(Boehringer Mannheim,Indianapolis,IN)纯化质粒DNA,并用QIAquick PCR纯化试剂盒(Qiagen,Santa Clarita,CA)纯化PCR产物。
ECa28-1(p28-7)基因的克隆
用Universal GenomeWalker试剂盒(CLONTECH,Palo Alto,CA)根据厂商提供的方法测定p28-7基因的全长序列。用5种限制性酶(DraI,EcoRV,PvuII,ScaI,StuI)完全消化犬埃里希体(Jake分离物)基因组DNA,产生钝端DNA。将试剂盒中提供的接头(AP1)与犬埃里希体DNA的每一端连接。将该基因组文库用作模板,采用与p28-7序列已知部分互补的引物,和对接头AP1特异性的引物,通过PCR来寻找p28-7基因的未知DNA序列。用于基因组步查的p28-7特异性引物是从衍生自引物793(SEQ ID NO:16)和1330(SEQ ID NO:17)PCR扩增p28-7的已知DNA序列设计的。引物394((5′-GCATTTCCACAGGATCATAGGTAA-3′;核苷酸687-710,SEQ ID NO.21)和394C(5′-TTACCTATGATCCTGT GGAAATGC-3;核苷酸710-687,SEQ ID NO.22))用于与提供的引物AP1连接,通过PCR扩增p28-7基因的未知5′和3′区。对相应于用引物394C和AP1扩增的p28-7基因5′区(2000bp)的PCR产物用引物793C(5′-GAGTA ACCAACAGCTCCTGC-3′,SEQ ID No.23)单向测序。用同一引物双向测序对应于以引物394和AP1扩增的p28-7基因3′区(580bp)的PCR产物。对邻接开放阅读框的5′和3′区的非编码区进行测序,设计与这些区域互补的引物EC28OM-F(5′-TCTACTTTGCACTTCC ACTATTGT-3′,SEQ ID NO.24)和EC28OM-R(5′-ATTCTTTTGCCACTATTT TTCTTT-3′,SEQ ID NO.25)以扩增整个p28-7基因。
DNA测序
用ABI Prism 377 DNA测序仪(Perkin-Elmer Applied Biosystems,Foster City,CA)对DNA进行测序。用引物EC28OM-F(SEQ ID No.24)和EC28OM-R(SEQ IDNo.25),95℃5分钟,30轮95℃30秒,62℃1分钟,72℃2分钟,和72℃延伸10分钟的热循环方案通过PCR扩增7种犬埃里希体分离物(来自North Carolina的4种,Oklahoma,Florida和Louisiana各一种)的整个p28-7基因。得到的PCR产物用相同引物双向测序。
实施例2
犬埃里希体ECa28-1(p28-7)基因的PCR扩增、克隆、测序和表达
表达载体
用引物-EC28OM-F和EC28OM-R PCR扩增了整个犬埃里希体p28-7基因,并克隆入pCR2.1-TOPO TA克隆载体,获得所需组的限制性酶切割位点(Invitrogen,Carlsbad,CA)。用BstX1从pCR2.1-TOPO上切下插入物,连接入pcDNA3.1真核表达载体(Invitrogen,Carlsbad,CA),称为pcDNA3.1/EC28用于后面的研究。扩增pcDNA3.1/EC28质粒,用KpnI-XbaI双消化切下基因,并定向连接到pThioHis原核表达载体(Invitrogen,Carlsbad,CA)中。该克隆(称为pThioHis/EC28)在大肠杆菌BL21中产生了一个重组的硫氧还蛋白融合蛋白。
实施例2
犬埃里希体ECa28-1(p28-7)基因的PCR扩增、克隆、测序和表达
表达载体
用引物EC28OM-F和EC28OM-R PCR扩增了全长的犬埃里希体p28-7基因,并克隆入pCR2.1-TOPO TA克隆载体,以获得所需的限制性酶切位点组(Invitrogen,Carlsbad,CA)。用BstX1从pCR2.1-TOPO上切下插入物,连接入pcDNA3.1真核表达载体(Invitrogen,Carlsbad,CA),称为pcDNA3.1/EC28,用于随后的研究。扩增pcDNA3.1/EC28质粒,用KpnI-XbaI双消化切下该基因,并定向连接入pThioHis原核表达载体中(Invitrogen,Carlsbad,CA)。该克隆(称为pThioHis/EC28)在大肠杆菌BL21中产生重组的硫氧还蛋白融合蛋白。离心粗纯化呈不溶相的重组融合蛋白。在天然条件下用镍-NTA旋转柱(Qiagen,Santa Clarita,CA)从溶解的细胞裂解物中,纯化得到对照的硫氧还蛋白融合蛋白。
蛋白质印迹分析
在4-15%Tris-HCl梯度凝胶(Bio-Rad,Hercules,CA)上对重组犬埃里希体p28-7融合蛋白进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE),并用半干转移槽(Bio-Rad,Hercules,CA)转移到纯硝酸纤维素(Schleicher & Schuell,Keene,NH)膜上。将该膜与1∶5000稀释的受犬埃里希体感染的恢复期犬抗血清保温1小时,洗涤,然后与抗犬IgG(H&L)碱性磷酸酶偶联的亲和层析纯化的二抗(1∶1000)保温1小时(Kirkegaard & Perry Laboratories,Gaithersburg,MD)。结合的抗体用5-溴-4-氯-3-吲哚磷酸/氮蓝四唑(BCIP/NBT)底物(Kirkegaard & Perry Laboratories,Gaithersburg,MD)。
Southern印迹分析
为了确定与p28-7基因同源的多基因是否存在于犬埃里希体基因组中,用标准方法(Sambrook等,1989)进行了基因组的Southem印迹分析。用各限制性酶BanII、EcoRV、HaeII、KpnI和SpeI完全消化犬埃里希体基因组DNA,它们不在p28-7基因内切割,AseI在核酸34、43和656处切开p28-7。通过用引物EC28OM-F和EC28OM-R和地高辛(DIG)-标记的脱氧核苷酸三磷酸(dNTP)(BoehringerMannheim,Indianapolis,IN)作PCR扩增产生探针,并用AseI消化。用琼脂糖凝胶电泳分离消化的探针(566-bp),凝胶纯化然后用于杂交。完全消化的基因组犬埃里希体DNA电泳并转移到尼龙膜(Boehringer Mannheim,Indianapolis,IN)上,与p28-7基因DIG-标记的探针在DIG Easy hyb缓冲液中,根据厂商的方案(BoehringerMannheim,Indianapolis,IN)40℃杂交16小时。用抗-DIG碱性磷酸酶-偶联抗体和发光底物(Boehringer Mannheim,Indianapolis,IN)检测结合的探针,并曝光BioMax科学显影胶片(Eastman Kodak,Rochester,NY)。
序列分析和比较
从国家生物技术信息中心(NCBI)获得恰菲埃里希体p28和反刍类考德里氏体map-1 DNA序列。用LASERGENE软件(DNASTAR,Inc.Madison,WI)进行核苷酸和推测的氨基酸序列和蛋白质以及系统发生分析。用McGeoch和von Heijine的方法,使用PSORT程序(McGeoch,1985;von Heijne,1986)进行翻译后加工分析,以识别信号序列。
用设计用于扩增该全长基因的引物对7种不同犬埃里希体株的p28-7进行序列分析。分析揭示该基因的序列在North Carolina(4种),Louisiana,Florida和Oklahoma的分离物中是保守的。
结果
用Jotun-Hein算法排列对比了恰菲埃里希体p28和反刍类考德里氏体map-1的核酸序列产生具有高同源(>90%)区域的共有序列。这些同源区(反刍类考德里氏体map-1核苷酸313-332和823-843;恰菲埃里希体p28的307-326和814-834)被定为PCR扩增的引物退火位点的目标。用引物793(5-GCAGGAGCTGTTGGTTACTC-3′)(SEQ ID NO.16)和1330(5′-CCTTCCTCCAAGTTCTATGCC-3′)(SEQID NO.17)完成了犬埃里希体p28-7基因的PCR扩增,得到518bp的PCR产物。用引物793和1330扩增犬埃里希体DNA,热循环方案为95℃2分钟,95℃30秒、62℃1分钟、72℃2分钟的30轮循环,然后72℃延伸10分钟,保持在4℃。通过直接用引物793和1330对产物测序获得犬埃里希体PCR产物的核酸序列。
序列分析揭示有一个编码170个氨基酸的蛋白质的开放阅读框,犬埃里希体PCR扩增获得的518bp序列和恰菲埃里希体p28基因的DNA序列的排列对比,揭示70%以上的相似性,表明这些基因是同源的。
用引物394和793C进行接头PCR,以确定全基因序列的5′和3′区段。引物394产生4种PCR产物(3kb、2kb、1拷贝和0.8kb),对0.8kb的产物用引物394和AP1进行双向测序。推测的序列与518bp产物的3′末端重叠,将开放阅读框延伸12个bp至终止密码子。还对p28-7基因3′末端的另一个625bp非编码序列进行了测序。
用引物394C和提供的引物AP1扩增p28-7基因的5′末端。这些引物扩增得到3种PCR产物(3.3、3-kb和2kb)。用引物793C对2-kb片段进行单向测序。序列提供了p28-7基因的推测的起始密码子,并完整了编码278个氨基酸的蛋白质的834bp开放阅读框。产生p28-7基因5′非编码区的其它144bp的可阅读序列。从邻近p28-7基因的互补非编码区设计引物EC28OM-F和EC28OM-R。
用相同引物直接对用这些引物扩增的PCR产物进行测序。图1显示了犬埃里希体p28-7基因(SEQ ID NO:1)的完整DNA序列。用这些引物扩增的p28-7 PCR片段含有整个开放阅读框和来自5′非编码引物区的17个其它氨基酸。将基因直接亚克隆入pThioHis表达载体,并用该构建物转化大肠杆菌BL21。表达的p28-7硫氧还蛋白融合蛋白是不可溶的。表达的蛋白质具有与硫氧还蛋白结合的另114个氨基酸,5个氨基酸是肠激酶识别位点,32个氨基酸来自多克隆位点和N-末端的5′非编码引物区。来自犬埃里希体感染的犬恢复期抗血清识别表达的重组融合蛋白,但不和硫氧还蛋白对照反应(图2)。
实施例3
犬埃里希体p28-7基因的序列同源性
用Clustal法排列对比了犬埃里希体p28-7(834bp)和包括信号序列的恰菲埃里希体omp-1基因家族(p28-7、omp-1A、B、C、D、E和F)的核酸序列,以检测这些基因之间的同源性(排列未显示)。在犬埃里希体p28-7、恰菲埃里希体p28和omp-1F之间核酸同源性相等保守(68.9%)。恰菲埃里希体omp-1家族中的其它推定的外膜蛋白基因,omp-1D(68.2%)、omp-1E(66.7%)、omp-1C(64.1%)、反刍类考德里氏体map-1(61.8%)、犬埃里希体28kDa蛋白1基因(60%)和28kDa蛋白2基因(部分)(59.5%)也与p28-7是同源的。恰菲埃里希体omp-1B与犬埃里希体p28-7的核酸同源性(45.1%)最少。
犬埃里希体p28-7(SEQ ID NO:2)和恰菲P28的预测氨基酸序列的排列对比,揭示了导致4个可变区(VR)的氨基酸取代。鉴定了犬埃里希体P28-7和恰菲埃里希体OMP-1家族可变区的氨基酸序列和位置中的取代或缺失(图3)。包括信号肽的氨基酸比较揭示,犬埃里希体P28-7与恰菲埃里希体OMP-1家族的OMP-1F具有最高的同源性(68%),然后是恰菲P28(65.5%)、OMP-1E(65.1%)、OMP-1D(62.9%)、OMP-1C(62.9%)、反刍类考德里氏体MAP-1(59.4%)、犬埃里希体28kDa蛋白质1(55.6%)和28kDa蛋白质2(部分)(53.6%)和OMP-1B(43.2%)。基于氨基酸序列的系统发生关系显示,犬埃里希体P28-7和反刍类考德里氏体MAP-1、恰菲埃里希体OMP-1蛋白以及犬埃里希体28kDa蛋白1和2(部分)是相关的(图4)。
实施例4
犬埃里希体P28-7的预测的表面可能性和免疫反应性
用疏水和亲水概况图分析犬埃里希体P28-7,预测P28-7上的表面暴露区域(图6)。在犬埃里希体P28-7中鉴定出8个含有3-9个氨基酸的主要表面暴露区域,与恰菲埃里希体P28上的表面暴露区域的情况相似(图6)。犬埃里希体P28-7上较大的表面暴露区域中的5个,位于蛋白质的N末端区域。在犬埃里希体P28-7的全部4个可变区中发现了表面暴露的亲水区域。用Rothbard-Taylor算法(Rothbard和Taylor,1988)在P28-7中预测了10个T-细胞基序,用Jameson-Wolf抗原性算法(图6)(Jamesor和Wolf,1988)预测了犬埃里希体P28-7的高抗原性。在犬埃里希体P28-7和恰菲埃里希体P28之间观察到抗原性和T-细胞基序的相似性。
实施例5
犬埃里希体p28-7基因同源基因组拷贝的检测
用限制性酶BanII,EcoRV,HaeII,KpnI,SpeI(在p28-7基因中没有限制性内切酶位点)和AseI(在核苷酸34、43和656有内部限制性内切酶位点)分别完全消化犬埃里希体DNA,进行基因组Southern印迹分析,揭示存在至少3种同源p28-7基因拷贝(图5)。虽然犬埃里希体p28-7具有内部AseI内部限制位点,用于杂交实验的DIG-标记的探针针对AseI消化基因产生的一个DNA片段内的基因区。用AseI消化产生3个条带(约566bp、850bp和3kb),它们与p28-7DNA探针杂交,表示基因组中存在与p28-7同源的多基因。用EcoRV和SpeI消化,产生与p28-7基因探针杂交的两个条带。
实施例6
犬埃里希体ECa28SA2(p28-5)、ECa28SA3(p28-6)的PCR扩增
多基因基因座的基因和鉴定
为了特异性扩增ECa28SA2(p28-5)下游的可能未知基因,用对p28-5特异性的引物46f(5′-ATATACTTCCTACCTAATGTCTCA-3′,SEQ ID No.18)和引物1330(SEQ ID NO:17)进行扩增,1330针对p28-7基因3′末端的保守区。凝胶纯化扩增产物,克隆入TA克隆载体(Invitrogen,Santa Clarita,CA)。对克隆进行双向测序,使用引物:来自载体的M13反向、46f、ECa28SA2(5′-AGTGCAGAGTCTTCGGTTTC-3′,SEQ ID No.19),ECa5.3(5′-GTTACTTGCGGAGGACAT-3′,SEQ ID No.20)。用热循环方案95℃2分钟,30轮的95℃30秒,48℃1分钟,72℃1分钟,然后72℃延伸10分钟,维持在4℃。
用这些引物扩增得到一含有2个开放阅读框的2-kb PCR产物。第一个开放阅读框含有p28-5基因的已知区域和该基因先前未测序的3′部分。在p28-5下游发现另一个不相同,但同源的28kDa蛋白基因,称为ECa28SA3(p28-6)。
用对应于p28-6内区域的特异性引物,称为ECaSA3-2(5′-CTAGGATTAGGTTATAGTATAAGTT-3′,SEQ ID No.26)和与p28-7内某区域退火的引物793C(SEQ ID NO:23)扩增基因p28-6和-p28-7之间的基因间区。用热循环方案95℃2分钟,30轮的95℃30秒,48℃1分钟,72℃1分钟,然后72℃延伸10分钟,维持在4℃扩增DNA。
扩增得到一800bp PCR产物,它含有p28-6的3′末端,p28-6和p28-7(28NC3)之间的基因间区,和p28-7的5′末端,与先前分离的基因座连接(图8)。p28-5的849bp的开放阅读框编码283氨基酸蛋白,p28-6具有编码280个氨基酸蛋白质的840bp开放阅读框。p28-6和p28-7之间的基因间非编码区域长345bp(图7和8)。
实施例7
犬埃里希体p28-4、p28-5、p28-6、p28-7和p28-8蛋白质的核酸和氨基酸同源性
用Clustal法排列对比了全部5种犬埃里希体28kDa蛋白质基因的核酸和氨基酸序列,以检测这些基因之间的同源性。核酸同源性是58-75%,在犬埃里希体28-kDa蛋白质基因成员之间观察到67-72%相似的氨基酸同源性。
转录启动子区域
通过与共有的大肠杆菌启动子区和恰菲埃里希体的启动子比较,分析28kDa蛋白质基因之间的基因间区的启动子序列(Yu等,1997;McClure,1985)。在对应于基因p28-5、p28-6、p28-7和p28-8(Eca28-2)的4个基因间序列中鉴定出推定的启动子序列,包括RBS,-10和-35区(图10)。p28-4(ECa28SA1)的上游非编码区是未知的,没有分析。
N-末端信号序列
氨基酸序列分析揭示,全长犬埃里希体p28-7具有30.5kDa的推测分子量,整个p28-6具有30.7kDa的推测分子量。两条蛋白质具有23个氨基酸的推测N-末端信号肽(MNCKKILITTALMSLMYYAPSIS,SEQ ID No.27),与恰菲埃里希体P28(MNYKKILITSALISLISSLPGV SFS,SEQ ID NO.28),和OMP-1蛋白家族预测的相似(Yu等,1999a;Ohashi等,1998b)。
在p28-7的氨基酸21、22和23处发现信号肽酶的优选切割位点(SIS;Ser-X-Ser)(Oliver,1985)。在氨基酸位置25处还存在另一个推定的切割位点(MNCKKILITTALISLMYSIPSISSFS,SEQ ID NO.29),与恰菲埃里希体P28的预测切割位点(SFS)相同,这导致具有27.7kDa预测分子量的成熟p28-7。预测p28-5先前报道的部分序列的信号切割位点在氨基酸30。然而,信号序列分析预测p28-4具有不可切割的信号序列。
总结
从包括犬埃里希体、恰菲埃里希体、反刍类考德里氏体的多个立克次氏体鉴定并克隆了具有相似分子量的蛋白质(Reddy等,1998;Jongejan等,1993;Ohashi等,1998)。先前描述了恰菲埃里希体中具有6个同源p28基因的一个基因座,和犬埃里希体中的2个基因座,各含有一些同源28kDa蛋白质基因。
本发明显示了与恰菲埃里希体omp-1多基因家族以及反刍类考德里氏体map-1基因同源的犬埃里希体编码成熟28-kDa蛋白质的基因的克隆、表达和特征确定。鉴定出两种新的28kDa蛋白质基因p28-7和p28-6。在本发明中对于另一种犬埃里希体28kDa蛋白质基因,先前部分测序(Reddy等,1998)的p28-5完全测序。还公开了含有5种犬埃里希体28kDa蛋白质基因(p28-4、p28-5、p28-6、p28-7和p28-8)的犬埃里希体中的一个基因座的鉴定和特征确定。
犬埃里希体28kDa蛋白质与恰菲埃里希体OMP-1家族以及反刍类考德里氏体MAP-1蛋白是同源的。最同源的犬埃里希体28kDa蛋白(p28-6、p28-7和p28-8)在该基因座中依次排列。这些蛋白质的同源性是67.5-72.3%。这些28kDa蛋白质中的趋多样是27.3-38.6%。犬埃里希体28kDa蛋白质p28-4和p28-5同源性最小,为50.9-59.4%,多样性是53.3-69.9%。基因间的不同主要在于4个超变区,提示这些区域是表面暴露的,受到免疫系统的选择压力。报道了p28-7在7种犬埃里希体分离物中的保守性(McBride等,1999),提示犬埃里希体在北美可能是纯系的。相反,报道了恰菲埃里希体中p28的显著多样性(Yu等,1999a)。
全部犬埃里希体28kDa蛋白质似乎是从30kD蛋白质翻译后加工成成熟28kD蛋白的。近来,在恰菲埃里希体P28上鉴定出一条信号序列(Yu等,1999a),N-末端氨基酸测序证实,蛋白质翻译后加工,切下信号序列产生成熟蛋白(Ohashi等,1998)。OMP-1F和OMP-1E的前导序列已被提出是前导信号肽(Ohashi等,1998)。在恰菲埃里希体OMP-1F、OMP-1E和P28上鉴定的信号序列与犬埃里希体28kDa蛋白的前导序列是同源的。p28基因的启动子序列未被实验所确定,但通过与RBS、大肠杆菌和其它埃里希体的-10和-35启动子区的共有序列比较,鉴定了可能的启动子区(Yu等,1997;McClure,1985)。这些启动子区将使每个基因可能被转录和翻译,提示这些基因可以在宿主中差异表达。在犬中持续感染可能与p28基因的差异表达有关,导致体内抗原改变,因此使生物规避免疫应答。
发现犬埃里希体28kDa蛋白基因显示与恰菲埃里希体omp-1基因家族和反刍类考德里氏体map-1基因的核酸和氨基酸序列同源性。先前研究已证明犬埃里希体30kDa蛋白,它与针对恰菲埃里希体的恢复期抗血清反应,但相信抗原是不同的(Rikihisa等,1994)。根据犬埃里希体28kDa蛋白质4个可变区中氨基酸取代比较所得发现,支持该可能性。这些发现一起提示负责犬埃里希体和恰菲埃里希体P28之间的抗原差异的氨基酸位于这些可变区中,可轻易接触免疫系统。
报道了免疫反应性肽位于反刍类考德里氏体、恰菲埃里希体、犬埃里希体28kDa蛋白的可变区中(Reddy等,1998)。犬埃里希体和恰菲P28的分析揭示,所有可变区具有预测的表面暴露的氨基酸。在犬中的研究显示,缺乏犬埃里希体和恰菲埃里希体的交叉保护(Dawson和Ewing,1992)。该观察可能与这些埃里希体品种的P28可变区中以及其它免疫学重要抗原的抗原性差异有关。另一个研究发现,恰菲埃里希体感染的病人的恢复期人抗血清识别恰菲埃里希体的29/28kDa蛋白,也与犬埃里希体的同源蛋白质反应(Chen等,1997)。犬埃里希体28kDa蛋白和恰菲埃里希体P28上的同源和交叉反应性表位似乎被免疫系统识别。
犬埃里希体28kDa蛋白可能是重要的免疫保护性抗原。几个报道证明,犬埃里希体的30kDa抗原显示强免疫反应性(Rikihisa等,1994;Rikihisa等,1992)。人和犬恢复期抗血清中的抗体与恰菲埃里希体和犬埃里希体的该大小范围内的蛋白质反应,提示它们可能是重要的免疫保护性抗原(Rikihisa等,1994;Chen等,1994;Chen等,1997)。另外,针对30、24和21kDa蛋白的抗体在对犬埃里希体的免疫应答中很早产生(Rikihisa等,1994;Rikihisa等,1992),提示这些蛋白质可能在疾病急性阶段的免疫应答中尤其重要。近来,在恰菲埃里希体中鉴定出分子量为28kDa的编码外膜蛋白的同源基因家族,用重组恰菲埃里希体P28的免疫小鼠产生针对同源攻击的免疫力(Ohashi等,1998)。恰菲埃里希体P28被证明存在于外膜,免疫电子显微镜将P28定位在该微生物的表面,因此提示它可能作为粘附素起作用(Ohashi等,1998)。可能在该研究中鉴定的恰菲埃里希体的28kDa蛋白具有相同的位置,可能起到相同作用。
不同犬埃里希体株p28-7的比较揭示,该基因明显完全保守。用恰菲埃里希体的研究显示多样性的免疫学和分子证据。恰菲埃里希体感染的病人具有对29/28kDa蛋白质可变化的免疫反应性,提示其具有抗原多样性(Chen等,1997)。近来已产生了分子证据,支持恰菲埃里希体p28基因中有抗原多样性(Yu等,1999a)。5种恰菲埃里希体分离物中的比较,揭示两种分离物(Sapulpa和St.Vincent)是100%相同的,但其它三种(Arkansas,Jax,91HE17)具有多样性,在氨基酸水平上达13.4%。犬埃里希体p28-7的保守提示,美国发现的犬埃里希体株是基因相同的,因此犬埃里希体28kDa蛋白是美国犬埃里希体病的吸引人的候选疫苗。美国以外的其它犬埃里希体分离物可提供关于犬埃里希体起源和进化的信息。28kDa蛋白的保守性使的它成为可靠血清诊断犬埃里希体病的重要的候选试剂。
现在还不知道多种同源性基因的作用;然而,犬中犬埃里希体的持续感染可能与由于同源28kDa蛋白基因表达变化导致的抗原性改变相关,因此使犬埃里希体能规避免疫监视。msp-3基因在着边无形体中的变化部分引起MSP-3蛋白的改变,导致持续感染(Alleman等,1997)。检测急性和慢性感染犬中犬埃里希体28kDa蛋白基因表达的研究,可提供28kDa蛋白基因家族在感染持续中的作用的理解。
实施例8
犬埃里希体p28-1、p28-2、p28-3和p28-9基因的鉴定
通过为p28-1(ECa28-75C)和p28-5(ECa28-5-818f)设计的基因特异性引物,双向延伸p28基因座,对上述串联排列的p28基因的5个基因座的DNA上游和下游的未知区域进行测序。进行多基因步查如下获得未知序列:用引物p28-5-818f(5’-TTA AAC ATA TGC CAC TTC GGA CTA-3’,SEQ ID No.34)扩增5个基因座1.9kp下游,并测序,产生900bp的扩增子,用1191(5’-TAT GAT CGT GTA AAATTG CTG TGA GTA T-3’,SEQ ID No.35),产生1kb的扩增子。用引物ECa28-75C(5’-TAC TGG CAC GTG CTG GAC TA-3’,SEQ ID No.36)对5个基因座DNA上游的3.67kbp进行扩增和测序,产生1.6kbp扩增子;用ECa5’-1600(5’-CAC CAA TAAATG CAG AGA CTT C-3’,SEQ ID No.37),产生1.6kbp扩增子;用3125(5’-AATCCA TCA TTT CTC ATT ACA GTG TG-3’,SEQ ID No.38),产生800bp扩增子。9个串联排列基因的该基因座含有4个新的p28基因和上述5个p28基因,命名为p28-1到p28-9(图11)。
用Clustal法排列对比了犬埃里希体p28基因的核酸和氨基酸序列,以检测这些基因之间的同源性。这些蛋白质的同源性为67.5-75%,这些P28蛋白中的多样性为26.9-38%。犬埃里希体P28蛋白P28-1、P28-2和P28-9与其它p28基因同源性最小,为37-49%,多样性为53-77%。9个p28基因的核酸同源性是28-72%。图12显示了基于犬埃里希体p28氨基酸序列的系统发生关系。
核苷酸序列和登录号
完全的9基因犬埃里希体(Jake株)p28基因座的核酸和氨基酸序列的GenBank登录号是AF082744。该登录号原来是给p28-7的,但已用9基因p28基因座的序列,包括p28-7更新。本研究中描述的其它犬埃里希体分离物的p28-7的核酸和氨基酸序列的GenBank登录号是:Louisiana,AF082745;Oklahoma,AF082746;Demon,AF082747;DJ,AF082748;Fuzzy,AF082749;Florida,AF082750。
用犬埃里希体感染的犬的恢复期血清的免疫印迹观察到28kDa范围内的许多条带(Rikihisa等,1994),多个p28蛋白的表达可能是该观察结果的解释。Southern印迹研究提示,基因组中除了该基因座中的5个成员外,存在其它p28基因(McBride等,1999;Ohashi等,1998b)。
在该研究中,鉴定出含有编码同源性,但非相同的p28基因的9个串联排列的犬埃里希体p28基因的一个基因座。9个基因的基因座包括4个新p28基因(图13-16)和上述报道的5个串联排列的p28基因。8个p28基因位于一条DNA链上,一个p28基因在互补链上。9个p28基因成员中核酸同源性是37-75%,氨基酸同源性是28-72%。
发现犬埃里希体P28与其它物种,例如恰菲埃里希体的28kDa蛋白像其与自身一样密切相关(McBride等,2000)。发现蛋白质中的区别主要在几个主要超变区中,提示这些区域是表面暴露的,受到免疫系统的选择压力(McBride等,2000)。
报道了7种地理学上不同的分离物中犬埃里希体p28基因(p28-7)的保守性(McBride等,1999),提示犬埃里希体在北美可能是高度保守的。类似的,犬埃里希体的120kDa糖蛋白在美国的分离物中也是保守的(Yu等,1997)。相反,恰菲埃里希体的120kDa和28kDa蛋白质基因在分离物中是多样的(Yu等,1999a;Chen等,1997)。恰菲埃里希体28kDa蛋白质基因的多样性似乎是由于超变区中的点突变所致,该突变可能是由于选择性免疫压力(Yu等,1999a)。这些资料提示犬埃里希体可能是较近被引入北美的,这可能是分离物中观察到的保守的原因。犬埃里希体分离物中p28基因的保守可提供机会来开发疫苗和血清诊断性抗原,它们对于疾病预防和血清诊断将特别有效。P28的混合物可提供最可靠的血清诊断试验,但用一种P28血清诊断对于免疫诊断是有用的(Ohashi等,1998b;McBride等,1999)。
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本说明书提到的任何专利或出版物指出了本发明涉及的领域中技术人员的水平。这些专利和出版物在此引入以供参考,程度相同,如同各独立出版物是特别和单独表明引入以供参考的一样。
本领域技术人员将易于理解,本发明适用于实施提到的目的和获得提到的结果和优点,以及与之相关的。本文描述的实施例、方法、程序、处理、分子和特定的化合物以优选例为代表,是示范性的,而不意味着限制本发明的范围。本领域技术人员对其中所作的改变和其它用途都将包括在权利要求所述的本发明的范围内。序列表<110>D.H.沃克(Walker,David H.)
J.W.麦克布里奇(McBride,Jere W.)
于学杰(Yu,Xue-Jie)<120>犬埃里希体的同源28kD免疫优势蛋白质基因及其用途<130>D6152CIP2/PCT<141>2001-09-12<150>09/660,587<151>2000-09-12<160>46<210>1<211>1607<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-7核酸序列<400>1attttattta ttaccaatct tatataatat attaaatttc tcttacaaaa 50atctctaatg ttttatacct aatatatata ttctggcttg tatctacttt 100gcacttccac tattgttaat ttattttcac tattttaggt gtaatatgaa 150ttgcaaaaaa attcttataa caactgcatt aatatcatta atgtactcta 200ttccaagcat atctttttct gatactatac aagatggtaa catgggtggt 250aacttctata ttagtggaaa gtatgtacca agtgtctcac attttggtag 300cttctcagct aaagaagaaa gcaaatcaac tgttggagtt tttggattaa 350aacatgattg ggatggaagt ccaatactta agaataaaca cgctgacttt 400actgttccaa actattcgtt cagatacgag aacaatccat ttctagggtt 450tgcaggagct atcggttact caatgggtgg cccaagaata gaattcgaaa 500tatcttatga agcattcgac gtaaaaagtc ctaatatcaa ttatcaaaat 550gacgcgcaca ggtactgcgc tctatctcat cacacatcgg cagccatgga 600agctgataaa tttgtcttct taaaaaacga agggttaatt gacatatcac 650ttgcaataaa tgcatgttat gatataataa atgacaaagt acctgtttct 700ccttatatat gcgcaggtat tggtactgat ttgatttcta tgtttgaagc 750tacaagtcct aaaatttcct accaaggaaa actgggcatt agttactcta 800ttaatccgga aacctctgtt ttcatcggtg ggcatttcca caggatcata 850ggtaatgagt ttagagatat tcctgcaata gtacctagta actcaactac 900aataagtgga ccacaatttg caacagtaac actaaatgtg tgtcactttg 950gtttagaact tggaggaaga tttaacttct aattttattg ttgccacata 1000ttaaaaatga tctaaacttg tttttawtat tgctacatac aaaaaaagaa 1050aaatagtggc aaaagaatgt agcaataaga gggggggggg ggaccaaatt 1100tatcttctat gcttcccaag ttttttcycg ctatttatga cttaaacaac 1150agaaggtaat atcctcacgg aaaacttatc ttcaaatatt ttatttatta 1200ccaatcttat ataatatatt aaatttctct tacaaaaatc actagtattt 1250tataccaaaa tatatattct gacttgcttt tcttctgcac ttctactatt 1300tttaatttat ttgtcactat taggttataa taawatgaat tgcmaaagat 1350ttttcatagc aagtgcattg atatcactaa tgtctttctt acctagcgta 1400tctttttctg aatcaataca tgaagataat ataaatggta acttttacat 1450tagtgcaaag tatatgccaa gtgcctcaca ctttggcgta ttttcagtta 1500aagaagagaa aaacacaaca actggagttt tcggattaaa acaagattgg 1550gacggagcaa cactaaagga tgcaagcwgc agccacacaw tagacccaag 1600tacaatg 1607<210>2<211>278<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-7蛋白氨基酸序列<400>2Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Ala Leu Ile Ser Leu
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95 100 105Ile Gly Tyr Ser Met Gly Gly Pro Arg Ile Glu Phe Glu Ile Ser
110 115 120Tyr Glu Ala Phe Asp Val Lys Ser Pro Asn Ile Asn Tyr Gln Asn
125 130 135Asp Ala His Arg Tyr Cys Ala Leu Ser His His Thr Ser Ala Ala
140 145 150Met Glu Ala Asp Lys Phe Val Phe Leu Lys Asn Glu Gly Leu Ile
155 160 165Asp Ile Ser Leu Ala Ile Asn Ala Cys Tyr Asp Ile Ile Asn Asp
170 175 180Lys Val Pro Val Ser Pro Tyr Ile Cys Ala Gly Ile Gly Thr Asp
185 190 195Leu Ile Ser Met Phe Glu Ala Thr Ser Pro Lys Ile Ser Tyr Gln
200 205 210Gly Lys Leu Gly Ile Ser Tyr Ser Ile Asn Pro Glu Thr Ser Val
215 220 225Phe Ile Gly Gly His Phe His Arg Ile Ile Gly Asn Glu Phe Arg
230 235 240Asp Ile Pro Ala Ile Val Pro Ser Asn Ser Thr Thr Ile Ser Gly
245 250 255Pro Gln Phe Ala Thr Val Thr Leu Asn Val Cys His Phe Gly Leu
260 265 270Glu Leu Gly Gly Arg Phe Asn Phe
275<210>3<211>849<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><221>mat_peptide<223>p28-5的核酸序列<400>3atgaattgta aaaaagtttt cacaataagt gcattgatat catccatata 50cttcctacct aatgtctcat actctaaccc agtatatggt aacagtatgt 100atggtaattt ttacatatca ggaaagtaca tgccaagtgt tcctcatttt 150ggaatttttt cagctgaaga agagaaaaaa aagacaactg tagtatatgg 200cttaaaagaa aactgggcag gagatgcaat atctagtcaa agtccagatg 250ataattttac cattcgaaat tactcattca agtatgcaag caacaagttt 300ttagggtttg cagtagctat tggttactcg ataggcagtc caagaataga 350agttgagatg tcttatgaag catttgatgt gaaaaatcca ggtgataatt 400acaaaaacgg tgcttacagg tattgtgctt tatctcatca agatgatgcg 450gatgatgaca tgactagtgc aactgacaaa tttgtatatt taattaatga 500aggattactt aacatatcat ttatgacaaa catatgttat gaaacagcaa 550gcaaaaatat acctctctct ccttacatat gtgcaggtat tggtactgat 600ttaattcaca tgtttgaaac tacacatcct aaaatttctt atcaaggaaa 650gctagggttg gcctacttcg taagtgcaga gtcttcggtt tcttttggta 700tatattttca taaaattata aataataagt ttaaaaatgt tccagccatg 750gtacctatta actcagacga gatagtagga ccacagtttg caacagtaac 800attaaatgta tgctactttg gattagaact tggatgtagg ttcaacttc 849<210>4<211>283<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>p28-5蛋白的氨基酸序列<400>4Met Asn Cys Lys Lys Val Phe Thr Ile Ser Ala Leu Ile Ser Ser
5 10 15Ile Tyr Phe Leu Pro Asn Val Ser Tyr Ser Asn Pro Val Tyr Gly
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50 55 60Lys Thr Thr Val Val Tyr Gly Leu Lys Glu Asn Trp Ala Gly Asp
65 70 75Ala Ile Ser Ser Gln Ser Pro Asp Asp Asn Phe Thr Ile Arg Asn
80 85 90Tyr Ser Phe Lys Tyr Ala Ser Asn Lys Phe Leu Gly Phe Ala Val
95 100 105Ala Ile Gly Tyr Ser Ile Gly Ser Pro Arg Ile Glu Val Glu Met
110 115 120Ser Tyr Glu Ala Phe Asp Val Lys Asn Pro Gly Asp Asn Tyr Lys
125 130 135Asn Gly Ala Tyr Arg Tyr Cys Ala Leu Ser His Gln Asp Asp Ala
140 145 150Asp Asp Asp Met Thr Ser Ala Thr Asp Lys Phe Val Tyr Leu Ile
155 160 165Asn Glu Gly Leu Leu Asn Ile Ser Phe Met Thr Asn Ile Cys Tyr
170 175 180Glu Thr Ala Ser Lys Asn lle Pro Leu Ser Pro Tyr Ile Cys Ala
185 190 195Gly Ile Gly Thr Asp Leu Ile His Met Phe Glu Thr Thr His Pro
200 205 210Lys Ile Ser Tyr Gln Gly Lys Leu Gly Leu Ala Tyr Phe Val Ser
215 220 225Ala Glu Ser Ser Val Ser Phe Gly Ile Tyr Phe His Lys Ile Ile
230 235 240Asn Asn Lys Phe Lys Asn Val Pro Ala Met Val Pro Ile Asn Ser
245 250 255Asp Glu Ile Val Gly Pro Gln Phe Ala Thr Val Thr Leu Asn Val
260 265 270Cys Tyr Phe Gly Leu Glu Leu Gly Cys Arg Phe Asn Phe
275 280<210>5<211>840<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><221>mat_peptide<223>p28-6的核酸序列<400>5atgaattgca aaaaaattct tataacaact gcattaatgt cattaatgta 50ctatgctcca agcatatctt tttctgatac tatacaagac gataacactg 100gtagcttcta catcagtgga aaatatgtac caagtgtttc acattttggt 150gttttctcag ctaaagaaga aagaaactca actgttggag tttttggatt 200aaaacatgat tggaatggag gtacaatatc taactcttct ccagaaaata 250tattcacagt tcaaaattat tcgtttaaat acgaaaacaa cccattctta 300gggtttgcag gagctattgg ttattcaatg ggtggcccaa gaatagaact 350tgaagttctg tacgagacat tcgatgtgaa aaatcagaac aataattata 400agaacggcgc acacagatac tgtgctttat ctcatcatag ttcagcaaca 450agcatgtcct ccgcaagtaa caaatttgtt ttcttaaaaa atgaagggtt 500aattgactta tcatttatga taaatgcatg ctatgacata ataattgaag 550gaatgccttt ttcaccttat atttgtgcag gtgttggtac tgatgttgtt 600tccatgtttg aagctataaa tcctaaaatt tcttaccaag gaaaactagg 650attaggttat agtataagtt cagaagcctc tgtttttatc ggtggacact 700ttcacagagt cataggtaat gaatttagag acatccctgc tatggttcct 750agtggatcaa atcttccaga aaaccaattt gcaatagtaa cactaaatgt 800gtgtcacttt ggcatagaac ttggaggaag atttaacttc 840<210>6<211>280<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>p28-6蛋白的氨基酸序列<400>6Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Ala Leu Met Ser Leu
5 10 15Met Tyr Tyr Ala Pro Ser Ile Ser Phe Ser Asp Thr Ile Gln Asp
20 25 30Asp Asn Thr Gly Ser Phe Tyr Ile Ser Gly Lys Tyr Val Pro Ser
35 40 45Val Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Arg Asn Ser
50 55 60Thr Val Gly Val Phe Gly Leu Lys His Asp Trp Asn Gly Gly Thr
65 70 75Ile Ser Asn Ser Ser Pro Glu Asn Ile Phe Thr Val Gln Asn Tyr
80 85 90Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105Ile Gly Tyr Ser Met Gly Gly Pro Arg Ile Glu Leu Glu Val Leu
110 115 120Tyr Glu Thr Phe Asp Val Lys Asn Gln Asn Asn Asn Tyr Lys Asn
125 130 135Gly Ala His Arg Tyr Cys Ala Leu Ser His His Ser Ser Ala Thr
140 145 150Ser Met Ser Ser Ala Ser Asn Lys Phe Val Phe Leu Lys Asn Glu
155 160 165Gly Leu Ile Asp Leu Ser Phe Met Ile Asn Ala Cys Tyr Asp Ile
170 175 180Ile Ile Glu Gly Met Pro Phe Ser Pro Tyr Ile Cys Ala Gly Val
185 190 195Gly Thr Asp Val Val Ser Met Phe Glu Ala Ile Asn Pro Lys Ile
200 205 210Ser Tyr Gln Gly Lys Leu Gly Leu Gly Tyr Ser Ile Ser Ser Glu
215 220 225Ala Ser Val Phe Ile Gly Gly His Phe His Arg Val Ile Gly Asn
230 235 240Glu Phe Arg Asp Ile Pro Ala Met Val Pro Ser Gly Ser Asn Leu
245 250 255Pro Glu Asn Gln Phe Ala Ile Val Thr Leu Asn Val Cys His Phe
260 265 270Gly Ile Glu Leu Gly Gly Arg Phe Asn Phe
275 280<210>7<21l>133<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>p28-5蛋白的部分氨基酸序列<400>7Met Asn Cys Lys Lys Val Phe Thr Ile Ser Ala Leu Ile Ser Ser
5 10 15Ile Tyr Phe Leu Pro Asn Val Ser Tyr Ser Asn Pro Val Tyr Gly
20 25 30Asn Ser Met Tyr Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro
35 40 45Ser Val Pro His Phe Gly Ile Phe Ser Ala Glu Glu Glu Lys Lys
50 55 60Lys Thr Thr Val Val Tyr Gly Leu Lys Glu Asn Trp Ala Gly Asp
65 70 75Ala Ile Ser Ser Gln Ser Pro Asp Asp Asn Phe Thr Ile Arg Asn
80 85 90Tyr Ser Phe Lys Tyr Ala Ser Asn Lys Phe Leu Gly Phe Ala Val
95 100 105Ala Ile Gly Tyr Ser Ile Gly Ser Pro Arg Ile Glu Val Glu Met
110 115 120Ser Tyr Glu Ala Phe Asp Val Lys Asn Gln Gly Asn Asn
125 130<210>8<211>287<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>p28-4蛋白的氨基酸序列<400>8Met Lys Tyr Lys Lys Thr Phe Thr Val Thr Ala Leu Val Leu Leu
5 10 15Thr Ser Phe Thr His Phe Ile Pro Phe Tyr Ser Pro Ala Arg Ala
20 25 30Ser Thr Ile His Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro Thr
35 40 45Ala Ser His Phe Gly Ile Phe Ser Ala Lys Glu Glu Gln Ser Phe
50 55 60Thr Lys Val Leu Val Gly Leu Asp Gln Arg Leu Ser His Asn Ile
65 70 75Ile Asn Asn Asn Asp Thr Ala Lys Ser Leu Lys Val Gln Asn Tyr
80 85 90Ser Phe Lys Tyr Lys Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105Ile Gly Tyr Ser Ile Gly Asn Ser Arg Ile Glu Leu Glu Val Ser
110 115 120His Glu Ile Phe Asp Thr Lys Asn Pro Gly Asn Asn Tyr Leu Asn
125 130 135Asp Ser His Lys Tyr Cys Ala Leu Ser His Gly Ser His Ile Cys
140 145 150Ser Asp Gly Asn Ser Gly Asp Trp Tyr Thr Ala Lys Thr Asp Lys
155 160 165Phe Val Leu Leu Lys Asn Glu Gly Leu Leu Asp Val Ser Phe Met
170 175 180Leu Asn Ala Cys Tyr Asp Ile Thr Thr Glu Lys Met Pro Phe Ser
185 190 195Pro Tyr Ile Cys Ala Gly Ile Gly Thr Asp Leu Ile Ser Met Phe
200 205 210Glu Thr Thr Gln Asn Lys Ile Ser Tyr Gln Gly Lys Leu Gly Leu
215 220 225Asn Tyr Thr Ile Asn Ser Arg Val Ser Val Phe Ala Gly Gly His
230 235 240Phe His Lys Val Ile Gly Asn Glu Phe Lys Gly Ile Pro Thr Leu
245 250 255Leu Pro Asp Gly Ser Asn Ile Lys Val Gln Gln Ser Ala Thr Val
260 265 270Thr Leu Asp Val Cys His Phe Gly Leu Glu Ile Gly Ser Arg Phe
275 280 285Phe Phe<210>9<211>281<212>PRT<213>恰菲埃里希体(Ehrlichia chaffeensis)<220><223>恰菲埃里希体P28的氨基酸序列<400>9Met Asn Tyr Lys Lys Val Phe Ile Thr Ser Ala Leu Ile Ser Leu
5 10 15Ile Ser Ser Leu Pro Gly Val Ser Phe Ser Asp Pro Ala Gly Ser
20 25 30Gly Ile Asn Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro Ser
35 40 45Ala Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Arg Asn Thr
50 55 60Thr Val Gly Val Phe Gly Leu Lys Gln Asn Trp Asp Gly Ser Ala
65 70 75Ile Ser Asn Ser Ser Pro Asn Asp Val Phe Thr Val Ser Asn Tyr
80 85 90Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105Ile Gly Tyr Ser Met Asp Gly Pro Arg Ile Glu Leu Glu Val Ser
110 115 120Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys Asn
125 130 135Glu Ala His Arg Tyr Cys Ala Leu Ser His Asn Ser Ala Ala Asp
140 145 150Met Ser Ser Ala Ser Asn Asn Phe Val Phe Leu Lys Asn Glu Gly
155 160 165Leu Leu Asp Ile Ser Phe Met Leu Asn Ala Cys Tyr Asp Val Val
170 175 180Gly Glu Gly Ile Pro Phe Ser Pro Tyr Ile Cys Ala Gly Ile Gly
185 190 195Thr Asp Leu Val Ser Met Phe Glu Ala Thr Asn Pro Lys Ile Ser
200 205 210Tyr Gln Gly Lys Leu Gly Leu Ser Tyr Ser Ile Ser Pro Glu Ala
215 220 225Ser Val Phe Ile Gly Gly His Phe His Lys Val Ile Gly Asn Glu
230 235 240Phe Arg Asp Ile Pro Thr Ile Ile Pro Thr Gly Ser Thr Leu Ala
245 250 255Gly Lys Gly Asn Tyr Pro Ala Ile Val Ile Leu Asp Val Cys His
260 265 270Phe Gly Ile Glu Leu Gly Gly Arg Phe Ala Phe
275 280<210>10<211>283<212>PRT<213>恰菲埃里希体(Ehrlichia chaffeensis)<220><223>恰菲埃里希体OMP-1B的氨基酸序列<400>10Met Asn Tyr Lys Lys Ile Phe Val Ser Ser Ala Leu Ile Ser Leu
5 10 15Met Ser Ile Leu Pro Tyr Gln Ser Phe Ala Asp Pro Val Thr Ser
20 25 30Asn Asp Thr Gly Ile Asn Asp Ser Arg Glu Gly Phe Tyr Ile Ser
35 40 45Val Lys Tyr Asn Pro Ser Ile Ser His Phe Arg Lys Phe Ser Ala
50 55 60Glu Glu Ala Pro Ile Asn Gly Asn Thr Ser Ile Thr Lys Lys Val
65 70 75Phe Gly Leu Lys Lys Asp Gly Asp Ile Ala Gln Ser Ala Asn Phe
80 85 90Asn Arg Thr Asp Pro Ala Leu Glu Phe Gln Asn Asn Leu Ile Ser
95 100 105Gly Phe Ser Gly Ser Ile Gly Tyr Ala Met Asp Gly Pro Arg Ile
110 115 120Glu Leu Glu Ala Ala Tyr Gln Lys Phe Asp Ala Lys Asn Pro Asp
125 130 135Asn Asn Asp Thr Asn Ser Gly Asp Tyr Tyr Lys Tyr Phe Gly Leu
140 145 150Ser Arg Glu Asp Ala Ile Ala Asp Lys Lys Tyr Val Val Leu Lys
155 160 165Asn Glu Gly Ile Thr Phe Met Ser Leu Met Val Asn Thr Cys Tyr
170 175 180Asp Ile Thr Ala Glu Gly Val Pro Phe Ile Pro Tyr Ala Cys Ala
185 190 195Gly Val Gly Ala Asp Leu Ile Asn Val Phe Lys Asp Phe Asn Leu
200 205 210Lys Phe Ser Tyr Gln Gly Lys Ile Gly Ile Ser Tyr Pro Ile Thr
215 220 225Pro Glu Val Ser Ala Phe Ile Gly Gly Tyr Tyr His Gly Val Ile
230 235 240Gly Asn Asn Phe Asn Lys Ile Pro Val Ile Thr Pro Val Val Leu
245 250 255Glu Gly Ala Pro Gln Thr Thr Ser Ala Leu Val Thr Ile Asp Thr
260 265 270Gly Tyr Phe Gly Gly Glu Val Gly Val Arg Phe Thr Phe
275 280<210>11<211>280<212>PRT<213>恰菲埃里希体(Ehrlichia chaffeensis)<220><223>恰菲埃里希体OMP-1C的氨基酸序列<400>11Met Asn Cys Lys Lys Phe Phe Ile Thr Thr Ala Leu Ala Leu Pro
5 10 15Met Ser Phe Leu Pro Gly Ile Leu Leu Ser Glu Pro Val Gln Asp
20 25 30Asp Ser Val Ser Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro
35 40 45Ser Ala Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Lys Asn
50 55 60Pro Thr Val Ala Leu Tyr Gly Leu Lys Gln Asp Trp Asn Gly Val
65 70 75Ser Ala Ser Ser His Ala Asp Ala Asp Phe Asn Asn Lys Gly Tyr
80 85 90Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105Ile Gly Tyr Ser Met Gly Gly Pro Arg Ile Glu Phe Glu Val Ser
110 115 120Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Gly Asn Tyr Lys Asn
125 130 135Asp Ala His Arg Tyr Cys Ala Leu Asp Arg Lys Ala Ser Ser Thr
140 145 150Asn Ala Thr Ala Ser His Tyr Val Leu Leu Lys Asn Glu Gly Leu
155 160 165Leu Asp Ile Ser Leu Met Leu Asn Ala Cys Tyr Asp Val Val Ser
170 175 180Glu Gly Ile Pro Phe Ser Pro Tyr Ile Cys Ala Gly Val Gly Thr
185 190 195Asp Leu Ile Ser Met Phe Glu Ala Ile Asn Pro Lys Ile Ser Tyr
200 205 210Gln Gly Lys Leu Gly Leu Ser Tyr Ser Ile Asn Pro Glu Ala Ser
215 220 225Val Phe Val Gly Gly His Phe His Lys Val Ala Gly Asn Glu Phe
230 235 240Arg Asp Ile Ser Thr Leu Lys Ala Phe Ala Thr Pro Ser Ser Ala
245 250 255Ala Thr Pro Asp Leu Ala Thr Val Thr Leu Ser Val Cys His Phe
260 265 270Gly Val Glu Leu Gly Gly Arg Phe Asn Phe
275 280<210>12<211>286<212>PRT<213>恰菲埃里希体(Ehrlichia chaffeensis)<220><223>恰菲埃里希体OMP-1D的氨基酸序列<400>12Met Asn Cys Glu Lys Phe Phe Ile Thr Thr Ala Leu Thr Leu Leu
5 10 15Met Ser Phe Leu Pro Gly Ile Ser Leu Ser Asp Pro Val Gln Asp
20 25 30Asp Asn Ile Ser Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Met Pro
35 40 45Ser Ala Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Arg Asn
50 55 60Thr Thr Val Gly Val Phe Gly Ile Glu Gln Asp Trp Asp Arg Cys
65 70 75Val Ile Ser Arg Thr Thr Leu Ser Asp Ile Phe Thr Val Pro Asn
80 85 90Tyr Ser Phe Lys Tyr Glu Asn Asn Leu Phe Ser Gly Phe Ala Gly
95 100 105Ala Ile Gly Tyr Ser Met Asp Gly Pro Arg Ile Glu Leu Glu Val
110 115 120Ser Tyr Glu Ala Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys
125 130 135Asn Glu Ala His Arg Tyr Tyr Ala Leu Ser His Leu Leu Gly Thr
140 145 150Glu Thr Gln Ile Asp Gly Ala Gly Ser Ala Ser Val Phe Leu Ile
155 160 165Asn Glu Gly Leu Leu Asp Lys Ser Phe Met Leu Asn Ala Cys Tyr
170 175 180Asp Val Ile Ser Glu Gly Ile Pro Phe Ser Pro Tyr Ile Cys Ala
185 190 195Gly Ile Gly Ile Asp Leu Val Ser Met Phe Glu Ala Ile Asn Pro
200 205 210Lys Ile Ser Tyr Gln Gly Lys Leu Gly Leu Ser Tyr Pro Ile Ser
215 220 225Pro Glu Ala Ser Val Phe Ile Gly Gly His Phe His Lys Val Ile
230 235 240Gly Asn Glu Phe Arg Asp Ile Pro Thr Met Ile Pro Ser Glu Ser
245 250 255Ala Leu Ala Gly Lys Gly Asn Tyr Pro Ala Ile Val Thr Leu Asp
260 265 270Val Phe Tyr Phe Gly Ile Glu Leu Gly Gly Arg Phe Asn Phe Gln
275 280 285Leu<210>13<211>278<212>PRT<213>恰菲埃里希体(Ehrlichia chaffeensis)<220><223>恰菲埃里希体OMP-1E的氨基酸序列<400>13Met Asn Cys Lys Lys Phe Phe Ile Thr Thr Ala Leu Val Ser Leu
5 10 15Met Ser Phe Leu Pro Gly Ile Ser Phe Ser Asp Pro Val Gln Gly
20 25 30Asp Asn Ile Ser Gly Asn Phe Tyr Val Ser Gly Lys Tyr Met Pro
35 40 45Ser Ala Ser His Phe Gly Met Phe Ser Ala Lys Glu Glu Lys Asn
50 55 60Pro Thr Val Ala Leu Tyr Gly Leu Lys Gln Asp Trp Glu Gly Ile
65 70 75Ser Ser Ser Ser His Asn Asp Asn His Phe Asn Asn Lys Gly Tyr
80 85 90Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Ala
95 100 105Ile Gly Tyr Ser Met Gly Gly Pro Arg Val Glu Phe Glu Val Ser
110 115 120Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys Asn
125 130 135Asp Ala His Arg Tyr Cys Ala Leu Gly Gln Gln Asp Asn Ser Gly
140 145 150Ile Pro Lys Thr Ser Lys Tyr Val Leu Leu Lys Ser Glu Gly Leu
155 160 165Leu Asp Ile Ser Phe Met Leu Asn Ala Cys Tyr Asp Ile Ile Asn
170 175 180Glu Ser Ile Pro Leu Ser Pro Tyr Ile Cys Ala Gly Val Gly Thr
185 190 195Asp Leu Ile Ser Met Phe Glu Ala Thr Asn Pro Lys Ile Ser Tyr
200 205 210Gln Gly Lys Leu Gly Leu Ser Tyr Ser Ile Asn Pro Glu Ala Ser
215 220 225Val Phe Ile Gly Gly His Phe His Lys Val Ile Gly Asn Glu Phe
230 235 240Arg Asp Ile Pro Thr Leu Lys Ala Phe Val Thr Ser Ser Ala Thr
245 250 255Pro Asp Leu Ala Ile Val Thr Leu Ser Val Cys His Phe Gly Ile
260 265 270Glu Leu Gly Gly Arg Phe Asn Phe
275<210>14<211>280<212>PRT<213>恰菲埃里希体(Ehrlichia chaffeensis)<220><223>恰菲埃里希体OMP-1F的氨基酸序列<400>14Met Asn Cys Lys Lys Phe Phe Ile Thr Thr Thr Leu Val Ser Leu
5 10 15Met Ser Phe Leu Pro Gly Ile Ser Phe Ser Asp Ala Val Gln Asn
20 25 30Asp Asn Val Gly Gly Asn Phe Tyr Ile Ser Gly Lys Tyr Val Pro
35 40 45Ser Val Ser His Phe Gly Val Phe Ser Ala Lys Gln Glu Arg Asn
50 55 60Thr Thr Thr Gly Val Phe Gly Leu Lys Gln Asp Trp Asp Gly Ser
65 70 75Thr Ile Ser Lys Asn Ser Pro Glu Asn Thr Phe Asn Val Pro Asn
80 85 90Tyr Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly
95 100 105Ala Val Gly Tyr Leu Met Asn Gly Pro Arg Ile Glu Leu Glu Met
110 115 120Ser Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys
125 130 135Asn Asp Ala His Lys Tyr Tyr Ala Leu Thr His Asn Ser Gly Gly
140 145 150Lys Leu Ser Asn Ala Gly Asp Lys Phe Val Phe Leu Lys Asn Glu
155 160 165Gly Leu Leu Asp Ile Ser Leu Met Leu Asn Ala Cys Tyr Asp Val
170 175 180Ile Ser Glu Gly Ile Pro Phe Ser Pro Tyr Ile Cys Ala Gly Val
185 190 195Gly Thr Asp Leu Ile Ser Met Phe Glu Ala Ile Asn Pro Lys Ile
200 205 210Ser Tyr Gln Gly Lys Leu Gly Leu Ser Tyr Ser Ile Ser Pro Glu
215 220 225Ala Ser Val Phe Val Gly Gly His Phe His Lys Val Ile Gly Asn
230 235 240Glu Phe Arg Asp Ile Pro Ala Met Ile Pro Ser Thr Ser Thr Leu
245 250 255Thr Gly Asn His Phe Thr Ile Val Thr Leu Ser Val Cys His Phe
260 265 270Gly Val Glu Leu Gly Gly Arg Phe Asn Phe
275 280<210>15<211>284<212>PRT<213>反刍类考德里氏体(Cowdria ruminantium)<220><223>反刍类考德里氏体MAP-1的氨基酸序列<400>15Met Asn Cys Lys Lys Ile Phe Ile Thr Ser Thr Leu Ile Ser Leu
5 10 15Val Ser Phe Leu Pro Gly Val Ser Phe Ser Asp Val Ile Gln Glu
20 25 30Glu Asn Asn Pro Val Gly Ser Val Tyr Ile Ser Ala Lys Tyr Met
35 40 45Pro Thr Ala Ser His Phe Gly Lys Met Ser Ile Lys Glu Asp Ser
50 55 60Arg Asp Thr Lys Ala Val Phe Gly Leu Lys Lys Asp Trp Asp Gly
65 70 75Val Lys Thr Pro Ser Gly Asn Thr Asn Ser Ile Phe Thr Glu Lys
80 85 90Asp Tyr Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala
95 100 105Gly Ala Val Gly Tyr Ser Met Asn Gly Pro Arg Ile Glu Phe Glu
110 115 120Val Ser Tyr Glu Thr Phe Asp Val Arg Asn Pro Gly Gly Asn Tyr
125 130 135Lys Asn Asp Ala His Met Tyr Cys Ala Leu Asp Thr Ala Ser Ser
140 145 150Ser Thr Ala Gly Ala Thr Thr Ser Val Met Val Lys Asn Glu Asn
155 160 165Leu Thr Asp Ile Ser Leu Met Leu Asn Ala Cys Tyr Asp Ile Met
170 175 180Leu Asp Gly Met Pro Val Ser Pro Tyr Val Cys Ala Gly Ile Gly
185 190 195Thr Asp Leu Val Ser Val Ile Asn Ala Thr Asn Pro Lys Leu Ser
200 205 210Tyr Gln Gly Lys Leu Gly Ile Ser Tyr Ser Ile Asn Pro Glu Ala
215 220 225Ser Ile Phe Ile Gly Gly His Phe His Arg Val Ile Gly Asn Glu
230 235 240Phe Lys Asp IIe Ala Thr Ser Lys Val Phe Thr Ser Ser Gly Asn
245 250 255Ala Ser Ser Ala Val Ser Pro Gly Phe Ala Ser Ala Ile Leu Asp
260 265 270Val Cys His Phe Gly Ile Glu Ile Gly Gly Arg Phe Val Phe
275 280<210>16<211>20<212>DNA<213>人工序列<220><221>primer_bind<222>反刍类考德里氏体MAP-1的核苷酸313-332,也是恰菲埃里希体P28的核苷酸307-326<223>PCR正向引物793<400>16gcaggagctg ttggttactc 20<210>17<211>21<212>DNA<213>人工序列<220><221>primer_bind<222>反刍类考德里氏体MAP-1的核苷酸823-843,也是恰菲埃里希体P28的核苷酸814-834<223>PCR反向引物1330<400>17ccttcctcca agttctatgc c 21<210>18<211>24<212>DNA<213>人工序列<220><221>primer_bind<223>引物46f,对p28-5基因特异性<400>18atatacttcc tacctaatgt ctca 24<210>19<211>20<212>DNA<213>人工序列<220><221>primer_bind<223>用于犬埃里希体28-kDa蛋白基因测序的引物<400>19agtgcagagt cttcggtttc 20<210>20<211>18<212>DNA<213>人工序列<220><221>primer_bind<223>用于犬埃里希体28-kDa蛋白质基因测序的引物<400>20gttacttgcg gaggacat 18<210>21<211>24<212>DNA<213>人工序列<220><221>primer_bind<222>犬埃里希体p28-7的核苷酸687-710<223>PCR引物394<400>21gcatttccac aggatcatag gtaa 24<210>22<211>24<212>DNA<213>人工序列<220><221>primer_bind<222>犬埃里希体p28-7的核苷酸710-687<223>PCR引物394C<400>22ttacctatga tcctgtggaa atgc 24<210>23<211>20<212>DNA<213>人工序列<220><221>primer_bind<223>与犬埃里希体p28-7的一个区域退火的引物793C,用于扩增基因p28-6和p28-7基因间区<400>23gagtaaccaa cagctcctgc 20<210>24<211>24<212>DNA<213>人工序列<220><221>primer_bind<222><223>与邻近p28-7开放阅读框的非编码区互补的引物EC280M-F<400>24tctactttgc acttccacta ttgt 24<210>25<211>24<212>DNA<213>人工序列<220><221>primer_bind<222><223>与邻近p28-7的开放阅读框互补的引物EC280M-R<400>25attcttttgc cactattttt cttt 24<210>26<211>25<212>DNA<213>人工序列<220><221>primer_bind<223>对应于p28-6内区域的引物ECaSA3-2,用于扩增基因p28-6和p28-7之间的基因间区NC3<400>26ctaggattag gttatagtat aagtt 25<210>27<211>23<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><221>PEPTIDE<223>p28-7和p28-6的预测的N-末端信号肽<400>27Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Ala Leu Met Ser Leu
5 10 15Met Tyr Tyr Ala Pro Ser Ile Ser
20<210>28<211>25<212>PRT<213>恰菲埃里希体(Ehrlichia chaffeensis)<220><223>恰菲埃里希体P28的N-末端信号肽的氨基酸序列<400>28Met Asn Tyr Lys Lys Ile Leu Ile Thr Ser Ala Leu Ile Ser Leu
5 10 15Ile Ser Ser Leu Pro Gly Val Ser Phe Ser
20 25<210>29<211>26<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>p28-7的推测切割位点的氨基酸序列<400>29Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Ala Leu Ile Ser Leu
5 10 15Met Tyr Ser Ile Pro Ser Ile Ser Ser Phe Ser
20 25<210>30<211>299<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>基因间非编码区1(28NC1)的核酸序列<400>30taatacttct attgtacatg ttaaaaatag tactagtttg cttctgtggt 50ttataaacgc aagagagaaa tagttagtaa taaattagaa agttaaatat 100tagaaaagtc atatgttttt cattgtcatt gatactcaac taaaagtagt 150ataaatgtta cttattaata attttacgta gtatattaaa tttcccttac 200aaaagccact agtattttat actaaaagct atactttggc ttgtatttaa 250tttgtatttt tactactgtt aatttacttt cactgtttct ggtgtaaat 299<210>31<211>345<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>基因间非编码区2(28NC2)的核酸序列<400>31taatttcgtg gtacacatat cacgaagcta aaattgtttt tttatctctg 50ctgtatacaa gagaaaaaat agtagtgaaa attacctaac aatatgacag 100tacaagttta ccaagcttat tctcacaaaa cttcttgtgt cttttatctc 150tttacaatga aatgtacact tagcttcact actgtagagt gtgtttatca 200atgctttgtt tattaatact ctacataata tgttaaattt ttcttacaaa 250actcactagt aatttatact agaatatata ttctgacttg tatttgcttt 300atacttccac tattgttaat ttattttcac tattttaggt gtaat 345<210>32<211>345<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>基因间非编码区3(28NC3)的核酸序列<400>32tgattttatt gttgccacat attaaaaatg atctaaactt gtttttatta 50ttgctacata caaaaaaaag aaaaatagtg gcaaaagaat gtagcaataa 100gagggggggg ggggactaaa tttaccttct attcttctaa tattctttac 150tatattcaaa tagcacaact caatgcttcc aggaaaatat gtttctaata 200ttttatttat taccaatcct tatataatat attaaatttc tcttacaaaa 250atctctaatg ttttatactt aatatatata ttctggcttg tatttacttt 300gcacttccac tattgttaat ttattttcac tattttaggt gtaat 345<210>33<211>355<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>基因间非编码区4(28NC4)的核酸序列<400>33taattttatt gttgccacat attaaaaatg atctaaactt gtttttawta 50ttgctacata caaaaaaaga aaaatagtgg caaaagaatg tagcaataag 100aggggggggg gggaccaaat ttatcttcta tgcttcccaa gttttttcyc 150gctatttatg acttaaacaa cagaaggtaa tatcctcacg gaaaacttat 200cttcaaatat tttatttatt accaatctta tataatatat taaatttctc 250ttacaaaaat cactagtatt ttataccaaa atatatattc tgacttgctt 300ttcttctgca cttctactat ttttaattta tttgtcacta ttaggttata 350ataaw 355<210>34<211>24<212>DNA<213>人工序列<220><223>引物p28-5-818f<400>34ttaaacatat gccacttcgg acta 24<210>35<211>28<212>DNA<213>人工序列<220><223>引物1191<400>35tatgatcgtg taaaattgct gtgagtat 28<210>36<211>20<212>DNA<213>人工序列<220><223>引物ECa28-75C<400>36tactggcacg tgctggacta 20<210>37<211>22<212>DNA<213>人工序列<220><223>引物ECa5’-1600<400>37caccaataaa tgcagagact tc 22<210>38<211>26<212>DNA<213>人工序列<220><223>引物3125<400>38aatccatcat ttctcattac agtgtg 26<210>39<211>879<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-1的核酸序列<400>39atgaataata aactcaaatt tactataata aacacagtat tagtatgctt 50attgtcatta cctaatatat cttcctcaaa ggccataaac aataacgcta 100aaaagtacta cggattatat atcagtggac aatataaacc cagtgtttct 150gttttcagta atttttcagt taaagaaacc aatgtcataa ctaaaaacct 200tatagcttta aaaaaagatg ttgactctat tgaaaccaag actgatgcca 250gtgtaggtat tagtaaccca tcaaatttta ctatccccta tacagctgta 300tttcaagata attctgtcaa tttcaatgga actattggtt acacctttgc 350tgaaggtaca agagttgaaa tagaaggttc ttatgaggaa tttgatgtta 400aaaaccctgg aggctataca ctaagtgatg cctatcgcta ttttgcatta 450gcacgtgaaa tgaaaggtaa tagttttaca cctaaagaaa aagtttctaa 500tagtattttt cacactgtaa tgagaaatga tggattatct ataatatctg 550ttatagtaaa tgtttgctac gatttctctt tgaacaattt gtcaatatcg 600ccttacatat gtggaggagc aggggtagat gctatagaat tcttcgatgt 650attacacatt aagtttgcat atcaaagcaa gctaggtatt gcttattctc 700taccatctaa cattagtctc tttgctagtt tatattacca taaagtaatg 750ggcaatcaat ttaaaaattt aaatgtccaa catgttgctg aacttgcaag 800tatacctaaa attacatccg cagttgctac acttaatatt ggttattttg 850gaggtgaaat tggtgcaaga ttgacattt 879<210>40<211>293<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-1蛋白的氨基酸序列<400>40Met Asn Asn Lys Leu Lys Phe Thr Ile Ile Asn Thr Val Leu Val
5 10 15Cys Leu Leu Ser Leu Pro Asn Ile Ser Ser Ser Lys Ala Ile Asn
20 25 30Asn Asn Ala Lys Lys Tyr Tyr Gly Leu Tyr Ile Ser Gly Gln Tyr
35 40 45Lys Pro Ser Val Ser Val Phe Ser Asn Phe Ser Val Lys Glu Thr
50 55 60Asn Val Ile Thr Lys Asn Leu Ile Ala Leu Lys Lys Asp Val Asp
65 70 75Ser Ile Glu Thr Lys Thr Asp Ala Ser Val Gly Ile Ser Asn Pro
80 85 90Ser Asn Phe Thr Ile Pro Tyr Thr Ala Val Phe Gln Asp Asn Ser
95 100 105Val Asn Phe Asn Gly Thr Ile Gly Tyr Thr Phe Ala Glu Gly Thr
110 115 120Arg Val Glu Ile Glu Gly Ser Tyr Glu Glu Phe Asp Val Lys Asn
125 130 135Pro Gly Gly Tyr Thr Leu Ser Asp Ala Tyr Arg Tyr Phe Ala Leu
140 145 150Ala Arg Glu Met Lys Gly Asn Ser Phe Thr Pro Lys Glu Lys Val
155 160 165Ser Asn Ser Ile Phe His Thr Val Met Arg Asn Asp Gly Leu Ser
170 175 180Ile Ile Ser Val Ile Val Asn Val Cys Tyr Asp Phe Ser Leu Asn
185 190 195Asn Leu Ser Ile Ser Pro Tyr Ile Cys Gly Gly Ala Gly Val Asp
200 205 210Ala Ile Glu Phe Phe Asp Val Leu His Ile Lys Phe Ala Tyr Gln
215 220 225Ser Lys Leu Gly Ile Ala Tyr Ser Leu Pro Ser Asn Ile Ser Leu
230 235 240Phe Ala Ser Leu Tyr Tyr His Lys Val Met Gly Asn Gln Phe Lys
245 250 255Asn Leu Asn Val Gln His Val Ala Glu Leu Ala Ser Ile Pro Lys
260 265 270Ile Thr Ser Ala Val Ala Thr Leu Asn Ile Gly Tyr Phe Gly Gly
275 280 285Glu Ile Gly Ala Arg Leu Thr Phe
290 293<210>41<211>840<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-2的核酸序列<400>41atgaattata agaaaattct agtaagaagc gcgttaatct cattaatgtc 50aatcttacca tatcagtctt ttgcagatcc tgtaggttca agaactaatg 100ataacaaaga aggcttctac attagtgcaa agtacaatcc aagtatatca 150cactttagaa aattctctgc tgaagaaact cctattaatg gaacaaattc 200tctcactaaa aaagttttcg gactaaagaa agatggtgat ataacaaaaa 250aagacgattt tacaagagta gctccaggca ttgattttca aaataactta 300atatcaggat tttcaggaag tattggttac tctatggacg gaccaagaat 350agaacttgaa gctgcatatc aacaatttaa tccaaaaaac accgataaca 400atgatactga taatggtgaa tactataaac attttgcatt atctcgtaaa 450gatgcaatgg aagatcagca atatgtagta cttaaaaatg acggcataac 500ttttatgtca ttgatggtta atacttgcta tgacattaca gctgaaggag 550tatctttcgt accatatgca tgtgcaggta taggagcaga tcttatcact 600atttttaaag acctcaatct aaaatttgct taccaaggaa aaataggtat 650tagttaccct atcacaccag aagtctctgc atttattggt ggatactacc 700atggcgttat tggtaataaa tttgagaaga tacctgtaat aactcctgta 750gtattaaatg atgctcctca aaccacatct gcttcagtaa ctcttgacgt 800tggatacttt ggcggagaaa ttggaatgag gttcaccttc 840<210>42<211>280<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-2蛋白的氨基酸序列<400>42Met Asn Tyr Lys Lys Ile Leu Val Arg Ser Ala Leu Ile Ser Leu
5 10 15Met Ser Ile Leu Pro Tyr Gln Ser Phe Ala Asp Pro Val Gly Ser
20 25 30Arg Thr Asn Asp Asn Lys Glu Gly Phe Tyr Ile Ser Ala Lys Tyr
35 40 45Asn Pro Ser Ile Ser His Phe Arg Lys Phe Ser Ala Glu Glu Thr
50 55 60Pro Ile Asn Gly Thr Asn Ser Leu Thr Lys Lys Val Phe Gly Leu
65 70 75Lys Lys Asp Gly Asp Ile Thr Lys Lys Asp Asp Phe Thr Arg Val
80 85 90Ala Pro Gly Ile Asp Phe Gln Asn Asn Leu Ile Ser Gly Phe Ser
95 100 105Gly Ser Ile Gly Tyr Ser Met Asp Gly Pro Arg Ile Glu Leu Glu
110 115 120Ala Ala Tyr Gln Gln Phe Asn Pro Lys Asn Thr Asp Asn Asn Asp
125 130 135Thr Asp Asn Gly Glu Tyr Tyr Lys His Phe Ala Leu Ser Arg Lys
140 145 150Asp Ala Met Glu Asp Gln Gln Tyr Val Val Leu Lys Asn Asp Gly
155 160 165Ile Thr Phe Met Ser Leu Met Val Asn Thr Cys Tyr Asp Ile Thr
170 175 180Ala Glu Gly Val Ser Phe Val Pro Tyr Ala Cys Ala G1y Ile Gly
185 190 195Ala Asp Leu Ile Thr Ile Phe Lys Asp Leu Asn Leu Lys Phe Ala
200 205 210Tyr Gln Gly Lys Ile Gly Ile Ser Tyr Pro Ile Thr Pro Glu Val
215 220 225Ser Ala Phe Ile Gly Gly Tyr Tyr His Gly Val Ile Gly Asn Lys
230 235 240Phe Glu Lys Ile Pro Val Ile Thr Pro Val Val Leu Asn Asp Ala
245 250 255Pro Gln Thr Thr Ser Ala Ser Val Thr Leu Asp Val Gly Tyr Phe
260 265 270Gly Gly Glu Ile Gly Met Arg Phe Thr Phe
275 280<210>43<211>828<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-3的核酸序列<400>43atgaactgta aaaaaattct tataacaact acattggtat cactaacaat 50tcttttacct ggcatatctt tctccaaacc aatacatgaa aacaatacta 100caggaaactt ttacattatt ggaaaatatg taccaagtat ttcacatttt 150gggaactttt cagctaaaga agaaaaaaac acaacaactg gaatttttgg 200attaaaagaa tcatggactg gtggtatcat ccttgataaa gaacatgcag 250cttttaatat cccaaattat tcatttaaat atgaaaataa tccattttta 300ggatttgcag gggtaattgg ctattcaata ggtagtccaa gaatagaatt 350tgaagtatca tacgagacat tcgatgtaca aaatccagga gataagttta 400acaatgatgc acataagtat tgtgctttat ccaatgattc cagtaaaaca 450atgaaaagtg gtaaattcgt ttttctcaaa aatgaaggat taagtgacat 500atcactcatg ttaaatgtat gttatgatat aataaacaaa agaatgcctt 550tttcacctta catatgtgca ggcattggta ctgacttaat attcatgttt 600gacgctataa accataaagc tgcttatcaa ggaaaattag gttttaatta 650tccaataagc ccagaagcta acatttctat gggtgtgcac tttcacaaag 700taacaaacaa cgagtttaga gttcctgttc tattaactgc tggaggactc 750gctccagata atctatttgc aatagtaaag ttgagtatat gtcattttgg 800gttagaattt gggtacaggg tcagtttt 828<210>44<211>276<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-3蛋白的氨基酸序列<400>44Met Asn Cys Lys Lys Ile Leu Ile Thr Thr Thr Leu Val Ser Leu
5 10 15Thr Ile Leu Leu Pro Gly Ile Ser Phe Ser Lys Pro Ile His Glu
20 25 30Asn Asn Thr Thr Gly Asn Phe Tyr Ile Ile Gly Lys Tyr Val Pro
35 40 45Ser Ile Ser His Phe Gly Asn Phe Ser Ala Lys Glu Glu Lys Asn
50 55 60Thr Thr Thr Gly Ile Phe Gly Leu Lys Glu Ser Trp Thr Gly Gly
65 70 75Ile Ile Leu Asp Lys Glu His Ala Ala Phe Asn Ile Pro Asn Tyr
80 85 90Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Gly Val
95 100 105Ile Gly Tyr Ser Ile Gly Ser Pro Arg Ile Glu Phe Glu Val Ser
110 115 120Tyr Glu Thr Phe Asp Val Gln Asn Pro Gly Asp Lys Phe Asn Asn
125 130 135Asp Ala His Lys Tyr Cys Ala Leu Ser Asn Asp Ser Ser Lys Thr
140 145 150Met Lys Ser Gly Lys Phe Val Phe Leu Lys Asn Glu Gly Leu Ser
155 160 165Asp Ile Ser Leu Met Leu Asn Val Cys Tyr Asp Ile Ile Asn Lys
170 175 180Arg Met Pro Phe Ser Pro Tyr Ile Cys Ala Gly Ile Gly Thr Asp
185 190 195Leu Ile Phe Met Phe Asp Ala Ile Asn His Lys Ala Ala Tyr Gln
200 205 210Gly Lys Leu Gly Phe Asn Tyr Pro Ile Ser Pro Glu Ala Asn Ile
215 220 225Ser Met Gly Val His Phe His Lys Val Thr Asn Asn Glu Phe Arg
230 235 240Val Pro Val Leu Leu Thr Ala Gly Gly Leu Ala Pro Asp Asn Leu
245 250 255Phe Ala Ile Val Lys Leu Ser Ile Cys His Phe Gly Leu Glu Phe
260 265 270Gly Tyr Arg Val Ser Phe
275<210>45<211>813<212>DNA<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-9的核酸序列<400>45atgaattaca aaagatttgt tgtaggtgtt acgctgagta catttgtttt 50tttcttatct gatggtgctt tttctgatgc aaatttttct gaagggagga 100gaggacttta tataggtagt cagtataaag ttggtattcc caattttagt 150aatttttcag ctgaagaaac aattcctggt attacaaaaa agatttttgc 200gttaggtctt gataagtctg agataaatac tcacagcaat tttacacgat 250catatgaccc tacttatgca agcagttttg cagggtttag tggtatcatt 300ggatattatg ttaatgactt tagggtagaa tttgaaggtt cttatgagaa 350ttttgaacct gaaagacaat ggtaccctga gaatagccaa agctacaaat 400tttttgcttt gtctcgaaat gctacaaata gtgataataa gtttatagta 450ctagagaata acggcgttgt tgacaagtct cttaatgtaa atgtttgtta 500tgatattgct agtggtagta ttcctttagc accttatatg tgtgctggtg 550ttggtgcaga ttatataaag tttttaggta tatcattgcc taagttttct 600tatcaagtta agtttggtgt caactaccct ctaaatgtta atactatgtt 650gtttggtggg ggttattacc ataaggttgt aggtgatagg catgagagag 700tagaaatagc ttaccatcct actgcattat ctgacgttcc tagaactact 750tcagcttctg ctactttaaa tactgattat tttggttggg agattggatt 800tagatttgcg cta 813<210>46<211>271<212>PRT<213>犬埃里希体(Ehrlichia canis)<220><223>犬埃里希体p28-9蛋白的氨基酸序列<400>46Met Asn Tyr Lys Arg Phe Val Val Gly Val Thr Leu Ser Thr Phe
5 10 15Val Phe Phe Leu Ser Asp Gly Ala Phe Ser Asp Ala Asn Phe Ser
20 25 30Glu Gly Arg Arg Gly Leu Tyr Ile Gly Ser Gln Tyr Lys Val Gly
35 40 45Ile Pro Asn Phe Ser Asn Phe Ser Ala Glu Glu Thr Ile Pro Gly
50 55 60Ile Thr Lys Lys Ile Phe Ala Leu Gly Leu Asp Lys Ser Glu Ile
65 70 75Asn Thr His Ser Asn Phe Thr Arg Ser Tyr Asp Pro Thr Tyr Ala
80 85 90Ser Ser Phe Ala Gly Phe Ser Gly Ile Ile Gly Tyr Tyr Val Asn
95 100 105Asp Phe Arg Val Glu Phe Glu Gly Ser Tyr Glu Asn Phe Glu Pro
110 115 120Glu Arg Gln Trp Tyr Pro Glu Asn Ser Gln Ser Tyr Lys Phe Phe
125 130 135Ala Leu Ser Arg Asn Ala Thr Asn Ser Asp Asn Lys Phe Ile Val
140 145 150Leu Glu Asn Asn Gly Val Val Asp Lys Ser Leu Asn Val Asn Val
155 160 165Cys Tyr Asp Ile Ala Ser Gly Ser Ile Pro Leu Ala Pro Tyr Met
170 175 180Cys Ala Gly Val Gly Ala Asp Tyr Ile Lys Phe Leu Gly Ile Ser
185 190 195Leu Pro Lys Phe Ser Tyr Gln Val Lys Phe Gly Val Asn Tyr Pro
200 205 210Leu Asn Val Asn Thr Met Leu Phe Gly Gly Gly Tyr Tyr His Lys
215 220 225Val Val Gly Asp Arg His Glu Arg Val Glu Ile Ala Tyr His Pro
230 235 240Thr Ala Leu Ser Asp Val Pro Arg Thr Thr Ser Ala Ser Ala Thr
245 250 255Leu Asn Thr Asp Tyr Phe Gly Trp Glu Ile Gly Phe Arg Phe Ala
260 265 270Leu271
Claims (20)
1.一种编码犬埃里希体30kDa蛋白质的分离的DNA序列,其特征在于,所述蛋白质与抗犬埃里希体血清具有免疫反应性。
2.如权利要求1所述的DNA序列,其特征在于,所述蛋白质含有选自SEQ ID No.2,4,6,40,42,44和46的氨基酸序列。
3.如权利要求2所述的DNA序列,其特征在于,所述蛋白质具有N-末端信号序列。
4.如权利要求3所述的DNA序列,其特征在于,所述蛋白质是经翻译后修饰成28kDa蛋白质。
5.如权利要求1所述的DNA序列,其特征在于,所述DNA含有选自SEQ ID No.1,3,5,39,41,43和45的序列。
6.如权利要求1所述的DNA序列,其特征在于,所述DNA包含在犬埃里希体的一个基因座中。
7.如权利要求6所述的DNA序列,其特征在于,所述基因座是长10,677bp的一个多基因基因座。
8.如权利要求7所述的DNA序列,其特征在于,所述基因座含有编码犬埃里希体同源性28kDa蛋白质的基因。
9.如权利要求8所述的DNA序列,其特征在于,所述犬埃里希体的同源性28kDa蛋白选自p28-1,p28-2,p28-3,p28-4,p28-5,p28-6,p28-7,p28-8和p28-9。
10.一种含有权利要求1所述的DNA序列的载体。
11.如权利要求10所述的载体,其特征在于,所述载体是一种表达载体,当所述载体被引入细胞时,能够表达选自SEQ ID No.1,3,5,39,41,43和45的序列编码的肽或多肽。
12.一种重组蛋白质,其特征在于,该蛋白质含有选自SEQ ID No.2,4,6,40,42,44和46的氨基酸序列。
13.如权利要求12所述的氨基酸序列,其特征在于,所述氨基酸序列由含有选自SEQ ID No.1,3,5,39,41,43和45的序列的核酸区段所编码。
14.一种宿主细胞,其特征在于,该细胞含有选自SEQ ID No.1,3,5,39,41,43和45的核酸区段。
15.一种产生权利要求12所述的重组蛋白质的方法,其特征在于,该方法包括步骤:
获得含有表达区的载体,所述表达区含有与启动子可操纵性连接的、编码选自SEQ ID No.2,4,6,40,42,44和46的氨基酸序列的序列;
将所述载体转染入细胞;和
在有效表达所述表达区的条件下培养所述细胞。
16.一种抗体,其特征在于,该抗体与含有选自SEQ ID No.2,4,6,40,42,44和46的氨基酸序列的多肽具有免疫反应性。
17.一种抑制个体内犬埃里希体感染的方法,其特征在于,该方法包括步骤:
在接触前或怀疑已接触,或已感染犬埃里希体时鉴定个体;和
以抑制犬埃里希体感染的有效量施用含有犬埃里希体28kDa抗原的组合物。
18.如权利要求17所述的方法,其特征在于,所述28kDa抗原是重组蛋白质,它含有选自SEQ ID No.2,4,6,40,42,44和46的氨基酸序列。
19.如权利要求18所述的方法,其特征在于,所述重组蛋白质由含有选自SEQID No.1,3,5,39,41,43和45的序列的基因编码。
20.如权利要求17所述的方法,其特征在于,所述含有28kDa抗原的组合物分散于药物学上可接受的载体中。
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US09/660,587 US6392023B1 (en) | 1999-03-03 | 2000-09-12 | Homologous 28-kilodalton immunodominant protein genes of Ehrlicha canis and uses thereof |
US09/660,587 | 2000-09-12 |
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CNA2008100927197A Pending CN101307312A (zh) | 2000-09-12 | 2001-09-12 | 犬埃里希体的同源28kD免疫优势蛋白质基因及其用途 |
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US6043085A (en) * | 1998-08-27 | 2000-03-28 | Research Development Foundation | Ehrlichia canis 120-kDa immunodominant antigenic protein and gene |
US6544517B1 (en) * | 1998-09-18 | 2003-04-08 | The Ohio State University Research Foundation | Outer membrane protein of Ehrlichia canis and Ehrlichia chaffeensis |
US6392023B1 (en) * | 1999-03-03 | 2002-05-21 | Research Development Foundation | Homologous 28-kilodalton immunodominant protein genes of Ehrlicha canis and uses thereof |
US6458942B1 (en) * | 1998-11-30 | 2002-10-01 | Research Development Foundation | 28-kDa immunoreactive protein gene of Ehrlichia canis and uses thereof |
AU762315B2 (en) | 1998-11-30 | 2003-06-19 | Research Development Foundation | Homologous 28-kilodalton immunodominant protein genes of ehrlichia canis and uses thereof |
US6685886B2 (en) * | 1998-12-17 | 2004-02-03 | Genencor International, Inc. | Agitation system for a fluid bed processing system and a method thereof |
US6214400B1 (en) | 1999-10-14 | 2001-04-10 | Lyco Manufacturing Inc. | Method for processing food product |
US6308529B1 (en) | 1999-10-20 | 2001-10-30 | Morris & Associates, Inc. | Poultry chiller with open auger |
US6658886B1 (en) | 2003-01-24 | 2003-12-09 | Morris & Associates, Inc. | Poultry chiller having an integral sump compartment |
-
2000
- 2000-09-12 US US09/660,587 patent/US6392023B1/en not_active Expired - Fee Related
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2001
- 2001-03-16 US US09/811,007 patent/US6660269B2/en not_active Expired - Fee Related
- 2001-09-12 BR BR0113818-9A patent/BR0113818A/pt not_active IP Right Cessation
- 2001-09-12 AU AU9092601A patent/AU9092601A/xx active Pending
- 2001-09-12 AU AU2001290926A patent/AU2001290926B2/en not_active Ceased
- 2001-09-12 IL IL15487001A patent/IL154870A0/xx unknown
- 2001-09-12 JP JP2002527225A patent/JP2004508823A/ja not_active Withdrawn
- 2001-09-12 CA CA002421952A patent/CA2421952A1/en not_active Abandoned
- 2001-09-12 KR KR1020037003641A patent/KR100837581B1/ko not_active IP Right Cessation
- 2001-09-12 CN CNA018184561A patent/CN1473166A/zh active Pending
- 2001-09-12 RU RU2003106809/13A patent/RU2288952C2/ru not_active IP Right Cessation
- 2001-09-12 EP EP01970986A patent/EP1317474A4/en not_active Withdrawn
- 2001-09-12 CN CNA2008100927197A patent/CN101307312A/zh active Pending
- 2001-09-12 WO PCT/US2001/028759 patent/WO2002022782A2/en active IP Right Grant
- 2001-09-12 NZ NZ524678A patent/NZ524678A/en unknown
- 2001-09-12 MX MXPA03002145A patent/MXPA03002145A/es not_active Application Discontinuation
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2002
- 2002-01-31 US US10/062,920 patent/US7309583B2/en not_active Expired - Fee Related
- 2002-01-31 US US10/062,051 patent/US20030073095A1/en not_active Abandoned
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2003
- 2003-03-07 ZA ZA2003/01886A patent/ZA200301886B/en unknown
- 2003-12-09 US US10/731,554 patent/US7344719B2/en not_active Expired - Fee Related
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105203755A (zh) * | 2009-11-20 | 2015-12-30 | 爱贝斯股份有限公司 | 用于检测埃里希体属抗体的肽、装置和方法 |
US10444231B2 (en) | 2012-10-11 | 2019-10-15 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
US10948487B2 (en) | 2012-10-11 | 2021-03-16 | Zoetis Services Llc | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
US9851352B2 (en) | 2014-04-04 | 2017-12-26 | Abaxis, Inc. | Compositions and methods for identifying Ehrlichia species |
US11204351B2 (en) | 2014-04-04 | 2021-12-21 | Zoetis Services Llc | Compositions and methods for identifying Ehrlichia species |
Also Published As
Publication number | Publication date |
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US6392023B1 (en) | 2002-05-21 |
KR100837581B1 (ko) | 2008-06-13 |
KR20030042461A (ko) | 2003-05-28 |
EP1317474A2 (en) | 2003-06-11 |
CA2421952A1 (en) | 2002-03-21 |
RU2288952C2 (ru) | 2006-12-10 |
US20030096250A1 (en) | 2003-05-22 |
MXPA03002145A (es) | 2004-05-04 |
NZ524678A (en) | 2004-11-26 |
US6660269B2 (en) | 2003-12-09 |
WO2002022782A2 (en) | 2002-03-21 |
ZA200301886B (en) | 2005-01-26 |
US20030073095A1 (en) | 2003-04-17 |
US20030185849A1 (en) | 2003-10-02 |
AU2001290926B2 (en) | 2006-12-14 |
US7309583B2 (en) | 2007-12-18 |
BR0113818A (pt) | 2004-06-29 |
WO2002022782A3 (en) | 2002-05-30 |
EP1317474A4 (en) | 2005-11-16 |
US7344719B2 (en) | 2008-03-18 |
AU9092601A (en) | 2002-03-26 |
CN101307312A (zh) | 2008-11-19 |
IL154870A0 (en) | 2003-10-31 |
JP2004508823A (ja) | 2004-03-25 |
US20040247616A1 (en) | 2004-12-09 |
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