CN110168110A - 基于噬菌体的疏螺旋体属病的检测及其方法 - Google Patents
基于噬菌体的疏螺旋体属病的检测及其方法 Download PDFInfo
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Abstract
本发明涉及检测广义伯氏疏螺旋体(Borrelia burgdorferi sensu lato)的方法或用于检测与回归热(RF)相关的疏螺旋体属(Borrelia)的方法、用于进行这样的方法的试剂盒和治疗受试者中的广义伯氏疏螺旋体或RF感染的方法。还提供了对疏螺旋体属特异性的噬菌体的用途。
Description
发明领域
本发明涉及检测广义伯氏疏螺旋体(Borrelia burgdorferi sensu lato)或用于检测与回归热(Relapsing Fever)(RF)相关的疏螺旋体属(Borrelia)的方法、用于进行这样的方法的试剂盒和治疗受试者中的广义伯氏疏螺旋体或RF感染的方法。还提供了对疏螺旋体属特异性的噬菌体的用途。
背景
广义(s.l.)伯氏疏螺旋体是螺旋体门的疏螺旋体属的一组细菌物种,并且其已知是莱姆病(LD)中的病原体。在广义(s.l.)伯氏疏螺旋体中,在欧洲的莱姆病患者中通常被发现的三种主要物种是狭义伯氏疏螺旋体(B.burgdorferi sensu stricto(s.s.))、阿氏疏螺旋体(B.afzelii)和伽氏疏螺旋体(B.garinii)。而狭义伯氏疏螺旋体在美国和在欧洲都引起LD。其他较不经常遇到的LD物种包括斯氏疏螺旋体(Borrelia spielmanii)、法雷斯疏螺旋体(Borrelia valaisiana)、比塞蒂疏螺旋体(Borrelia bissettii)、卢西塔尼亚疏螺旋体(Borrelia lusitaniae)、芬兰疏螺旋体(Borrelia finlandensis)、巴伐利亚疏螺旋体(Borrelia bavariensis)、日本疏螺旋体(Borrelia japonica)、西尼伽疏螺旋体(Borrelia sinica)、斯氏疏螺旋体(Borrelia spielmanii)、塔卢基疏螺旋体(Borreliatanukii)、土德疏螺旋体(Borrelia turdi)、Borrelia Yangtze、梅奥疏螺旋体(Borreliamayonii)、Borrelia carolinensis、安德森疏螺旋体(Borrelia andersonii)、孤星疏螺旋体(Borrelia lonestari)和美国疏螺旋体(Borrelia Americana)。
引起LD的细菌通过被细菌感染的蜱的叮咬(并且较不频繁地通过其他昆虫)传播至人类。在美国这是最经常报告的蜱媒疾病,并且疾病控制和预防中心(CDC)每年记录大约30000个证实的LD病例。然而,根据CDC,每年疑似的疏螺旋体属感染的数目是约300000个。在欧洲,过去20年来,LD病例的数目已经稳步地增加,估计每年约85000例。在英格兰和威尔士,据估计每年约3000个新的LD的病例被诊断。
临床诊断LD通常是有问题的,因为LD的症状容易与其他疾病混淆。感染的表现的范围可以从明显的皮疹、疲劳、肌肉疼痛、头痛到严重的关节炎、神经状况和心脏状况。因此,疏螺旋体属感染的基于实验室的确定在LD诊断中发挥关键作用。目前的基于实验室的诊断方法集中在基于对该细菌的人类免疫应答间接地确定患者的身体中存在或不存在该细菌。直接检测患者中疏螺旋体属的存在的当前的方法的灵敏度低,并且因此被认为无诊断价值。
与所有已知方法学相关的第一个问题是由于这样的事实的结果,即细菌仅在感染的早期容易地从血液样品可得到。在感染的晚期,细菌倾向于迁移至神经系统,在神经系统它们可以保持隔离(remain sequestered),并且难以用常规诊断工具定位和鉴定。因此,在晚期通过试图检测细菌的存在来鉴定感染通常必须包括脑脊液或类似物的分析,而不是更简单的血液样品的分析。
用于鉴定细菌的存在的目前的“金标准”技术包括使来自疑似感染莱姆病的个体的样品的悬浮细菌培养物生长,并且之后表征该培养物中的细菌。通常,表征基于细菌的形态学特征的显微术评估。然而,在已经从培养物获得足够地大和发育的培养的群体以证实细菌的存在前,使培养物生长需要长达7周。即便如此,这样的测试也不能够区分莱姆病和其他蜱媒细菌感染。例如,与回归热(RF)相关的细菌形态学上类似于引起LD的细菌。LD和RF也具有重叠的临床症状。因此,当事实上患者患有RF时,该患者可能通过该方法被诊断为患有LD;这是一个问题,因为治疗方法学对于每种疾病可能不同。
第二种诊断方法学包括基于抗体的程序。对被感染的宿主的自身抗体(所述自身抗体作为宿主中对感染的免疫应答的一部分而产生)(即免疫应答抗体)特异性的抗体(即诊断抗体)被用于检测存在或不存在感染。然而,不幸的是,此类基于抗体的方法学仅具有短的机会窗口以精确地鉴定感染的存在。在LD的早期(感染后2-3周),宿主中作为其对感染的自身免疫反应的一部分的抗体应答通常地不足以发展成被诊断抗体清楚地检测。在晚期LD中,免疫应答抗体的产生被细菌抑制,并且因此再次,抗体信号可能太弱而不能以合理的精确度被检测。因此,估计基于抗体的诊断方法错误地将54%的患者鉴定成未被感染的。
第三种已知的诊断方法包括细菌的基于PCR的检测。由于患者的样品中通常极低的相关细菌的浓度,这类方法具有不良的灵敏度的水平。已经计算出,在美国,当患者的脑脊液样品被测试时,仅三分之一的患有LD的患者显示出阳性PCR结果。处于LD的早期的一半的患者显示出对于在血液样品中的细菌的存在的阴性PCR得出的结果。
作为目前的诊断和治疗LD的困难的实例,兽医学专业人员报告被他们使用的和基于使用诊断抗体的测试通常地提供对于在马中的广义伯氏疏螺旋体感染的大量的假阳性结果。结果,通常地认可大量的马被不必要地用抗生素诸如土霉素治疗。
因此,存在对于检测LD的病原体广义伯氏疏螺旋体的存在的替代方法的需要。由于诊断回归热(其也由疏螺旋体属物种引起,并且经常被错误地诊断为LD,并且反之亦然)面临的类似的困难,还存在对于检测疏螺旋体属的存在的替代方法的需要,疏螺旋体属是回归热的病原体(本文中被称为回归热疏螺旋体属(RF疏螺旋体属)),诸如宫本疏螺旋体(Borrelia miyamotoi)和赫姆斯疏螺旋体(Borrelia hermsii)。
发明概述
噬菌体(bacteriophage)(或噬菌体(phage))是可以感染细菌并在细菌中增殖的病毒。通常,它们由包裹在蛋白质外壳(即衣壳)中的核酸的核组成。感染广义伯氏疏螺旋体和RF疏螺旋体属的噬菌体被了解得非常少。缺乏关于疏螺旋体属噬菌体的研究努力/产出主要是由于以下两个障碍。首先,在体外生长疏螺旋体属菌株需要复杂的培养基以模拟其体内条件。这伴有对高度熟练的、“专门训练的”科学家的需求,这些科学家需要拥有关于疏螺旋体属和噬菌体二者的良好的专业知识水平,意味着关于疏螺旋体属噬菌体的学术输出已经被抑制。其次,通常使用的噬菌体表征方法,诸如噬斑测定,不能简单地转化为疏螺旋体属噬菌体研究,因为疏螺旋体属先前没有被观察到在固体琼脂表面上生长以形成可以在其上观察噬菌体的融合细胞生长(confluent cell growth)。本发明人认为他们是第一个持续地生长疏螺旋体属的有活力的菌苔的人,在所述疏螺旋体属的有活力的菌苔上可以进行疏螺旋体属噬菌体的研究。
对于适用于在检测存在或不存在细菌感染中使用的剂,该剂应该能够特异性鉴定与感染相关的细菌。有益地,该剂应该还能够以尽可能最小的侵入方式检测细菌感染。迄今为止,噬菌体还没有被表明在鉴定存在或不存在广义伯氏疏螺旋体感染中是有用的。即使它们已经被表明是有用的,由于对已知感染广义伯氏疏螺旋体的噬菌体的有限的了解,存在的信息不足以确定噬菌体是否可以在这类诊断方法中使用。本领域已经通过引导方法学到直接鉴定细菌或鉴定针对细菌直接产生的免疫应答抗体,继续开发所有相关的诊断。
然而,在大量实验后,发明人已经出乎意料地发现,可以发现对其广义伯氏疏螺旋体或回归热疏螺旋体属宿主特异性的噬菌体。作为结果,成功的方法学已经被提供用于鉴定这类细菌的感染,并且基于鉴定这类噬菌体的存在或不存在。
回归热疏螺旋体属是为回归热的病原体的任何疏螺旋体属物种,例如,宫本疏螺旋体、赫姆斯疏螺旋体、回归热疏螺旋体(Borrelia recurrentis)、麝鼩疏螺旋体(Borrelia crocidurae)、中非疏螺旋体(Borrelia duttoni)、西班牙疏螺旋体(Borreliahispanica)、帕克疏螺旋体(Borrelia parkeri)和墨西哥疏螺旋体(Borrelia turicatae)或其任何组合。
因此,在本发明的第一方面中,提供了确定样品中存在或不存在广义伯氏疏螺旋体或RF疏螺旋体属的方法,该方法包括以下步骤:
a)检测样品中对广义伯氏疏螺旋体或RF疏螺旋体属特异性的噬菌体;和
b)在检测到噬菌体的基础上确定样品中存在广义伯氏疏螺旋体或RF疏螺旋体属,或在没有检测到噬菌体的基础上确定样品中不存在广义伯氏疏螺旋体或RF疏螺旋体属。
本发明人已经出乎意料地发现广义伯氏疏螺旋体和RF疏螺旋体属,甚至在隔离状态中的那些,分配了(dispense)大量的噬菌体。因此,例如,即使当细菌被隔离并且如此难以检测(在血液中很少或没有细菌被发现)时,来自被感染的受试者的血液样品确实包含起源于细菌的大量的噬菌体或噬菌体片段。因此,本发明的特别有益的方面是,本发明的方法可以对非神经元样品实施,诸如血液样品(例如全血、血清、血浆)、尿液样品、粪便样品、皮肤样品、淋巴样品或其组合,并且仍然提供对细菌感染的存在或不存在有用的结论,而与感染的阶段无关;这是用已知诊断方法学不可能做到的。
对广义伯氏疏螺旋体或RF疏螺旋体属特异性的噬菌体
发明人已经发现,例如噬菌体的末端酶基因的系统发育分析揭示了末端酶基因序列和疏螺旋体属物种的身份之间的紧密相关性(图1)。引起LD的疏螺旋体属物种的独立亚组以统计学上显著的自展值(boot strap value)与其他疏螺旋体属菌株良好分离。这证明了末端酶基因是鉴定莱姆病疏螺旋体属菌株中的良好分子标志物。如在图1中示出的,这也是RF疏螺旋体属的情况。例如,用本文提供的测试,区分引起莱姆病的疏螺旋体属(广义伯氏疏螺旋体)和RF疏螺旋体属是可能的。
已经对其他噬菌体基因进行了相同的分析。例如,穴蛋白和内溶菌素。基于穴蛋白(图2)、内溶菌素(图3)、整合酶(图4)和门蛋白(portal protein)(图5)的系统发育分析也示出了这些基因在检测疏螺旋体属物种方面的潜力。
因此,特异性可以被定义为区分广义伯氏疏螺旋体的噬菌体和来自其他疏螺旋体属物种的噬菌体的方法。例如,核苷酸引物或抗体,所述核苷酸引物或抗体优先与广义伯氏疏螺旋体的噬菌体结合;或不与来自另一个疏螺旋体属物种的核苷酸序列或氨基酸序列交叉反应。该方法还可以区分RF疏螺旋体属的噬菌体和来自其他物种的噬菌体。
噬菌体
噬菌体(bacteriophage),通常也被称为噬菌体(phage),是感染细菌并在细菌中复制的病毒。本文描述的噬菌体可以是原噬菌体、温和/溶源性噬菌体、噬菌体样颗粒(诸如质粒)或裂解性噬菌体。
原噬菌体/温和噬菌体/溶源性噬菌体是由双链或单链DNA或RNA制成的噬菌体颗粒。噬菌体基因组可以被插入和整合到环状细菌DNA染色体中,或作为染色体外质粒存在。这是噬菌体的潜伏形式,其中病毒基因存在于细菌中而不引起细菌细胞的破坏,并且有时可以为细菌宿主的整体适应性提供竞争优势。
裂解性噬菌体或烈性噬菌体包含病毒DNA/RNA,该病毒DNA/RNA与宿主细菌DNA分开存在,并且与宿主细菌DNA分开复制。裂解性噬菌体在破坏被感染的细胞及其膜时被释放。
样品
样品可以来源于许多起源。因此,样品可以包含以下或由以下组成:血浆、血清、全血、脑脊液、尿液、粪便物质、皮肤、脑组织、神经胶质细胞、淋巴、汗液或羊膜液或其任何组合。
因此,样品可以是在感染后的任何时间取自受试者并且持续地提供存在感染的正确的确定的样品。最出乎意料地,样品可以在感染后不久(即早期感染)获得,或在感染已经充分发展并且细菌已经变得隔离后(即晚期感染)获得。这证明了与本发明相关的优于已知方法学的技术优势。早期感染将通常被认为是自感染后不到2周或3周。晚期感染将通常被认为当被感染的受试者开始患有神经障碍(即脑雾(brain fog))时发生,其可以是从感染后6个月起。
可以从其获得样品的受试者是患有LD或RF的任何动物。例如,受试者可以是人类、马(equine)、犬(canine)、猫(feline)、羊(ovine)、山羊(caprine)、蜱、虱子或其任何组合。因此,受试者可以是人类、昆虫或动物。
噬菌体的检测
“检测”意指确定存在或不存在噬菌体和对广义伯氏疏螺旋体或回归热疏螺旋体属特异性的检测分子(例如分别对噬菌体核酸或蛋白质特异性的引物或抗体)之间的相互作用。这些方法可以离体或在体外进行。检测也可以是体内的,例如通过染料探针进行。
特异性结合对莱姆病疏螺旋体属或RF疏螺旋体属特异性的噬菌体的检测分子可以被加标签(tagged)或标记(labelled)。例如,检测可以包括使用能够使用例如分光光度法、流式细胞术或显微术检测的剂(标记)。示例性标记包括放射性同位素(诸如3H、14C、15N、35S、90V、99Tc、111Ln、125I或131I)、荧光团(诸如荧光素、异硫氰酸荧光素、罗丹明等)、发色团、配体、化学发光剂、生物发光剂(诸如萤光素酶、绿色荧光蛋白(GFP)或黄色荧光蛋白)、可以产生可检测的反应产物的酶(诸如辣根过氧化物酶、萤光素酶、碱性磷酸酶、β-半乳糖苷酶)及其组合。
噬菌体也可以被用任何DNA染料诸如SYBR绿、SYBR金、EB标记。下一代测序技术,诸如短读段测序(short-read sequencing)技术(例如454、Illumina、SOLiD和Ion Torrent)和长读段测序(long-read sequencing)技术(例如PacBio测序)也可以在通过全基因组测序鉴定噬菌体中使用。
噬菌体核酸的检测
噬菌体的检测可以包括检测噬菌体基因或基因片段。
在莱姆病诊断的情况中,噬菌体基因可以通过对广义伯氏疏螺旋体宿主特异性的噬菌体基因组的区域的核酸序列定义(即核酸序列是仅在靶宿主(例如广义伯氏疏螺旋体)中、或在对这类宿主特异性的噬菌体中、或其部分中发现的噬菌体基因或噬菌体基因的部分)。可选地,在RF诊断的情况中,噬菌体基因可以通过对RF疏螺旋体属宿主特异性的噬菌体基因组的区域的核酸序列定义(即核酸序列是仅在靶宿主(例如RF疏螺旋体属)中、或在对这类宿主特异性的噬菌体中、或其部分中发现的噬菌体基因或噬菌体基因的部分)。
本申请人已经发现合适的噬菌体基因是编码穴蛋白、内溶菌素、整合酶、衣壳、门蛋白或末端酶蛋白的基因或其组合。
以下描述了对广义伯氏疏螺旋体噬菌体特异性的核酸序列。
例如,噬菌体基因可以是广义伯氏疏螺旋体末端酶基因,当是这种情况时,基因可以是根据SEQ ID NO.1-10的序列的核酸,或能够编码根据SEQ ID NO.36-45的序列的蛋白质的基因。任选地,末端酶基因可以是与SEQ ID NO.1-10具有大于或等于70%-100%序列同源性的核酸,或是能够编码与SEQ ID NO.36-45具有大于70%-100%序列同源性的蛋白质的基因。例如,与SEQ ID NO.1-10或SEQ ID NO.36-45大于或等于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性。关于同源基因和蛋白质,优选地这些基因和蛋白质保留末端酶的功能。
可选地或另外地,噬菌体基因可以是一个或更多个穴蛋白(SEQ ID NO.17-25)、内溶菌素(SEQ ID NO.26-34)、整合酶(SEQ ID NO.16)、衣壳蛋白和/或门蛋白(SEQ IDNO.11-15)基因。可选地,噬菌体基因可以是能够编码根据SEQ ID NO.52-60的序列的穴蛋白(holin protein)或内溶菌素蛋白(SEQ ID NO.61-69)或整合酶蛋白(SEQ ID NO 51)或衣壳蛋白和/或门蛋白(SEQ ID NO.46-50)的基因。任选地,被检测的基因可以是与SEQ IDNO.11-34中的任一个具有大于70%-100%序列同源性的核酸,或是能够编码与SEQ IDNO.36-69具有大于70%-100%序列同源性的蛋白质的基因。例如,与SEQ ID NO.11-34或SEQ ID NO.36-69大于或等于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性。关于同源基因和蛋白质,优选地这些基因和蛋白质保留穴蛋白、门蛋白、内溶菌素、整合酶或衣壳蛋白的功能。
作为百分比序列同源性的替代,噬菌体基因的同源物可以可选地被定义为具有添加、取代和/或缺失1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、40个或50个连续或非连续核苷酸的同源物,优选地同时保留被基因编码的蛋白质的功能,添加、取代和缺失是相对于SEQ ID NO.1-34的未修饰的序列。
检测还可以包括检测穴蛋白、内溶菌素、整合酶、门蛋白、衣壳蛋白或末端酶蛋白中任一个的片段或其组合。例如,检测50个-1300个碱基对片段。例如,50个、100个、150个、200个、250个、300个、350个、400个、450个、500个、550个、600个、650个、700个、750个、800个、850个、900个、950个、1000个、1050个、1100个、1150个、1200个、1250个或1300个连续碱基对或基于这些值的任何范围的碱基对。任选地,待检测的基因片段可以是与SEQ IDNO.1-34的任何片段具有等于或大于70%-100%序列同源性的核酸。例如,与SEQ ID NO.1-34等于或大于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性,优选地同时保留由基因编码的蛋白质的功能。
例如,被检测的末端酶基因的片段可以是:
或与SEQ ID NO.35具有等于或大于70%-100%序列同源性的任何序列。例如,与SEQ ID NO.35等于或大于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性。
以下描述了对RF疏螺旋体属噬菌体特异性的核酸序列。
噬菌体基因可以是RF疏螺旋体属末端酶基因,当是这种情况时,基因可以是根据SEQ ID NO.84或SEQ ID NO.86的序列的核酸,或是能够编码根据SEQ ID NO.85或SEQ IDNO.87的序列的蛋白质的基因。任选地,末端酶基因可以是与SEQ ID NO.84或SEQ ID NO.86具有等于或大于70%-100%序列同源性的核酸;或是能够编码与SEQ ID NO.85或SEQ IDNO.87具有等于或大于70%-100%序列同源性的蛋白质的基因。例如,与SEQ ID NO.84或SEQ ID NO.86等于或大于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性,优选地同时保留由基因编码的末端酶蛋白的功能。
可选地或另外地,噬菌体基因可以是一个或更多个穴蛋白(SEQ ID NO.97-100)、内溶菌素(SEQ ID NO.101)、整合酶(SEQ ID NO.103)、衣壳蛋白和/或门蛋白(SEQ IDNO.102)基因。可选地,噬菌体基因可以是能够编码根据SEQ ID NO.104-107的序列的穴蛋白或内溶菌素蛋白(SEQ ID NO.108)或整合酶蛋白(SEQ ID NO.110)或衣壳蛋白和/或门蛋白(SEQ ID NO.109)的基因。任选地,被检测的基因可以是与SEQ ID NO.97-103中的任一个具有大于70%-100%序列同源性的核酸,或是能够编码与SEQ ID NO.104-110具有大于70%-100%序列同源性的蛋白质的基因。例如,与SEQ ID NO.97-103或SEQ ID NO.104-110大于或等于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性。关于同源基因和蛋白质,优选地这些基因和蛋白质保留穴蛋白、门蛋白、内溶菌素、整合酶或衣壳蛋白的功能。
作为百分比序列同源性的替代,噬菌体基因的同源物可以可选地被定义为具有添加、取代和/或缺失1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、40个或50个连续或非连续核苷酸的同源物,优选地同时保留由基因编码的蛋白质的功能,添加、取代和缺失是相对于SEQ ID NO.84、SEQ ID NO.86或SEQ ID NO.97-103的未修饰的序列。
检测还可以包括检测穴蛋白、内溶菌素、整合酶、门蛋白、衣壳蛋白或末端酶蛋白中任一个的片段或其组合。例如,检测50个-1300个碱基对片段。例如,50个、100个、150个、200个、250个、300个、350个、400个、450个、500个、550个、600个、650个、700个、750个、800个、850个、900个、950个、1000个、1050个、1100个、1150个、1200个、1250个或1300个连续碱基对或基于这些值的任何范围的碱基对。任选地,待检测的基因片段可以是与SEQ IDNO.84、SEQ ID NO.86或SEQ ID NO.97-103的任何片段具有等于或大于70%-100%序列同源性的核酸。例如,与SEQ ID NO.84、SEQ ID NO.86或SEQ ID NO.97-103的任何片段等于或大于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性,优选地同时保留由基因编码的蛋白质的功能。
以下描述适用于对广义伯氏疏螺旋体或RF疏螺旋体属特异性的噬菌体的整个噬菌体基因或基因片段的检测。
从样品分离核酸
为了辅助分子生物学工具实施检测的步骤,步骤a)之前可以是从样品分离核酸的步骤,并且步骤a是对分离的核酸实施的。技术人员可用的能够从样品分离核酸的任何方法将适于在本申请中使用。不希望被进一步限制,但为了清楚起见,从样品分离核酸可以使用苯酚氯仿提取和异丙醇沉淀,或使用基于膜的核酸分离试剂盒(诸如试剂盒;QIAAmp DNA stool mini)进行。
本发明人已经设计了用于从包含疏螺旋体属的样品分离核酸的有效方法。因此,还提供了从疏螺旋体属提取噬菌体DNA的方法,该方法包括:a)在氢氧化铵中孵育疏螺旋体属;和b)将苯酚-氯仿添加至疏螺旋体属和氢氧化铵混合物。
该方法还可以包括离心以去除细菌碎片。去除细菌碎片后所得的上清液可以与乙酸钠混合。然后异丙醇可以被添加至上清液和乙酸钠混合物。然后可以进行另外的离心步骤以沉淀噬菌体DNA。
可以使用与苯酚-氯仿相同的体积作为氢氧化铵的体积。
部分a)可以在50℃-150℃的温度进行。例如,70℃-130℃、80℃-120℃或90℃-110℃。
氢氧化铵浓度可以是0.5M-1M。例如,0.6M、0.7M、0.8M、0.9M。
另外的示例性细节在标题“PCR和测序”的实施例部分中提供。
核酸的检测
技术人员已知的并且能够检测基因(任选地在分离的核酸中)的存在或不存在的任何方法可以适用于在当前要求保护的发明的步骤a)中使用。因为噬菌体基因可以以相对低的拷贝数存在,为了使检测更容易,在本发明的一个实施方案中,检测的方法包括使分离的核酸经历噬菌体基因的扩增,例如通过实时聚合酶链式反应(qPCR)进行。
可选地或另外地,噬菌体基因的检测可以通过核酸序列分析、通过检测被插入到扩增产物中的标记的核苷酸、或通过检测对噬菌体基因特异性的探针的杂交(例如,通过使用标记的探针检测的杂交)证实。
除了基于Taqman的qPCR平台外,可以被使用的核酸检测的其他方法包括基于SYBR绿的实时PCR测定、涉及将相同的qPCR混合物分到大量的单个孔中的数字PCR(digitalPCR)。终点PCR产物使用泊松统计学分析根据每个孔中存在(评分成‘1’)和不存在(评分成‘0’)荧光信号确定。其他可能的方法包括环介导的等温扩增(LAMP),一种不需要热循环仪的等温核酸扩增技术。LAMP可以用于靶向末端酶、穴蛋白、内溶菌素、整合酶、衣壳蛋白和门蛋白;和基于DNA杂交的方法,诸如荧光原位杂交(FISH),荧光原位杂交(FISH)通过使用荧光标记的短DNA链(噬菌体基因)作为与其在基因组DNA上的互补部分杂交的探针来进行DNA/DNA杂交而工作。
检测可以包括使用能够使用例如分光光度法、流式细胞术或显微术检测的剂(标记)。示例性标记包括放射性同位素(诸如3H、14C、15N、35S、90V、99Tc、111Ln、125I或131I)、荧光团(诸如荧光素、异硫氰酸荧光素、罗丹明等)、发色团、配体、化学发光剂、生物发光剂(诸如萤光素酶、绿色荧光蛋白(GFP)或黄色荧光蛋白)、可以产生可检测的反应产物的酶(诸如辣根过氧化物酶、萤光素酶、碱性磷酸酶、β-半乳糖苷酶)及其组合。
以下涉及对广义伯氏疏螺旋体噬菌体特异性的引物。
扩增的步骤可以包括使用正向引物,所述正向引物选自由以下核酸组成的组:包含或由SEQ ID NO.70:
5’GTGAACTTATATCAAAC3’组成的核酸。
可选地,或另外地,扩增的步骤包括使用反向引物,所述反向引物选自由以下核酸组成的组:包含或由SEQ ID NO.71:
5’ATAATCTTCTTTTCATT3’组成的核酸。
可选地或另外地,扩增的步骤可以包括使用SEQ ID NO.73-77和/或SEQ IDNO.79-83的引物中的任一个或更多个。
在噬菌体基因的鉴定通过使用杂交探针实现的情况中,合适的探针可以是能够特异性结合噬菌体基因或其部分的任何探针。例如,适当的探针可以是上文提到的包含SEQID NO.70-77和/或SEQ ID NO.79-83或由SEQ ID NO.70-77和/或SEQ ID NO.79-83组成的引物中的任一个或更多个、或包含SEQ ID NO.1-34或由SEQ ID NO.1-34组成的核酸、或其片段、其任何同源物、或其特异性结合部分、或其任何组合。
以下涉及对RF疏螺旋体属噬菌体特异性的引物。
扩增的步骤可以包括使用包含SEQ ID NO.88-96或由SEQ ID NO.88-96组成的任何引物、其任何同源物、或其特异性结合部分、或其任何组合。
在噬菌体基因的鉴定通过使用杂交探针实现的情况中,合适的探针可以是能够特异性结合噬菌体基因或其部分的任何探针。例如,适当的探针可以是上文提到的包含SEQID NO.88-96或由SEQ ID NO.88-96组成的引物中的任一个或更多个、或包含SEQ IDNO.84、SEQ ID NO.86、和/或SEQ ID NO.97-103或由SEQ ID NO.84、SEQ ID NO.86、和/或SEQ ID NO.97-103组成的核酸、或其片段、其任何同源物、或其特异性结合部分、或其任何组合。
检测还可以包括检测噬菌体RNA或RNA片段。例如,50个、100个、150个、200个、250个、300个、350个、400个、450个、500个、550个、600个、650个、700个、750个、800个、850个、900个、950个、1000个、1050个、1100个、1150个、1200个、1250个或1300个碱基对或基于这些值的任何范围的碱基对的RNA片段。
例如,噬菌体基因的存在还可以通过检测对应的mRNA而被检测。这可以通过进行逆转录酶实时PCR而实现,所述逆转录酶实时PCR通过对从总RNA合成的cDNA进行实时PCR。例如,通过引物与mRNA的杂交。在PCR方法,例如RT-PCR中使用的方法学将是本领域技术人员将是周知的。
除了PCR外,存在基于探针的方法,诸如RNA印迹、原位杂交。还存在可以在通过测序总RNA检测噬菌体RNA中使用的RNA测序方法。
一些方法可能需要从样品分离RNA。这类分离技术在本领域中是已知的,并且可以使用从制造商(诸如Qiagen)商购可得的RNA分离试剂盒、或来自Promega的Maxwell病毒总核酸纯化试剂盒、或以上描述的关于基因序列检测的分离方法。
噬菌体蛋白质的检测
检测还可以包括检测噬菌体蛋白质或蛋白质片段。
检测疏螺旋体属噬菌体的其他方法包括检测噬菌体特异性蛋白质。
在LD的情况中,噬菌体蛋白质是对广义伯氏疏螺旋体宿主特异性的噬菌体蛋白质(即氨基酸序列或其部分仅在靶宿主,例如广义伯氏疏螺旋体中被发现,或在对这样的宿主特异性的噬菌体中被发现)。
用于LD检测的合适的蛋白质包括穴蛋白(SEQ ID NO.52-60)、内溶菌素(SEQ IDNO.61-69)、整合酶(SEQ ID NO.51)、门蛋白(SEQ ID NO.46-50)、衣壳蛋白和/或末端酶(SEQ ID NO.36-45)。
任选地,检测的氨基酸序列可以与SEQ ID NO.36-69具有等于或大于70%-100%序列同源性,或可以由与SEQ ID NO.1-34具有等于或大于70%-100%序列同源性的基因编码。例如,与SEQ ID NO.36-69或SEQ ID NO.1-34等于或大于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性。关于同源基因和蛋白质,优选地这些基因和蛋白质保留穴蛋白、门蛋白、内溶菌素、整合酶或衣壳蛋白的功能。
在RF的情况中,噬菌体蛋白质是对RF疏螺旋体属宿主特异性的噬菌体蛋白质(即氨基酸序列或其部分仅在靶宿主(例如RF疏螺旋体属)中被发现,或在对这样的宿主特异性的噬菌体中被发现)。
用于RF检测的合适的蛋白质包括末端酶(SEQ ID NO.85或SEQ ID NO.87)、穴蛋白(SEQ ID NO.104-107)、内溶菌素(SEQ ID NO.108)、门蛋白(SEQ ID NO.109)和/或整合酶(SEQ ID NO.110)。
任选地,被检测的氨基酸序列可以与SEQ ID NO.85、SEQ ID NO.87或SEQ IDNO.104-110具有等于或大于70%-100%序列同源性,或可以由与SEQ ID NO.84、SEQ IDNO.86或SEQ ID NO.97-103具有等于或大于70%-100%序列同源性的基因编码。例如,与SEQ ID NO.84-87或SEQ ID NO.97-110等于或大于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性。关于同源基因和蛋白质,优选地这些基因和蛋白质保留穴蛋白、门蛋白、内溶菌素、整合酶或衣壳蛋白的功能。
作为百分比序列同源性的替代,同源物可以被定义为具有添加、取代和/或缺失1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、40个或50个连续或非连续氨基酸的蛋白质,优选地同时保留蛋白质的功能,添加、取代和缺失是相对于SEQ ID NO.85、SEQ IDNO.87或SEQ ID NO.104-110的未修饰的序列的。
检测还可以包括检测穴蛋白、内溶菌素、整合酶、门蛋白、衣壳蛋白或末端酶蛋白的任一个的片段或其组合。例如,通过对广义伯氏疏螺旋体特异性的抗体检测在这些蛋白质中的任一个上的表位。表位可以是线性表位。例如,片段可以包含至少5个至10个连续氨基酸、10个至15个连续氨基酸、15个至20个连续氨基酸或20个至30个或更多个连续氨基酸的氨基酸残基的链段(stretch)。或者,表位可以是对噬菌体蛋白质特异性的非连续表位。例如,包含5个、10个、15个或20个氨基酸的非连续表位。
任选地,待检测的蛋白质片段可以是与SEQ ID NO.36-69、SEQ ID NO.85、SEQ IDNO.87或SEQ ID NO.104-110的任何片段具有等于或大于70%-100%序列同源性的氨基酸。例如,与SEQ ID NO.36-69、SEQ ID NO.85、SEQ ID NO.87或SEQ ID NO.104-110的任何片段等于或大于70%、75%、80%、85%、90%或95%、96%、97%、98%、99%或99.5%序列同源性,同时保留蛋白质的功能。
蛋白质特异性检测可以通过本领域中已知的任何方法进行。例如,免疫组织化学(IHC)可以用于检测蛋白质表达。蛋白质印迹和ELSIA也是用于检测蛋白质表达和分泌的有用的方法。抗体或能够选择性结合噬菌体蛋白质的其他蛋白质或分子可以用于检测。蛋白质也可以通过MALDI-TOF质谱法检测。蛋白质测序,例如通过质谱法或Edman降解,也可以用于检测。10个、20个、30个、40个、50个、60个、70个、80个、90个、100个、110个、120个、130个、140个、150个、160个、170个、180个、190个、200个、300个、400个、500个氨基酸或这些数目的任何范围的线性片段可以被测序。
噬菌体蛋白质可以使用具有对噬菌体蛋白质的结合特异性的一级抗体检测。一级抗体可以用可检测的部分标记,或可以与半抗原(诸如生物素等)缀合,其中半抗原是通过可检测地标记的对应(cognate)半抗原结合分子(例如链霉亲和素辣根过氧化物酶)可检测的。可选地,可以使用特异性结合一级抗体的二级抗体,并且替代地,该二级抗体可以是如以上针对一级抗体描述的可检测的。
噬菌体抗体(具有对噬菌体蛋白质的结合特异性的抗体)的结合特异性可以使用例如蛋白质印迹确定。
术语抗体是指特异性结合特定抗原或与特定抗原免疫学上反应的免疫球蛋白分子或其组合,并且包括多克隆、单克隆、遗传工程化和以其他方式修饰的形式的抗体,不限于嵌合抗体、人源化抗体、杂缀合抗体(heteroconjugate antibody)(例如双特异性抗体、双抗体(diabody)、三抗体(triabody)和四抗体(tetrabody))、单链Fv抗体(scFv)或包含足以赋予对多肽的特异性抗原结合的免疫球蛋白的至少一部分的多肽。抗体片段包括蛋白水解抗体片段诸如F(ab’)2片段、Fab’片段、Fab’-SH片段、Fab片段、FV、rIgG、重组抗体片段诸如sFv片段、dsFv片段、双特异性sFv片段、双特异性dsFv片段、互补决定区(CDR)片段、骆驼抗体(camelid antibody)和由软骨鱼和硬骨鱼产生的抗体及其分离的结合结构域。Fab片段是由VL、VH、CL和CH1结构域组成的单价片段;F(ab’)2片段是包含通过在铰链区的二硫桥连接的两个Fab片段的二价片段,Fd片段由VH和CH1结构域组成;FV片段由抗体的单臂的VL和VH结构域组成;并且dAb片段由VH结构域组成。单链抗体(scFv)是其中VL和VH区通过使它们能够被制造成单个蛋白质链的合成接头配对以形成单价分子的抗体。双抗体是二价双特异性抗体,其中VH和VL结构域在单个多肽链上被表达,但使用太短以致于不允许在相同的链上的两个结构域之间配对的接头,从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合位点。嵌合抗体是包含来自一种抗体的一个或更多个区域和来自一种或更多种其他抗体的一个或更多个区域的抗体。抗体可以具有一个或更多个结合位点。如果存在多于一个结合位点,则结合位点可以彼此相同或可以不同。例如,天然存在的免疫球蛋白具有两个相同的结合位点,单链抗体或Fab片段具有一个结合位点,而双特异性或双功能抗体具有两个不同的结合位点。
可选地,能够选择性结合的其他蛋白质也可以用于检测。
例如,噬菌体蛋白质可以使用以下检测:适体(例如,呈现特定的序列依赖性形状和以高亲和力和特异性结合噬菌体蛋白质的单链核酸分子(诸如,DNA或RNA))、镜像适体(SPIEGELMERTM)、工程化非免疫球蛋白结合蛋白,例如基于支架的非免疫球蛋白结合蛋白,包括纤连蛋白(ADNECTINSTM)、CTLA-1(EVIBODIESTM)、脂质运载蛋白(ANTICALINSTM)、蛋白A结构域(AFFIBODIESTM)等。
同源性
关于核酸序列,同源性可以被定义为编码具有类似的功能的蛋白质的核苷酸序列。关于蛋白质序列,同源性可以被定义为具有类似的功能的蛋白质。例如,来自另一种广义伯氏疏螺旋体或RF疏螺旋体属物种的具有相同的功能的蛋白质。
百分比同源性可以被定义为相同残基的百分比和具有类似的物理化学性质的保守的残基的百分比。序列之间的同源性的程度可以使用本领域中已知的任何合适的方法确定(参见例如,Smith和Waterman,Adv.Appl.Math.,2:482[1981];Needleman和Wunsch,J.MoI.Biol,48:443[1970];Pearson和Lipman,Proc.Natl.Acad.Sci.USA 85:2444[1988];程序诸如Wisconsin Genetics Software Package(Genetics Computer Group,Madison,WI)中的GAP、BESTFIT、FASTA和TFASTA;和Devereux等人,Nucl.Acid Res.,12:387-395[1984])。
以上同源性的百分比可以同等地仅适用于百分比序列同一性。例如,末端酶核酸序列和另一种末端酶序列之间相同的核酸残基的百分比,所述另一种末端酶序列可以例如来自不同的疏螺旋体属物种。
作为百分比序列同源性的替代,噬菌体基因的同源物可以被定义为具有添加、取代或缺失1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、40个或50个连续或非连续核苷酸的基因,优选地同时保留被基因编码的蛋白质的功能,添加、取代和缺失是相对于未修饰的序列的。
序列
在本发明的另外的方面中,提供了噬菌体基因、引物、核酸和杂交探针;和如以上定义的氨基酸序列。
作为参考,序列表中从各种广义伯氏疏螺旋体分离的各种基因序列的表在下文随对应的蛋白质一起提供。
还提供了用于基因序列检测的引物的SEQ ID NO.:
以下提供了序列表中从各种疏螺旋体属物种分离的各种基因序列的表,这些疏螺旋体属物种是RF的病原体:
还提供了用于基因序列检测的引物的SEQ ID NO.:
根据以上针对检测方法的描述,还提供了这些核酸和蛋白质序列的同源物和片段。
还提供了以上序列、同源物和片段作为用于LD或RF的诊断标志物的用途。
物种检测
广义伯氏疏螺旋体可以是LD的病原体中的任一种。例如阿氏疏螺旋体、斯氏疏螺旋体、法雷斯疏螺旋体、伽氏疏螺旋体、芬兰疏螺旋体、狭义伯氏疏螺旋体、比塞蒂疏螺旋体、巴伐利亚疏螺旋体、日本疏螺旋体、卢西塔尼亚疏螺旋体、西尼伽疏螺旋体、斯氏疏螺旋体、塔卢基疏螺旋体、土德疏螺旋体、法雷斯疏螺旋体、Borrelia Yangtze、梅奥疏螺旋体、Borrelia carolinensis、安德森疏螺旋体、孤星疏螺旋体和美洲疏螺旋体中的任一种,或其组合。例如,它可以是阿氏疏螺旋体、狭义伯氏疏螺旋体、伽氏疏螺旋体或其任何组合。例如,它可以是狭义伯氏疏螺旋体或狭义伯氏疏螺旋体与以上中的任一种的任何组合。
RF疏螺旋体属可以是RF病原体中的任何一种。例如,宫本疏螺旋体、赫姆斯疏螺旋体、回归热疏螺旋体、麝鼩疏螺旋体、中非疏螺旋体、西班牙疏螺旋体、帕克疏螺旋体和墨西哥疏螺旋体中的任一种或其任何组合。
出乎本发明人意料地是,当进一步分析广义伯氏疏螺旋体物种特异性噬菌体基因时,这些基因不仅显示出对广义伯氏疏螺旋体物种的特异性,还显示出对在该组中的物种的特异性。RF疏螺旋体属特异性噬菌体基因也被发现是对特定物种特异性的。
已知的方法没有一种可以区分疏螺旋体属的不同物种。这可以是重要的优势,因为知道存在何种的疏螺旋体属的物种可以教导治疗方案,并且从而优化治疗的成功(因为一些疏螺旋体属的物种比其他疏螺旋体属的物种对特定抗生素的应答更好)。
因此,该方法可以确定样品中存在或不存在广义伯氏疏螺旋体或RF疏螺旋体属的物种,该方法包括以下步骤:
a)检测样品中存在或不存在广义伯氏疏螺旋体或RF疏螺旋体属物种特异性噬菌体基因;
b)在检测到物种特异性噬菌体基因的基础上确定样品中存在广义伯氏疏螺旋体或RF疏螺旋体属的物种,或在没有检测到物种特异性噬菌体基因的基础上确定样品中不存在广义伯氏疏螺旋体或RF疏螺旋体属的物种。
为了辅助分子生物学工具实施检测的步骤,步骤a)之前可以是从样品分离核酸的步骤,并且步骤a)是对分离的核酸实施的。技术人员可用的能够从样品分离核酸的任何方法将是适于在本申请中使用的。不希望被进一步限制,但为了清楚起见,从样品分离核酸可以使用苯酚氯仿提取和异丙醇沉淀,或使用基于膜的核酸分离试剂盒(诸如试剂盒;QIAAmp DNA stool mini)进行。
例如,当细菌是狭义伯氏疏螺旋体时,狭义伯氏疏螺旋体特异性噬菌体基因可以是以上讨论的那些基因中的任一个。例如,基因可以是末端酶基因(即噬菌体基因),其可以是根据SEQ ID NO 1的末端酶基因。任选地,基因是与SEQ ID No.1具有等于或大于70%-99.5%序列同源性的核酸。用于扩增狭义伯氏疏螺旋体特异性末端酶基因的正向引物是包含SEQ ID NO 70的核酸或由SEQ ID NO 70组成的核酸。反向引物可以是包含SEQ ID NO 71的核酸或由SEQ ID NO 71组成的核酸。在该基因的鉴定是通过使用杂交探针实现的情况中,合适的探针可以是前文提到的引物、包含SEQ ID NO 1-10或SEQ ID NO 35的核酸或由SEQ ID NO 1-10或SEQ ID NO 35组成的核酸、其任何同源物、或其特异性结合的部分中的任一种、或其任何组合。
例如,当细菌是宫本疏螺旋体时,特异性噬菌体基因可以是以上讨论的那些基因中的任一种。例如,基因可以是末端酶基因(即噬菌体基因),所述末端酶基因可以是根据SEQ ID NO 84的末端酶基因。任选地,基因是与SEQ ID No.84具有等于或大于95%、96%、97%、98%、99%或99.5%序列同源性的核酸。用于扩增宫本疏螺旋体特异性末端酶基因的正向引物是包含SEQ ID NO 89或SEQ ID NO 92的核酸或由SEQ ID NO 89或SEQ ID NO 92组成的核酸。反向引物可以是包含SEQ ID NO 90或SEQ ID NO 93的核酸或由SEQ ID NO 90或SEQ ID NO 93组成的核酸。在该基因的鉴定是通过使用杂交探针实现的情况中,合适的探针将是包含SEQ ID NO 84的核酸或由SEQ ID NO 84组成的核酸、或包含SEQ ID NO.88-93或由SEQ ID NO.88-93组成的任何引物、其任何同源物、或其特异性结合部分、或其任何组合。
试剂盒
在本发明的另外的方面中,存在用于确定样品中存在或不存在广义伯氏疏螺旋体和/或RF疏螺旋体属的试剂盒,试剂盒可以包含一种或更多种前文提到的引物,用于与疏螺旋体属特异性噬菌体基因的核酸序列特异性杂交。引物或优选地引物对(即正向引物和反向引物)的选择将由靶广义伯氏疏螺旋体或RF疏螺旋体属物种指导。试剂盒还可以包含杂交探针,所述杂交探针可以是前文提到的引物,或任何前文提到的探针。再次,探针的选择将在待由试剂盒待鉴定的靶物种的基础上选择。用于确定广义伯氏疏螺旋体的存在的试剂盒或实际上用于鉴定这类细菌的特异性物种的试剂盒的试剂盒的杂交探针的选择可以从以上讨论的适当的杂交探针的细节推导出来。作为核酸探针的替代物,结合噬菌体特异性蛋白质的抗体可以包括在试剂盒中。这类抗体在以上讨论。
监测感染的进展
该方法可以应用于监测来自广义伯氏疏螺旋体或RF疏螺旋体属;或基于本发明的较早方面的教导的这类细菌的物种的感染的进展。
在本发明的此方面中使用的术语与在本发明的较早方面中使用的术语相同,并且因此重新参考本发明的较早方面中使用的术语。
在本发明的另外的方面,提供了监测来自广义伯氏疏螺旋体或RF疏螺旋体属的感染的进展的方法。该方法包括以下步骤:
a)确定从受试者获得的第一样品中的噬菌体的量;
b)检测在第二时间点从受试者获得的第二样品中的噬菌体的量;
c)比较在步骤a)中鉴定的第一样品中的噬菌体的量和在步骤b)中鉴定的噬菌体的量。
该方法可以包括在步骤a)之前,从在第一时间段从受试者获得的第一样品分离第一群体的核酸的步骤。该方法可以包括在步骤b)之前,从在第二时间段从受试者获得的第二样品分离第二群体的核酸的步骤。
治疗
检测来自受试者的样品中广义伯氏疏螺旋体的存在证实该受试者患有LD的诊断。用于广义伯氏疏螺旋体感染的治疗是莱姆病的治疗。
检测来自受试者的样品中RF疏螺旋体属的存在证实该受试者患有RF的诊断。
描述的任何方法可以包括施用抗生素或多于一种抗生素的步骤。这样的方法能够确定抗生素治疗广义伯氏疏螺旋体或RF感染或物种特异性广义伯氏疏螺旋体或RF感染的能力。
以下涉及LD的治疗:
用于感染的推荐的治疗是包括在相同的时间使用一种、两种或有时三种抗生素的联合疗法。这可以呈连续治疗或同步地联合长期抗生素治疗的形式。抗生素诸如四环素类可以单独地或与羟氯喹组合用于治疗。这可以包括同时施用抗生素。
推荐的用于使用的主要抗生素是:1)青霉素类;2)头孢菌素类;3)大环内酯类;4)氟喹诺酮类(fluroquinolones)和/或5)环素类(cyclines)。
以上任何抗生素可以被组合或连续地使用。
例如,头孢菌素类可以单独地或与米诺环素组合使用。该治疗可以包括在两种抗生素之间交替。
多西环素和/或米诺环素可以与阿奇霉素和/或羟氯喹组合。基于以上类别的抗生素或其他抗生素的其他组合是可能的。
在本发明的此方面中使用的术语与在本发明的较早方面中使用的相同的术语相同,并且因此重新参考本发明的较早方面中使用的相同的术语。
以下涉及RF的治疗:
对于回归热(RF),治疗使用如用于LD的相同类别的抗生素。用于感染的治疗可以是包括在相同的时间使用的一种、两种或有时三种抗生素的联合疗法。这可以是呈连续治疗或同步地联合长期抗生素治疗的形式。以上抗生素中的任何一种都可以被组合或连续地使用。
例如,口服治疗可以包括每日单剂量的来自以上1-5类中的任一个的任何抗生素。例如,口服治疗可以由以下组成:每日单剂量的四环素500mg、多西环素200mg,或当四环素类被禁用时,红霉素500mg。由于单序列(single-sequence)后报告的20%或更高的复发,治疗持续时间可以长达7天-10天或更长。在成人中,当口服疗法不被耐受时,可以使用多西环素、红霉素、四环素或普鲁卡因青霉素G的静脉内疗法。
普鲁卡因青霉素G可以在患有LBRF的成年患者中以600,000IU的单一剂量,或在患有RF的患者中以600,000IU每日被施用。
例如,在本发明的另外的方面中,提供了治疗由广义伯氏疏螺旋体或RF疏螺旋体属、或广义伯氏疏螺旋体或RF疏螺旋体属的物种引起的感染的方法,该方法包括以下步骤:
a)使用根据本发明的第一方面的方法鉴定广义伯氏疏螺旋体或RF疏螺旋体属感染,或广义伯氏疏螺旋体或RF疏螺旋体属感染的物种;
b)选择适用于治疗广义伯氏疏螺旋体或RF疏螺旋体属感染,或确定的广义伯氏疏螺旋体或RF疏螺旋体属的物种的至少一种抗生素;
c)向在步骤a中被鉴定为被广义伯氏疏螺旋体或RF疏螺旋体属感染的受试者施用选择的一种或更多种抗生素。
在本发明的另外的方面中,提供了治疗广义伯氏疏螺旋体或RF疏螺旋体属、或广义伯氏疏螺旋体或RF疏螺旋体属的物种的感染的方法,该方法包括以下步骤:
a)选择适用于治疗广义伯氏疏螺旋体或RF疏螺旋体属感染、或确定的广义伯氏疏螺旋体或RF疏螺旋体属的物种的至少一种抗生素;
b)通过根据本发明的第一方面的方法,向已经被鉴定为患有广义伯氏疏螺旋体或RF疏螺旋体属感染,或广义伯氏疏螺旋体或RF疏螺旋体属感染的物种的受试者施用选择的一种或更多种抗生素。
可选地,该方法可以被应用于研究目的,例如,对广义伯氏疏螺旋体或RF疏螺旋体属的生理学作用的研究。当确定受试者是否已经被广义伯氏疏螺旋体或RF疏螺旋体属感染是重要的时,该方法还可以应用在临床情况中。因此,在本发明的一个实施方案中,该方法是诊断受试者中的广义伯氏疏螺旋体或RF疏螺旋体属或广义伯氏疏螺旋体或RF疏螺旋体属的物种的感染的方法,该方法包括以下步骤:
a)检测样品中存在或不存在噬菌体或物种特异性噬菌体;
b)通过检测到样品中的噬菌体或物种特异性噬菌体确定受试者被广义伯氏疏螺旋体或RF疏螺旋体属或其物种感染,或通过没有检测到样品中的噬菌体或其物种确定受试者未被广义伯氏疏螺旋体或RF疏螺旋体属或其物种感染。
在本发明的此方面中使用的术语与在本发明的较早方面中使用的相同的术语是相同的,并且因此重新参考本发明的较早方面中使用的相同术语。例如,在本发明的每种方法中使用的样品可以在感染后不久(即早期感染)获得,或在感染已经充分发展并且细菌已经变得隔离后(即晚期感染)获得。
用于本发明的所有方法的检测步骤可以是定量步骤,即噬菌体基因、RNA或蛋白质的量被计算。用于本发明的所有方法的方法可以包括从受试者采集样品的步骤。其中,当来自受试者的样品被确定为对广义伯氏疏螺旋体的存在呈阳性时,本发明的所有方法可以包括选择受试者进行针对LD的治疗;或当来自受试者的样品被确定为对RF疏螺旋体属的存在呈阳性时,本发明的所有方法可以包括选择受试者进行针对RF的治疗。
现在将通过参考附图,通过实例的方式描述本发明,在附图中:
图1示出了基于末端酶蛋白的系统发育分析,示出了莱姆病疏螺旋体属物种是彼此紧密相关的,但与其他疏螺旋体属菌株天然地分离。比例尺代表0.2个氨基酸变化/位点。
图2示出了基于穴蛋白的系统发育分析,示出了莱姆病疏螺旋体属物种是彼此紧密相关的,但与其他疏螺旋体属菌株天然地分离。比例尺代表0.2个氨基酸变化/位点。
图3示出了基于内溶菌素蛋白的系统发育分析,示出了莱姆病疏螺旋体属物种是彼此紧密相关的,但与其他疏螺旋体属菌株天然地分离。前10个(从阿氏疏螺旋体至法雷斯疏螺旋体)是莱姆病疏螺旋体属菌株,接下来的8个(从赫姆斯疏螺旋体至中非疏螺旋体)是回归热疏螺旋体属菌株。比例尺代表0.2个氨基酸变化/位点。
图4示出了基于门蛋白的系统发育分析,示出了莱姆病疏螺旋体属物种是彼此紧密相关的,但与其他疏螺旋体属菌株天然地分离。比例尺代表0.2个氨基酸变化/位点。
图5示出了基于整合酶蛋白的系统发育分析,示出了莱姆病疏螺旋体属物种是彼此紧密相关的,但与其他疏螺旋体属菌株天然地分离。比例尺代表0.2个氨基酸变化/位点。
图6示出了基于细菌掺入(bacterial spiked)的人类血液的分析,本发明的基于噬菌体的方法的检测限值。
图7示出了与标准的基于细菌16S的方法相比,本发明的基于噬菌体的方法的检测限值。
图8示出了根据本发明的基于噬菌体的方法对广义伯氏疏螺旋体的四个物种的特异性。
图9示出了携带末端酶基因片段的质粒DNA的一系列的5次十倍稀释(106个拷贝下降至102个拷贝)。对于每次稀释进行了10次技术重复。如示出的,从4次稀释的质粒DNA样品下降至102个拷贝观察到阳性扩增。
图10示出了跨质粒DNA模板的浓度和Ct值之间的线性关系,具有强相关系数(R2=0.99)。此外,BbFAM的扩增效率是100%。
图11示出了针对不同背景(莱姆病阳性、莱姆病阴性、莱姆病临界线、健康志愿者)的222个血清样品的基于噬菌体的测定的性能。
现在将参考以下实施例描述本发明,这些实施例仅仅是通过说明的方式。以下实施例并不意图完全限定或以其他方式限制本发明的范围。
实施例
莱姆病
广义伯氏疏螺旋体分离株
广义(s.l.)伯氏疏螺旋体菌株的实验室培养物由Sven教授(University,Sweden)提供。两个宫本疏螺旋体菌株由CDC(疾病控制和预防中心,USA)提供。实验室菌株被维持在BSKII培养基中。常规表征被使用相差显微镜进行。
PCR和测序
DNA使用结合氢氧化铵和苯酚氯仿的新方法从血清样品提取。将600μL的样品在1.2ml 0.7M氢氧化铵的存在下,在100℃孵育持续5min,然后将管打开,在100℃孵育持续10min。在管被冷却至室温后,将样品用相同体积的苯酚-氯仿(1:1)提取。在RT的5min孵育时间后,将溶液在18000g离心持续10min。将澄清的上清液转移到新的2ml管中,并与0.1体积的3M乙酸钠混合。然后该悬浮液与0.7体积的室温的异丙醇混合。DNA通过在4℃在21000g离心持续10min沉淀下来。在倾析上清液后,添加1.5ml的室温的70%乙醇,随后是在4℃在21000g离心持续10min。所得的DNA沉淀被短暂地风干持续5min,并溶解在50μl-100μl的合适的缓冲液(诸如洗脱缓冲液,EB,其为10mM Tris-C1,pH 8.5)中。
针对所有已知的疏螺旋体属噬菌体末端酶基因序列中的保守区域手动设计PCR引物。引物扩增了来自8个实验室的所有莱姆病广义伯氏疏螺旋体菌株的194bp产物。PCR在LabCycler(SensoQuest GmbH,Germany)中以50μl的总体积进行,所述50μl的总体积包含0.25mM dNTP、3mM MgCl2、2μM引物、50ng的模板DNA、0.5单位的Taq聚合酶(Bioline)和5μl 10×Taq缓冲液(Bioline)。扩增条件是:94℃持续2min,30个循环的94℃持续45秒、48℃持续45秒、72℃持续1min,在72℃最终延伸10min。PCR产物使用Qiagen凝胶提取试剂盒凝胶纯化,并且经历TOPO TA克隆(Invitrogen)。测序由GATC Biotech进行。测序结果使用Chromas 2.33编辑,使用核苷酸BLAST(NCBI)搜索。
广义伯氏疏螺旋体物种鉴定
先前报道的多位点序列分型(Multi locus sequence typing)(MLST)方案用于区分疏螺旋体属的不同基因型。
系统发育分析
系统发育分析使用程序分子进化遗传学分析(Molecular EvolutionaryGeneticsAnalysis)(MEGA)软件包4.1版(β)构建(Tamura等人,2007;Kumar等人,2008)。MEGA 4.1(β)中的Alignment Explorer/CLUSTAL用于比对DNA序列。对核苷酸数据集进行NJ和MP分析;对于NJ,使用最大复合似然模型(maximum composite likelihood model),并且对于MP,使用具有搜索水平为3的近邻交换(close-neighbour-interchange)。对进化枝的支持使用1000个重复在MEGA中实施的自展分析评价。树以λ噬菌体(NC_001416)作为外群(outgroup)生根。
核酸杂交
“杂交”是指两个核酸序列通过氢键合彼此缔合。通常,一个序列将被固定在固体支持物上,并且另一个序列将是在溶液中游离的。然后,这两个序列将在有利于氢键合的条件下彼此接触地放置
影响这种键合的因素包括:溶剂的类型和体积;反应温度;杂交的时间;搅拌;阻断液相序列与固体支持物的非特异性附接的剂(Denhardt试剂或BLOTTO);序列的浓度;使用增加序列的缔合率的化合物(硫酸葡聚糖或聚乙二醇);和杂交后洗涤条件的严格度。
“严格度”是指杂交反应中有利于非常类似的序列优于不同的序列缔合的条件。例如,应该选择所研究的杂交体的计算的Tm以下约120℃至200℃的温度和盐浓度的组合。温度和盐条件通常可以在初步实验中凭经验确定,其中固定在过滤器上的基因组DNA的样品与感兴趣的序列杂交并且然后在不同严格度的条件下被洗涤。参见Sambrook等人.9.50页。
当进行例如DNA印迹时考虑的变量是(1)被印迹的DNA的复杂性和(2)探针和被检测的序列之间的同源性。待研究的一个或更多个片段的总量可以变化10的数量级,从对于质粒或噬菌体消化物的0.1mg至1mg,到对于在高度复杂的真核基因组中的单拷贝基因的10-9g至10-8g。对于较低复杂性多核苷酸,可以使用基本上较短的印迹、杂交和暴露时间,较少量的起始多核苷酸和较低的探针的特异性活性。例如,单拷贝酵母基因可以用以下检测:仅1小时的暴露时间、用1mg的酵母DNA起始、印迹持续2小时和用108cpm/mg的探针杂交持续4-8小时。
对于单拷贝哺乳动物基因,保守方法将是以下:用10mg的DNA起始、印迹过夜和在10%硫酸葡聚糖的存在下使用大于108cpm/mg的探针杂交过夜,产生24小时的暴露时间。
几个因素可以影响探针和感兴趣的片段之间的DNA-DNA杂交体的解链温度(Tm),并且因此,影响用于杂交和洗涤的适当条件。在许多情况中,探针不是与片段100%同源的。其他通常遇到的变量包括杂交序列的长度和总G+C含量和杂交缓冲液的离子强度和甲酰胺含量。所有的这些因素的作用可以通过单个方程被近似:其中Ci是盐浓度(单价离子),并且n是以碱基对计的杂交体的长度(从Meinkoth&Wahl(1984)Anal.Biochem.138:267-284稍微地修改)。
在设计杂交实验中,影响核酸杂交的一些因素可以方便地被改变。杂交和洗涤的温度和洗涤期间的盐浓度是最简单地调节的。当杂交的温度增加(即严格度),不同源的链之间发生杂交变得较不可能,并且作为结果,背景降低。如果放射标记的探针与固定的片段不完全同源(如频繁地在基因家族和物种间杂交实验中的情况),杂交温度必须被降低,并且背景将升高。洗涤的温度以类似的方式影响杂交条带的强度和背景的程度。洗涤的严格度也随着降低盐浓度而增加。
一般而言,在50%甲酰胺的存在下的适宜的杂交温度,对于与靶片段95%至100%同源的探针而言是42℃,对于与靶片段90%至95%同源性的探针而言是37℃,并且对于与靶片段85%至90%同源性的探针而言是32℃。如果探针和靶片段之间的同源性是未知的,最简单的方法是用非严格的杂交和洗涤条件二者开始。如果在放射自显影后观察到非特异性条带或高背景,则过滤器可以在高严格度洗涤并再次曝光。如果暴露所需要的时间使该方法不切实际,那么应该并行测试几个杂交和/或洗涤严格度。
核酸探针测定
实施例1:基于噬菌体的测试的效力,如在掺入的血液中示出的
将已知数目的狭义伯氏疏螺旋体B31菌株的细胞(1000个、100个、10个和1个细胞/单元)添加至1ml的商购可得的健康人类全血中(3个重复)。总DNA分别使用Qiagen血液和组织试剂盒从100μl的掺入的血液提取。然后,将提取的DNA用作用于末端酶基因的PCR扩增的模板(使用的引物是如在SEQ ID No.70和SEQ ID No.71—正向和反向—中示出的)。
如在图6中示出的凝胶图中清楚地证明的,PCR产物在来自掺入了B31菌株的100个或更多个细胞的血液样品的DNA中被观察到。这示出了将需要100个疏螺旋体属细胞以产生阳性末端酶PCR测试。这也证明了末端酶PCR检测限值是100个疏螺旋体属/ml血液,该浓度反映了莱姆病患者中的正常的疏螺旋体属浓度。无末端酶PCR产物可以从由无疏螺旋体属掺入的全血提取的总DNA被观察到(见仅标记着血液的泳道)。未发现检测的存在的错误信号。
与已知方法学的比较
如以上描述的,本发明是使用靶向广义伯氏疏螺旋体的末端酶PCR对具有已知浓度的伯氏疏螺旋体菌株B31的样品实施的。将本测试的灵敏度与基于细菌的PCR进行比较,我们的末端酶PCR靶向基于广义伯氏疏螺旋体噬菌体的PCR。本发明的测试示出了与已知测试相比的显著更高的灵敏度,并且可以检测浓度在~1个细菌/ml的细菌(见表1)。
表1:
结论:基于噬菌体的PCR对浓度为少于1个细菌/ml的莱姆病疏螺旋体属悬浮液是阳性的,而细菌PCR是阴性的。
实施例2:本发明的方法与常规的16sPCR方法的比较研究
将4个单独的伯氏疏螺旋体B31培养物稀释至10个疏螺旋体属/ml,然后将100μl的这些稀释的培养物经历使用Qiagen血液和组织试剂盒的DNA提取。然后将所得的提取的DNA用作用于末端酶基因的PCR扩增的模板(使用的引物如SEQ ID No.70和SEQ ID No.71—正向和反向—示出的),并且用作用于16S(对于核糖体RNA的细菌基因,通常作为用于检测存在或不存在细菌的分子诊断工具)的PCR扩增的模板。如可以在图7中示出的凝胶图中观察到的,用末端酶PCR观察到两个强阳性结果,而用16S PCR仅鉴定出一个弱阳性。最左侧的两条泳道是PCR阴性(即不包括来源于培养物的起始材料),最右侧的泳道是PCR阳性(即包括高浓度的细菌细胞)。
结论:该实施例证明了末端酶PCR的效率高于16S PCR的效率。
实施例3:根据本发明的测试对不同广义伯氏疏螺旋体物种的证明
针对不同疏螺旋体属基因型的集合(不同的分离株)进行末端酶PCR。
图8的凝胶图示出了该PCR的结果:B31=伯氏疏螺旋体B31;数字是如在下表中示出的疏螺旋体属菌株:
分离株名称 | 科学名称 | |
3 | VS185 P9 | 狭义伯氏疏螺旋体 |
4 | NE218 | 法雷斯疏螺旋体 |
5 | ACA1 | 阿氏疏螺旋体 |
6 | UK过滤的 | 狭义伯氏疏螺旋体 |
7 | 190 P9 | 伽氏疏螺旋体 |
8 | 中国23 | 狭义伯氏疏螺旋体 |
表2.
这证明了本发明的末端酶PCR技术可以扩增广义伯氏疏螺旋体组的四个关键菌株,所述四个关键菌株是伯氏疏螺旋体、伽氏疏螺旋体、阿氏疏螺旋体和法雷斯疏螺旋体。该末端酶PCR技术还被应用于其他细菌,诸如艰难梭菌(Clostridium difficile)、泰国伯克氏菌属(Burkholderia thailandensis)、大肠杆菌(E.coli)、沙门氏菌属(Salmonella)、军团菌属(legionellae)和嗜血杆菌(haemophilius)菌株。这些细菌均没有用末端酶引物生成任何PCR产物。
进行进一步分析以确定该方法区分莱姆病疏螺旋体属和回归热疏螺旋体属菌株的效率。
引物和TaqMan探针(表3)基于伯氏疏螺旋体末端酶基因序列(GenBank登录号NC_000948.1)使用工具(IDT)设计。为了确保引物/探针组合(被称为‘BbFAM’)的特异性,使用提交至GenBank的序列进行BLAST分析。具有e值<0.01的所有命中是以伯氏疏螺旋体为主的莱姆病疏螺旋体属物种,以下疏螺旋体属菌株中的每一个具有一个命中:梅奥疏螺旋体、伽氏疏螺旋体、阿氏疏螺旋体、比塞蒂疏螺旋体和法雷斯疏螺旋体。此外,针对所有可得的细菌物种进行了“计算机PCR”(http://insilico.ehu.es/PCR/),仅从伯氏疏螺旋体的质粒部分观察到正确大小的PCR产物。这证明了引物/探针组合可以检测莱姆病疏螺旋体属菌株。将TaqMan探针的5’用6-羧基荧光素(FAM)荧光染料标记,并且将3’用具有ZENTM猝灭剂和Iowa Black FQ的双猝灭剂标记(5’FAM/ZEN/3’IBFQ)。与包含单个猝灭剂的探针相比,这些双猝灭的探针产生更少的背景和增加的信号。两个引物、探针和PrimeTime基因表达主混合物由IDT提供。
表3用于末端酶实时PCR(BbFAM)的引物和探针的序列
如在表4中观察到的,针对从不同基因型的疏螺旋体属菌株提取的DNA测试引物/探针。所有测试的莱姆病疏螺旋体属菌株(1-6)产生阈值循环(Ct)值<30的阳性PCR。无Ct值可以从回归热疏螺体属菌株检测到。除了“计算机”PCR外,还进行了“湿实验”以确认当针对包括来自大肠杆菌、假单胞菌属(Pseudomonas)、梭菌属(Clostridium)、嗜血杆菌属(Haemophilus)、伯克氏菌属(Burkholderia)和沙门氏菌属的DNA的不同细菌DNA的范围进行BbFAM PCR时,无PCR产物被观察到。
表4针对不同莱姆病疏螺旋体属和回归热疏螺旋体属菌株的BbFAMPCR。
为了测试BbFAM PCR的稳健性、效率和检测限值(LOD),用BbFAM扩增一系列的5个十倍稀释的携带末端酶基因片段的质粒。为了构建用于实时PCR的标准品,制造了携带末端酶基因片段的质粒。该质粒用作阳性对照,并且帮助计算每个PCR中的拷贝数。如以下描述的构建末端酶质粒:
使用引物Blast针对末端酶基因序列(SEQ ID No 1)设计PCR引物。引物是FTer721:AGACTAAGATGCGGGCAAGA(SEQ ID NO.82)和RTer721:TTGCATCAAGAGCGTCATCA(SEQID NO.83)。生成了721bp的PCR产物。PCR在LabCycler(SensoQuest GmbH,Germany)中以50μl的总体积进行,所述50μl的总体积包含0.25mM dNTP、3mM MgCl2、3μM引物、50ng的模板DNA、0.5单位的Taq聚合酶(Bioline)和5μl 10×Taq缓冲液(Bioline)。扩增条件是:94℃持续2min,30个循环的94℃持续30秒、50℃持续30秒、72℃持续1min,在72℃最终延伸10min。将PCR产物用Qiagen凝胶提取试剂盒凝胶纯化,并且根据标准方案经历使用PCR克隆试剂盒的克隆。阳性克隆由PCR和测序证实。将所得的携带末端酶基因片段的质粒用Qiagen质粒试剂盒纯化,并用作阳性对照。DNA的浓度用Qubit荧光计(Invitrogen)测量。具有末端酶基因片段的质粒DNA的拷贝数根据以下式(在线可得)计算:
为了得出Taqman实时PCR的检测限值(LOD),首先测试携带末端酶片段的质粒DNA的10倍系列稀释,以评价Taqman PCR的性能。每个稀释(106-102)通过BbFAM PCR用5个重复测试(表5)。
接下来,将质粒DNA从1000个拷贝数稀释至100个、80个、60个、40个、20个、10个、5个和1个拷贝/PCR。对于每次稀释,使用10个重复。在SPSS中进行概率单位分析(probitanalysis),以计算具有95%概率的32个拷贝的质粒的LOD。
结果在以下两个表中概述:
拷贝数/PCR | 平均Ct |
10<sup>6</sup> | 20.63 |
10<sup>5</sup> | 24.32 |
10<sup>4</sup> | 28.03 |
10<sup>3</sup> | 31.12 |
10<sup>2</sup> | 34.27 |
表5携带末端酶基因片段的质粒DNA的Taqman实时PCR性能
表6Taqman实时PCR的LOD的确定(对于每个稀释,10个重复)
由具有95%概率的概率单位分析确定的LOD是30个拷贝的质粒DNA。
质粒DNA模板的拷贝数/PCR的范围从106至102。如图10中示出的,从所有质粒DNA样品中观察到阳性扩增。如图11中示出的,跨DNA模板的浓度和Ct值之间的线性关系具有强相关系数(R2=0.99)。此外,BbFAM的扩增效率是100%。这证明了PCR的高灵敏度和效率。
为了排除人类DNA干扰的可能性,从Sigman购买人类DNA和健康的全血。总DNA从健康的全血提取。使用BbFAM PCR检测两种DNA。从任何PCR都没有观察到Ct值,而靶向人类管家基因RNA酶P的阳性PCR产生阳性。这证实了人类DNA对BbFAM PCR没有影响。
结论:该实验证明了该末端酶PCR是对广义伯氏疏螺旋体特异性的,以及使用实时测序,该测试具有非常低的检测限值(LOD)。
用于穴蛋白、内溶菌素和用于扩增包含这两个基因的区域的PCR引物(SEQ IDNO.72-77;包含这两个基因的区域=SEQ ID NO.78)也已被证明能够扩增莱姆病疏螺旋体属的正确的区域。
实施例4:本发明的方法与在血清样品中实施的常规16sPCR方法的比较研究
5个莱姆病阳性血清样品(S1-S5)(如通过抗体和临床表现测试测试和证实的)经历了使用Qiagen血液和组织试剂盒的DNA提取。然后分别使用末端酶(上图)和16S(下图)PCR引物二者分析DNA(在末端酶的情况中,使用的引物是如SEQ ID No.70和SEQ ID No.71中的)。如在图9中示出的凝胶图中观察到的,对于末端酶(S1、S2、S4和S5)观察到4个PCR阳性结果,而对于16S(S2和S3)仅可以观察到2个阳性。这是用大规模临床试验进行实时PCR实验前的初步研究(见以下)。
结论:该实施例证明了末端酶PCR比基于16s的技术具有高得多的灵敏度。+=对照,高浓度的细菌细胞。
实施例5:临床结果
还针对222个血清样品研究了BbFAM PCR的灵敏度,所述222个血清样品全部来源于临床上证实的莱姆病患者(在这些患者中,91名患者具有ELISA和/或蛋白质印迹数据)。
222名患者中的207名示出了BbFAM PCR阳性,代表了93.2%的灵敏度。
222名患者中的91名已经通过ELISA和/或WB检查。
91名患者中,他们中的16名示出了ELISA和/或WB阳性,代表了17.6%的灵敏度。在这些91名患者中,85名示出了对BbFAM PCR阳性,代表了93.4%的灵敏度,这与基于222名患者计算的灵敏度非常一致。如果研究BbFAM PCR结果与ELISA/WB数据之间的相关性,将发现所有16名ELISA和/或WB阳性患者都显示出BbFAM PCR阳性。此外,来自91名患者队列(cohort)的表现出BbFAM PCR阴性的6名患者还显示出ELISA/WB阴性。91名队列中的绝大多数临床上证实的患者仅示出对BbFAM PCR阳性,但对ELISA/WB阴性,指示BbFAM PCR的高灵敏度和可靠性。为了比较BbFAM与目前的商用莱姆病PCR检测试剂盒的灵敏度,将GeneProof伯氏疏螺旋体PCR试剂盒应用于从222名队列随机选择的65个血清样品。65个血清样品中仅7个示出了对GeneProof试剂盒阳性(10.8%的灵敏度)。
总结
这是使用分子标志物研究疏螺旋体属噬菌体的分布和多样性的第一项研究。
由本发明人构建的噬菌体末端酶基因的系统发育树示出了,噬菌体末端酶基因是良好的系统发育标志物,因为疏螺旋体属噬菌体序列形成离散但遗传上多样的组,该组是与其他螺旋体清楚地分离的。莱姆病感染(即广义伯氏疏螺旋体感染)形成离散的良好支持的进化枝,并且这些进化枝与细菌良好相关。
对于莱姆病的总结
1)与细菌16S方法相比,以高得多的灵敏度鉴定LD的整体能力。此外,临床结果还证明,基于噬菌体末端酶的实时PCR比基于细菌16S的PCR(GeneProof PCR试剂盒)显著更灵敏。
2)涉及广义伯氏疏螺旋体的多个物种(表4:针对不同的莱姆病疏螺旋体属和回归热疏螺旋体属菌株的基于噬菌体末端酶的实时PCR)。
3)早期检测是可能的,因为基于噬菌体的测试可以检测低浓度的细菌。实时PCR具有30个拷贝的末端酶基因的低检测限值。
回归热
实施例6:回归热(赫姆斯疏螺旋体)与莱姆病的区分
引物和TaqMan探针(表8)基于赫姆斯疏螺旋体末端酶基因序列使用工具(IDT)设计。为了确保引物/探针组合(被称为‘BhFAM’)的特异性,使用提交至GenBank的序列进行BLAST分析。在赫姆斯疏螺旋体命中和下一个最接近的墨西哥疏螺旋体和帕克疏螺旋体命中之间,观察到从0.000004至0.004的大E值下降。
此外,针对所有可得的细菌物种进行了“计算机PCR”(http://insilico.ehu.es/PCR/),仅从赫姆斯疏螺旋体观察到正确大小的PCR产物。TaqMan探针的5’用6-羧基荧光素(FAM)荧光染料标记,并且3’用具有ZENTM猝灭剂和Iowa Black FQ的双猝灭剂标记(5’FAM/ZEN/3’IBFQ)。与包含单个猝灭剂的探针相比,这些双猝灭的探针产生较小的背景和增加的信号。两个引物、探针和PrimeTime基因表达主混合物由IDT提供。
表8靶向赫姆斯疏螺旋体中的末端酶基因的引物和探针的序列(BhFAM)
如在表9中可见的,针对从不同基因型的疏螺旋体属菌株提取的DNA测试引物/探针。从所有莱姆病疏螺旋体属菌株(1-6)和其他回归热疏螺旋体属菌株都没有观察到阳性。仅赫姆斯疏螺旋体生成了正确的PCR产物。除了“计算机”PCR外,还进行了“湿实验”以确认当针对包括来自大肠杆菌、假单胞菌属、梭菌属、嗜血杆菌属、伯克氏菌属和沙门氏菌属的DNA不同的细菌DNA的范围进行BhFAM PCR时,观察不到PCR产物。
表9针对不同莱姆病疏螺旋体属和回归热疏螺旋体属菌株的BhFAM PCR
实施例7:回归热(宫本疏螺旋体)与莱姆病的区分
两组引物和TaqMan探针基于两种宫本疏螺旋体末端酶基因使用具有人工检查的工具(IDT)设计(分别被称为“BmFAM”和“Bm-2FAM”,如在表10中示出的)。两组靶向定位在不同质粒上的不同版本的末端酶基因,因此它们在检测宫本疏螺旋体时是彼此互补的。为了确保引物/探针组合的特异性,使用提交至GenBank的序列进行BLAST分析。对于两组Taqman探针,观察到大的E值下降。例如,BmFAM和Bm-2FAM示出了,在宫本命中和下一个最接近的blast命中之间,E值分别从0.0002下降至0.79和从0.006下降至1.6。针对所有可得的细菌物种进行了“计算机PCR”(http://insilico.ehu.es/PCR/),仅从宫本疏螺旋体观察到正确大小的PCR产物。这证明了引物/探针组合在检测宫本疏螺旋体菌株中的特异性。TaqMan探针的5’用6-羧基荧光素(FAM)荧光染料标记,并且3’具有ZENTM猝灭剂和IowaBlack FQ的双猝灭剂标记(5’FAM/ZEN/3’IBFQ)。与包含单个猝灭剂的探针相比,这些双猝灭的探针产生较小的背景和增加的信号。引物、探针和PrimeTime基因表达主混合物由IDT提供。
表10靶向宫本疏螺旋体菌株中的末端酶基因的两组引物/探针(BmFAM和Bm-2FAM)
如在表11中可见的,针对从不同基因型的疏螺旋体属菌株提取的DNA测试引物/探针。无Ct值可以从所有莱姆病疏螺旋体属菌株(1-6)和回归热疏螺旋体属菌株检测到。PCR阳性仅可在宫本疏螺旋体DNA中观察到。此外,当针对包括来自大肠杆菌、假单胞菌属、梭菌属、嗜血杆菌属、伯克氏菌属和沙门氏菌属的的DNA的不同细菌DNA的范围进行两组引物/探针时,没有观察到PCR产物。
表11针对不同莱姆病疏螺旋体属和回归热疏螺旋体属菌株的BmFAM和Bm-2FAMPCR。
作为初步研究,来源于健康志愿者和莱姆病患者的43管的血液/血清样品被随机地选择,并且分别经历针对BmFAM和BbFAM的测试。结果如下:7支管(16.3%)示出对BmFAM阳性,而26支(60.5%)示出对BbFAM阳性。这证明了在群体中携带潜在低水平的宫本疏螺旋体。大规模临床验证正在进行中。
对于回归热的总结
1)基于噬菌体的测试可以特异性扩增RF疏螺旋体属菌株,并且不扩增LD疏螺旋体属菌株。这使得设计基于噬菌体末端酶的双重PCR以同时诊断LD和RF成为可能。
2)基于噬菌体的RF测试具有区分不同的RF疏螺旋体属菌株,诸如宫本疏螺旋体和赫姆斯疏螺旋体的能力。只要RF物种是已知的,这就将使临床医生能够更好地管理和开出抗生素。通过完全验证的基于噬菌体的RF测试,临床医生将更好地了解哪些疏螺旋体属物种引起症状。
3)作为这些成功的初始验证结果的结果,大规模临床验证目前正在进行。
序列表
<110> 菲利克斯研究与发展有限公司
莱斯特大学
<120> 检测方法
<130> P184103.WO.01
<150> GB1618565.4
<151> 2016-11-03
<160> 110
<170> PatentIn 3.5版
<210> 1
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体(Borrelia burgdorferi)的噬菌体(Phage from Borreliaburgdorferi)
<400> 1
gtgaacttat atcaaacaaa actttttaca acactacaaa aggaatacaa aaataaatat 60
ggagttgata tatcacaatt tgtaaagcta acaaattctt caattaattt tgataagttt 120
gaagaagaac agttaacttt aaaacaaaaa aatgtgataa aaagcattaa aaagaataat 180
gaaaagaaga ttatactcag cggaggcata gctagtggca aaacgtatct tgcatgttat 240
ctttttctaa aaagtttaat tgaaattaaa aagttatact ctagtgatac taataatttc 300
attataggga attcacaacg ttcagttgaa gttaatgttt tggggcaatt tgaaaagcta 360
tgtaaacttc ttaaaattcc ttatattcca agacatacaa ataattcata tattctgatt 420
gattcactac gtattaatct atatggagga gataaggcaa gtgattttga aagatttagg 480
ggaagtaatt cggcacttat ttttgttaat gaggctacaa ctttacacaa gcaaacttta 540
gaggaagtct taaaaagact aagatgcggg caagaaacta ttatttttga tactaatcct 600
gatcatccag aacactattt taaaaccgat tatattgata atatagcgac ctttaagaca 660
tataagttta caacttatga taatgtgcta cttagtaaag gatttgtcga aacacaagaa 720
aagctatata aagatatacc atcatataaa gcaagagttt tgttaggtga gtggatagca 780
agcactgatt caatttttac acaaataaat attactgatg attatgtatt tactagcccg 840
atagcatatt tagacccagc atttagtgtt ggcggggata acactgcatt atgtgttatg 900
gagcgagttg atgataagta ttatgctttt gtatttcaag accaaagacc agctaatgat 960
ccttatatta tgaatatggt aaagactgtt atagaaaatt tcaatgtgca tacactgtat 1020
ttagaggata gagataatac aaaaggtgct ggtggattga cccgtgaata catcttgcta 1080
agaagtaata taagccaata ttttagaatt gttccagtta agccaaagtc taataaattt 1140
agcagaataa caacgttaat tacgccgttt acttacaaaa aactttatat tacaaagtac 1200
agtagttctt ccgtatttaa tgatatttat tcgtataagg gggataataa aacccatgat 1260
gacgctcttg atgcaatatc tgcagcatat ttgatgttgt ctttaggata tagagagcga 1320
agtgttcact ttggcaatca aagatttttg taa 1353
<210> 2
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 2
gtgaacttat atcaaacaaa actttttaca acactacaaa aggaatacaa aaataaatat 60
ggagttgata tatcacaatt tgtaaagcta acaaattctt caattaattt tgataagttt 120
gaagaaaaac agttaacttt aaaacaaaaa aatgtgataa aaagtattaa aaagaataat 180
gaaaaaaaga ttatattcag cggcggcata gctagcggca aaacgtatct tgcatgctat 240
ctttttctca aaagtttaat tgaaaataaa aagttatatt ctagcgatac gaataatttt 300
attattggga attcacaacg ctcagttgaa gttaatgttt tgggacaatt tgaaaagcta 360
tgtaaacttc ttaaaattcc ttatattcca agacatacaa ataattcata tattctgatt 420
gattcactac gtattaatct atatggtgga gataaggcaa gtgattttga aagatttagg 480
ggaagtaatt cggcacttat ttttgttaat gaggctacaa ctttacacaa gcaaacttta 540
gaggaggtcc taaaaagact aagatgcggg caagaaacta ttatttttga tactaatcct 600
gatcatccag aacactattt taaaaccgat tatattgata atatagcgac atttaagaca 660
tataatttta caacttatga taatgttcta cttagtaaag gatttatcga aacacaagaa 720
aaactctata aagatatacc atcatataaa gcaagagttt tgctaggtga gtggatagca 780
agcactgatt caatttttac acaaataaat attactgatg attatgtatt tactagtcca 840
atagcatatt tagacccagc atttagtgtt ggaggagata acactgcatt atgtgttatg 900
gagcgagttg atgataagta ttatgctttt gtatttcaag accaacgacc agccaatgat 960
ccttatatta tgaatatggt aaagaccgtt atagaaaatt tcaatgtgca tacactgtat 1020
ttagaggata gagataatac aaaaggtgct ggtggattga cccgcgaata catcttacta 1080
agaaataata taagccaata ttttagaatt gttccagtta agccaaagtc taataaattt 1140
agcagaataa caacgttaat tacgccgttt acttataaga aactttacat tacaaagtac 1200
agtagttctt ctgtatttaa tgatatttat tcgtataagg gggatagcaa aacccatgat 1260
gatgctcttg atgcaatgtc tgcagcatat ttgatgttgt ctttaggata tagagagcga 1320
agtgttcact ttggcaatca aagatttttg taa 1353
<210> 3
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 3
gtgaacttat atcaaacaaa actttttaca acactacaaa aggaatacaa aaataaatat 60
ggagttgaca tatcacaatt tgtaaagcta acaaattctt caattaattt tgataagttt 120
gaaaaagaac agttaaccat aaaacaaaaa aatgttataa aaagtattca aaaaaataat 180
gaaaagaaga ttatactcag cggcggcata gctagcggca aaacgtatct tgcatgttat 240
ctttttctca aaagtttaat tgaaaataaa aagttatact ctggtgatat taataatttt 300
attattggga attcacaacg ctcagttgaa gttaatgttt tgggacaatt tgaaaagcta 360
tgtaaacttc ttaaaattcc ttatatccca agacatacaa ataattcata tatattaatt 420
gattcacttc gtattaatct atatggagga gataaggcaa gtgattttga aagatttaga 480
ggcagtaatt cggcacttat ttttgttaat gaggctacta ctttacacaa gcaaacttta 540
gaggaggtct taaaaagact taggtgcgga caagaaacta ttatttttga tactaatcct 600
gatcatccag aacactattt taaaaccgat tatattgata atatagcgac atttaagaca 660
tataatttta caacttatga taatgtgcta cttagtaaag gatttatcga aacacaagaa 720
aaactctata aagatatacc atcatataaa gcaagagttt tgctaggtga gtggatagca 780
agcaccgatt caatttttac acaaataaat attactaatg attatgtatt tactagcccg 840
atagcatatt tagacccagc atttagtgtt ggaggggata acactgcatt atgtgttatg 900
gagcgagttg atgataagta ttatgctttt gtatttcaag accaacgacc agccaatgat 960
ccttatatta tgaatatggt aaagaccgtt atagaaaatt tcaatgtgca tacactgtat 1020
ttagaggata gagataatac aaaaggtgct ggtggattga cccgcgaata catcttgcta 1080
agaaataata taagccaata ttttagaatt gttccagtta agccaaagtc taataaattt 1140
agcagaataa caacggtaat tacgccgttt acttataaga aactttacat tacaaagtac 1200
agtagttctt ctgtatttaa tgatatttat tcgtataagg gggatagcaa aacccatgat 1260
gatgctcttg atgcaatgtc tgcagcatat ttgatgttgt ctttaggata tagagagcga 1320
agtgttcact ttggcaatca aagatttttg taa 1353
<210> 4
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 4
gtgaacttat atcaaacaaa actttttaca acactacaaa aggaatacaa aaataaatat 60
ggagttgata tatcacaatt tgtaaagcta acaaattctt caattaattt tgataagttt 120
gaagaaaaac agttaacttt aaaacaaaaa aatgtgataa aaagtattaa aaagaataat 180
gaaaaaaaga ttatactcag cggcggcata gctagcggca aaacgtatct tgcatgctat 240
ctttttctca aaagtttaat tgaaaataaa aagttatatt ctagcgatac gaataatttt 300
attattggga attcacaacg ctcagttgaa gttaatgttt tgggacaatt tgaaaagcta 360
tgtaaacttc ttaaaattcc ttatattcca agacatacaa ataattcata tattctgatt 420
gattcactac gtattaatct atatggtgga gataaggcaa gtgattttga aagatttagg 480
ggaagtaatt cggcacttat ttttgttaat gaggctacaa ctttacacaa gcaaacttta 540
gaggaggtct taaaaagact aagatgcggg caagaaacta ttatttttga tactaatccc 600
gatcatccag aacactattt taaaaccgat tatattgata atatagcgac ctttaagaca 660
tataatttta caacttatga taatgtgcta cttagtaaag gatttgtcga aacacaagaa 720
aagctatata aagatatacc atcatataaa gcaagagttt tgctaggtga gtggatagca 780
agcactgatt caatttttac acaaataaat attactgatg attatgtatt tactagcccg 840
atagcatatt tagacccagc atttagtgtt ggcggggata acactgcatt atgtgttatg 900
gagcgagttg atgataagta ttatgctttt gtatttcaag accaaagacc agctaatgat 960
ccttatatta tgaatatggt aaagactgtt atagaaaatt tcaatgtgca tacactgtat 1020
ttagaggata gagataatac aaaaggtgct ggtggattga cccgtgaata catcttgcta 1080
agaagtaata taagccaata ttttagaatt gttccagtta agccaaagtc taataaattt 1140
agcagaataa caacgttaat tacgccgttt acttacaaaa aactttacat tacaaagtac 1200
agtagttctt ctgtatttaa tgatatttat tcgtataagg gggatagcaa aacccatgat 1260
gatgctcttg atgcaatgtc tgcagcatat ttgatgttgt ctttaggata tagagagcga 1320
agtgttcact ttggcaatca aagatttttg taa 1353
<210> 5
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 5
gtgaacttat atcaaacaaa actttttaca acactacaaa aggaatacaa aaataaatat 60
ggagttgata tatcgcaatt tataaagcta acaaattctt caattaattt tgataagttt 120
gaagaagaac agctaacttt aaaacaaaaa aatgtgataa aaagcattaa aaagaataat 180
gaaaagaaga ttatactcag tggaggtata gctagcggca aaacgtatct tgcatgttat 240
ctttttctca aaagtttaat tgaaaataaa aagctatatt ctagcgatac gaataatttc 300
attataggga attcacaacg ttcagttgaa gttaatgttt tgggacaatt tgaaaagcta 360
tgtaaacttc ttaaaattcc ttatattcca agacatacaa ataattcata tattttgatt 420
gattcactac gtattaatct atatggtgga gataaggcaa gtgattttga aagatttagg 480
ggaagtaatt cagcacttat ttttgtgaat gaggctacaa ctttacacaa gcaaacttta 540
gaggaagtct taaaaagact aaggtgtggg caagaaacta ttatttttga tactaacccc 600
gatcatccgg aacactattt taaaaccgat tatattgata atatagcaac ttttaagaca 660
tataatttta caacttatga taatgttcta cttagcaaag gatttatcga aactcaagaa 720
aagctatata aagatatacc atcatataaa gcaagagttt tgctaggtga atggatagca 780
agcaccgatt caatttttac acaaataaat attactaatg attatgtatt tactagcccg 840
atagcatatt tagacccagc atttagtgtt ggaggggata acactgcatt atgtgttatg 900
gagcgagttg atgataagta ttatgctttt gtatttcaag accaaagacc agccaatgac 960
ccgtatatta tgaatatggt taagaccgtt ttagagaatt ttaatgtaca tacactttat 1020
ttagaagata gagacaatac aaaaggtgct ggtggattga ctcgtgaata catgttgcta 1080
agaaataata tgggtcaata ttttagaatt gttccagtta agccaaagtc taataaattt 1140
agcagaataa caacgttaat tacgccgttt acttataaga aactttacat tacaaagtac 1200
agcagttctt ctgtatttaa tgatatttat tcgtataaag gagataacaa aacccatgat 1260
gatgctcttg atgcaatatc tgcagcatat ttgatgttgt ctttagggta tagagagaga 1320
agtgttcact ttggcaatca aagatttttg taa 1353
<210> 6
<211> 1352
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 6
gtgaacttat atcaaacaaa actttttaca acactacaaa aggaatacaa aaataaatat 60
ggagttgata tatcacaatt tgtaaagcta acaaattctt caattaattt tgataagttt 120
gaagaagaac agttaacttt aaaacaaaaa aatgtgataa aaagcattaa aaagaataat 180
gaaaagaaga ttatactcag cggaggcata gctagtggca aaacgtatct tgcatgttat 240
ctttttctaa aaagtttaat tgaaaataaa aagttatact ctagtgatac taataatttc 300
attataggga attcacaacg ttcagttgaa gttaatgttt tggggcaatt tgaaaagcta 360
tgtaaacttc ttaaaattcc ttatattcca agacatacaa ataattcata tattctgatt 420
gattcactac gtattaatct atatggagga gataaggcaa gtgattttga aagatttagg 480
ggaagtaatt cggcacttat ttttgttaat gaggctacaa ctttacacaa gcaaacttta 540
gaggaagtct taaaaagact aagatgcggg caagaaacta ttatttttga tactaatcct 600
gatcatccag aacactattt taaaaccgat tatattgata atatagcgac ctttaagaca 660
tataatttac aacttatgat aatgtgctac ttagtaaagg atttgtcgaa acacaagaaa 720
agctatataa agatatacca tcatataaag caagagtttt gttaggtgag tggatagcaa 780
gcactgattc aatttttaca caaataaata ttactgatga ttatgtattt actagcccga 840
tagcatattt agacccagca tttagtgttg gaggggataa cactgcatta tgtgttatgg 900
agcgagttga tgataagtat tatgcttttg tatttcaaga ccaaagacca gctaatgatc 960
cttatattat gaatatggta aagactgtta tagaaaattt caatgtgcat acactgtatt 1020
tagaggatag agataataca aaaggtgctg gtggattgac ccgtgaatac atcttgctaa 1080
gaagtaatat aagccaatat tttagaattg ttccagttaa gccaaagtct aataaattta 1140
gcagaataac aacgttaatt acgccgttta cttacaaaaa actttatatt acaaagtaca 1200
gtagttcttc cgtatttaat gatatttatt cgtataaggg ggataataaa acccatgatg 1260
acgctcttga tgcaatatct gcagcatatt tgatgttgtc tttaggatat agagagcgaa 1320
gtgttcactt tggcaatcaa agatttttgt aa 1352
<210> 7
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 7
gtgaacttat atcaaacaaa actttttaca acactacaaa aggaatacaa aaataaatat 60
ggagttgaca tatcacaatt tgtaaagcta gcaaattctt caattaattt tgataagttt 120
gaagaaaaac agttaacttt aaaacaaaaa aatgtgataa aaagtattaa aaagaataat 180
gaaaaaaaga ttatactcag cggaggcata gctagtggca aaacgtatct tgcatgttat 240
ctttttctaa aaagtttaat tgaaaataaa aagttatact ctagtgatac taataatttc 300
attattggga attcacaacg ctcagttgaa gttaatgttt taggacaatt tgaaaagcta 360
tgtaaacttc ttaaaattcc ttatatccca agacatacaa ataattcata tattctgatt 420
gattcactac gtattaatct atatggtgga gataaggcaa gtgattttga aagatttagg 480
ggaagtaatt cagcacttat ttttgttaat gaggctacaa ctttacacaa gcaaacttta 540
gaggaggtct taaaaagact aagatgcggg caagaaacta ttatttttga tactaatcct 600
gatcatccag aacactattt taaaaccgat tatattgata atatagcgac atttaagata 660
tataatttta caacttatga taatgtgcta cttagtaaag gatttatcga aacacaagaa 720
aaactctata aagatatacc atcatataaa gcaagagttt tgctaggtga gtggatagca 780
agcactgatt caatttttac acaaataaat attactgatg attatatatt tactagcccg 840
atagcatatt tagacccagc atttagtgtt ggaggggata acactgcatt atgtgttatg 900
gagcgagttg atgataagta ttatgctttt gtatttcaag accaacgacc agccaatgat 960
ccttatatta tgaatatggt aaagaccgtt atagaaaatt tcaatgtgca tacactgtat 1020
ttagaggata gagataatac aaaaggtgct ggtggattga cccgcgaata catcttgcta 1080
agaaataata taagccaata ttttagaatt gttccagtta agccaaagtc taataaattt 1140
agcagaataa caatgttaat tacgccgttt acttacaaaa aactttatat tacaaagtac 1200
agtagttctt ctgtatttaa tgatatttat tcgtataagg gggatagcaa aacccatgat 1260
gatgctcttg atgcaatatc tgcagcatat ttgatgttgt ctttaggata tagagagcga 1320
agtgttcact ttggcaatca aagatttttg taa 1353
<210> 8
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 8
gtgaacttat atcaaacaaa actttttaca atactacaaa aggaatacaa aaataaatat 60
ggagttgata tatcacaatt tgtaaagcta acaaattctt caattaattt tgataagttt 120
gaagaagagc agctaacttt aaaacaaaaa aatgtgataa aaagcattaa aaagaataat 180
gaaaaaaaga ttatactcag cggcggcata gctagcggca aaacgtatct tgcatgttat 240
ctttttataa aaagtttaat tgaaaataaa aagctatatt ctagcgatac gaataatttc 300
attataggga attcacaacg ctcagttgaa gttaatgttt tggggcaatt tgaaaagcta 360
tgcaaactgc ttaaaattcc ttatattcca agacatacaa ataattcata tattctgatt 420
gattcactgc gaattaatct acatggtgga gataaggcaa gtgattttga aagatttagg 480
ggaagtaatt cagcacttat ttttgttaat gaggctacaa ctttacacaa gcaaacttta 540
gaggaagtct taaaaagatt aagatgcggg caagaaacta ttatttttga tactaatcct 600
gatcatccag aacactattt taaaaccgat tatattgata atatagcgac ctttaagaca 660
tataatttta caacttatga taatgttcta cttagtaaag gatttatcga aacacaagaa 720
aagctctata aagatatacc atcatataaa gcaagagttt tgctagggga gtggatagca 780
agcaccgatt caatttttac acaaataaat attactaatg attatgtatt tactagcccg 840
atagcatatt tagacccagc atttagtgtt ggcggggata acactgcatt atgtgttatg 900
gagcgggttg atgataagta ttatgctttt gtatttcaag accaacgacc agccaatgac 960
ccgtatatta tgaatatggt taagaccgtt ttagaaaatt ttaatgtaca tacattttat 1020
ttagaagata gagacaatac aaaaggtgct ggtggattga cccgcgaata catcttgcta 1080
agaaataata tgggtcaata ttttagaatt gttccagtta agccaaagtc taataaattt 1140
agcagaataa cagcgttaat tacgccgttt atttataaga aactgtacat tacgaagtac 1200
agcagttctt ctgtatttaa tgatatttat tcgtataaag gagataacaa aacccatgat 1260
gatgctcttg atgcaatatc tgcagcatat ttgatgttgt ctttagggta tagagagaga 1320
agtgttcact ttggcaatca aagatttttg taa 1353
<210> 9
<211> 1353
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 9
ttgaaattct taagatcatt agcgtttctc aatcttcaga aaaagtttaa aaataagttt 60
aatattaaca ttttggatta tattaagcca aaaccaacca aaatttgttt taaagatttt 120
gaaaataaat acttaactgc taagcaaaag gaagttcttt ttgacataga aagtaacaat 180
tattcaaaag taatatttag cggtgggatt gcaagtggta agacgttttt agcttcatat 240
ttgcttgtta aaaagcttat tgaaaacaaa tctttttatg agcaagatac caataatttt 300
attataggca actcaattgg tttgttgatg acaaatactg taaagcaaat agagaaaatt 360
tgcagcttgc tgggaattga ttacgagaaa aagaaaagcg ggcagtcttt ttgtaagata 420
gcaggtctta agcttaatat ttacgggggt aaaaacagag atgctttttc caagattcgt 480
gggggcaata gtgcaataat ttatgtaaat gaagcaacag taattcacag agaaacttta 540
cttgaagtaa taaaaaggct tagaaagggt aaagaaatta ttatttttga tacaaatccg 600
gaaagccctg cgcattattt taaaaccgat tatattgaaa atacagatgt ttttaagaca 660
tataatttta caacttatga caatccttta aattcagcag attttattca aactcaggaa 720
aaactttaca ggcgttttcc cgcctatagg gctcgtgtgc tttacgggga gtggatttta 780
aatgaatcta cgctatttaa tgagatgatt ttcaatcaag attatgaatt taaaagccca 840
ataatgtaca ttgatcctgc attctcagtg ggtggagaca atacagctat ttgtgtttta 900
gagcgcactt ttgagaagtt ttatgcatat atttatcaag accaaaaacc agtaagtgat 960
agtttaatgc ttgcttccat tcaagtttta atagaaaatt tcaatgtaaa taccgtttac 1020
attgaggaga gagacagtac caagggggat ggaattttaa ctaaaacaat tcttttcttg 1080
agaaacaaaa gcagtcacta ttttaaagtt gcaccaataa aacctttaag caataaattt 1140
aaaagaatat gcgcgcttat tcctttgttt gaaagccgca aaatagaatt tttaaaaata 1200
ataagtaaaa atgtaatatc tgatatttac agctacaaag gagatggcaa aacaaaagac 1260
gatgcgcttg attctcttgc aaatgcgtat cttcttttaa cgctaaatta taaagaaaaa 1320
ttatttcatt tcggcaggtt taaatattta taa 1353
<210> 10
<211> 1191
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 10
atgagactaa ggcgacttcc agtatatgtt gatgcctaca aagaaaagcc caatgctgaa 60
attttcatat actactcaag taggggaact ggtaaaacct acgacattgc aactgttaat 120
ttagaaagaa aatttagtgc tgatggcgga gatacccttg caattagaaa aaagaaaaac 180
aaaacaacac aatcaataca caaagaaatt ttagaacttt taagcatata caacttaaga 240
aaatttttca atataagcaa agcaaaaatt gaaagtaaga gtttgatttt tgggaaaaaa 300
cgtgcttttg tttttgaagg ggggcatgat acaagagatt taaaatctta tgcgcatttt 360
aaggacttat ggctggaaga agccaatcag tttagcgctg atgatataga aatgcttgtc 420
cctacaatga gagaacaagg cggcagaatc tatatgtcaa gcaatccggt gcctaaatca 480
cattggctat acaaaaggta cttatcaaat caagacaatc ctgctgtgtg tataatcaaa 540
agcacttacc gtgacaaccc atttttaaat ggtggagacg tacaagcttg gcttgaaaaa 600
caaagacttg catatcatgg caatgacatt ggttttagaa ttgaggtttt aggagaagag 660
tttgattttg gtacagcaag gctaattaaa aaatttaatg tgtgtggtcc cgaaattctt 720
tccagagcta atggaagcta ttatacagga atacacgtta aaggcaacag aatttgtttt 780
ttagaaattc ttgttggaag aatttcctat cttccagttg taattattac aaacgcatgt 840
agtaaagttt tactatcaaa aactgattac caatccgaaa ttaataaatt taaaggggtt 900
tttgtattgc caacagcaag agaagaactc aaatatgtat tttctcgttt tggtagaggt 960
actttgcttg caaaaaaacg aaacttgtat tcactctcag actatttgat tccgtctaat 1020
cttaatgtgg taaacaaacc cgaaaccact gatgtaattt cagagttcaa cgaaactgaa 1080
tattattatg atgaatctag tgcagaagat agcgaagtta caaattttgt tatgcaaaaa 1140
gatttggtat acatcccggc atttctcaat gccatatcgg tttttagcta a 1191
<210> 11
<211> 1224
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 11
gtgtgtgatt taagaaaaac aaaactaata gataaaataa gttcactaga actatacaaa 60
tactcaatat tttttagaaa ttacattgaa aatgtagcag aagattgtct caagaacgga 120
cttattcttg agagtgctgc ccacaatgtt agtgaggttg aacttgctag gttaaaggta 180
cagcttaaga atgctctgct taattgtatt ataagctacc gttttcatgg gattggctat 240
gttttagtaa aaaccaaaga taccctaata gatctcgaac aacccgttaa tatagaatta 300
cctattggtt ttgaatacct tgattatgaa tatgtaagag atttgggagt tgattttgat 360
catataacct ataaagtaaa atccaacaat aagaacaatt ctttagacgc agttaaaata 420
cataaaagtc gacttatcat atatgaaaac tttgattata tcttaaaaag atatgttccg 480
tgttataccg aaagcttttt actagatatt tatttatttg aaaagatata cgtagaaata 540
gaaagacgta ttgaaaacca caatttcttg ttttataaag atgaatcttt agtacaacta 600
caagacgcac tctctagtgc aacaacttct ttaagtgcac ttactcagag caataatgat 660
aggggaagtg gcattttatc ttcttttttg agaaaacaaa attcaaacaa tcatagtaaa 720
gatatttcta atttaagaaa ccttaatgac tcattatcac aggagcttgc taggctaaaa 780
agcaatctaa ataatgaggg aatgttttat acggccaccc ctagtgctag tttagaggtt 840
attaaatacg atcttagcta cttaaaggag gctttagcat taattaaggc aaaaattggt 900
gcagatacta aagagccctt aaccagaagt tttaatgagc aggctaaagg actaggaaat 960
gatggtaaag gtgataggag taattattac gattttctca aaggtgtaca agaacaagtt 1020
gagaactctt gtaatttaaa acttacaaag tattttgggc ttgatatgaa gtttaattct 1080
ctgattatgt taagtgaaga acaaaaagtg gaaagagata taaagctaat tgagctttac 1140
agtaaatata accagcttat acaaagtagc tcatttaata atgaggagct agcgatgtta 1200
aaagagaaat tattctcatt ttga 1224
<210> 12
<211> 1224
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 12
gtgtgtgatt taagaaaaac aaaactaata gataaaataa gttcactaga actatacaaa 60
tactcaatat tttttagaaa ttacattgaa aatgtagcag aagattgtct caagaacgga 120
cttattcttg agagtgctgc ccacaatgtt agtgaggttg aacttgctag gttaaaggta 180
cagcttaaga atgctctgct taattgtatt ataagctacc gttttcatgg gattggctat 240
gttttagtaa aaaccaaaga taccctaata gatctcgaac aacccgttaa tatagaatta 300
cctattggtt ttgaatacct tgattatgaa tatgtaagag atttgggagt tgattttgat 360
catataacct ataaagtaaa atccaacaat aagaacaatt ctttagatgc agttaaaata 420
cataaaagtc gacttatcat atatgaaaac tttgattata tcttaaaaag atatgttccg 480
tgctataccg aaagcttttt actagatatt tatttatttg aaaagatata cgtagaaata 540
gaaagacgta ttgaaaacca caattttttg ttttacaaag atgaatcttt agtacaacta 600
caagacgcac tttctagtgc aacaacttct ttaagtgcac ttactcagag caataatgat 660
aggggaagtg gcattttatc ttcttttttg agaaaacaaa attcaaacaa tcatagtaaa 720
gatatttcta atttaagaaa ccttaatgac tcattgtcac aggagcttgc caggctaaaa 780
agcaatctaa ataatgaggg aatgttttat acagctactc ctagtgctag tttagaggtt 840
attaaatacg atcttagtta cttaaaggag gctttagcat taattaaggc aaaaattggt 900
gcagatacta aagagccctt aaccagaagt tttaatgaac aggctaaagg actgggaaat 960
gatggtaaag gtgataggag taattattac gattttctca aaggtgtaca agaacaagtt 1020
gagaactctt gtaatttaaa acttacaaag tattttggac ttgatatgaa gtttaattcg 1080
ctgattatgt taagtgaaga acaaaaagta gaaagagata taaagctaat tgagctttac 1140
agtaaatata accagcttat acaaagtagc tcatttaata atgaggagct agcgatgtta 1200
aaagagaaat tattctcatt ttga 1224
<210> 13
<211> 1224
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 13
gtgtgtgatt taagaaaaac aaaactaata gataaaataa gttcactaga actatacaaa 60
tactcaatat tttttagaaa ttacattgaa aatgtagcag aagattgtct caagaacgga 120
cttattcttg agagtgctgc tcacaatgtt agtgaggttg aacttgctag gttaaaggta 180
cagcttaaga atgctctgct taattgtatt ataagctacc gttttcatgg gattggctat 240
gttttagtaa aaaccaaaga taccctaata gatctcgaac aacccgttaa tatagaatta 300
cctattggtt ttgaatacct tgattatgaa tatgtaagag atttgggagt tgattttgat 360
catataacct ataaagtaaa atccaacaat aagaacaatt ctttagacgc agttaaaata 420
cataaaagtc gacttatcat atatgaaaac tttgattata tcttaaaaag atatgttccg 480
tgttataccg aaagcttttt actagatatt tatttatttg aaaagatata cgtagaaata 540
gaaagacgta ttgaaaacca caattttttg ttttacaaag atgaatcttt agtacaacta 600
caagacgcac tttctagtgc aacaacttct ttaagtgcac ttactcagag caataatgat 660
aggggaagtg gcattttatc ttcttttttg agaaaacaaa attcaaacaa tcatagtaaa 720
gatatttcta atttaagaaa ccttaatgac tcattatcac aggagcttgc taggctaaaa 780
agcaatctaa ataatgaggg aatgttttat acggccaccc ctagtgctag tttagaggtt 840
attaaatacg atcttagcta cttaaaggag gctttagcat taattaaggc aaaaattggt 900
gcagatacta aagagccctt aaccagaagt tttaatgagc aggctaaagg gctaggaaat 960
gatggtaaag gtgataggag taattattac gattttctca aaggtgtaca agaacaagtt 1020
gagaactctt gtaatttaaa acttacaaag tattttgggc ttgatatgaa gtttaattcg 1080
ctgattatgt taagtgaaga acaaaaagta gagagagata taaagctaat tgagctttac 1140
agtaaatata accagcttat acaaagtagc tcatttaata atgaggagct agcgatgtta 1200
aaagagaaat tattctcatt ttga 1224
<210> 14
<211> 1224
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 14
gtgtgtgatt taagaaaaac aaaactaata gataaaataa gttcactaga actatacaaa 60
tactcaatat tttttagaaa ttacattgaa aatgtagcag aagattgtct caagaacgga 120
cttattcttg agagtgctgc ccacaatgtt agtgaggttg aacttgctag gttaaaggta 180
cagcttaaga atgctctgct taattgtatt ataagctacc gttttcatgg gattggctat 240
gttttagtaa aaaccaaaga taccctaata gatctcgaac aacccgttaa tatagaatta 300
cctattggtt ttgaatacct tgattatgaa tatgtaagag atttgggagt tgattttgat 360
catataacct ataaagtaaa atccaacaat aagaacaatt ctttagacgc agttaaaata 420
cataaaagtc gacttatcat atatgaaaac tttgattata tcttaaaaag atatgttccg 480
tgttataccg aaagcttttt actagatatt tatttatttg aaaagatata cgtagaaata 540
gaaagacgta ttgaaaacca caatttcttg ttttataaag atgaatcttt agtacaacta 600
caagacgcac tctctagtgc aacaacttct ttaagtgcac ttactcagag caataatgat 660
aggggaagtg gcattttatc ttcttttttg agaaaacaaa attcaaacaa tcatagtaaa 720
gatatttcta atttaagaaa ccttaatgac tcattatcac aggagcttgc taggctaaaa 780
agcaatctaa ataatgaggg aatgttttat acggccaccc ctagtgctag tttagaggtt 840
attaaatacg atcttagcta cttaaaggag gctttagcat taattaaggc aaaaattggt 900
gcagatacta aagagccctt aaccagaagt tttaatgagc aggctaaagg actaggaaat 960
gatggtaaag gtgataggag taattattac gattttctca aaggtgtaca agaacaagtt 1020
gagaactctt gtaatttaaa acttacaaag tattttgggc ttgatatgaa gtttaattct 1080
ctgattatgt taagtgaaga acaaaaagtg gaaagagata taaagctaat tgagctttac 1140
agtaaatata accagcttat acaaagtagc tcatttaata atgaggagct agcgatgtta 1200
aaagagaaat tattctcatt ttga 1224
<210> 15
<211> 1227
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 15
atgattaacg aatttaaaat ggaaataaaa aacaataaat ccaaaatcaa tcctattgat 60
gtttacagat attcaatttt ttttagaaac tacatagaaa gtaccgcaga agatgcactt 120
aagaatggaa ttaatttaag cctgcttgaa aagtcttgct tgaaagaaag cagcaagtct 180
cttttggggt taaaggttca gttaaaagaa gctcttcttg aagcaatgat aagctacaga 240
tttaatggag caggttatat tttggtaaaa cccaaaagtg aagatgaaga tttaagcaaa 300
aaggttaaca gcgaattacc cacaggtttt aaatatttgg attttcaaaa aattattaac 360
aaaagagatt ctagttacgt ggagtacctt tctaactcaa aagacccgga tgattttgag 420
agagcaagag ttgtaaaaat agataaaagc agagtaataa tttatgaaaa ttatgattat 480
gttttaggcg agcaagaacc tgcttacacg caaagtttgc tgcttaacat ttgtctttta 540
gaacaaattt atttagaaat agaaaaaaga ataagaaatt ataatttttt gttttacaaa 600
gacgaacatt tggtgggact tgttgaatct ttagagattg caaaagaaga aattaatgtt 660
ttggcaaaca gcaaaggcaa aatattttct acctttttta aagctcaaga gccaaacaaa 720
agttttcaag cattaagtag cgtctctgac gagcttagca gagagcttag caagattaaa 780
aacactctaa acaatgatgg aattttttac acagcatcag agaatgcacg cctagaagta 840
ataaaatatg atttagagtt tttaaaagac gcatttgaac ttgttaaagc aaaaattggc 900
gctgatacca aagagccttt aaccagaagc tttaatgagc aagtcaaagg gcttggcagc 960
agcggcaagg gagataaaag caattattat gattatttaa aaggcgttca agaatctgtt 1020
gctaatgcgt gcaatctcaa gcttaacaaa tattacagat taaatatgaa gtttaatgag 1080
cttgaagctt taagctatga gcaaagactt caaaaagaca atttgcttct tgatgtttat 1140
tttaaatatt tagagctaat taaaaatgaa aatctaagcg acaaggcaaa agaaaattta 1200
aaagaacaat taagtttggt tatttaa 1227
<210> 16
<211> 777
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 16
atgaacacta ataattattt taatttaaat aatttcaaca tggattttat gctcaaacta 60
tttcaagatt atcaaaatgt gctaaatgaa aataaaattc ttaaaaattc actaaaaatt 120
tcttctaagc ctactaaaaa agcttcaaaa ccaactccta agttttattt gaccccaaaa 180
gctatcaaaa taattgaaaa atgcgttaaa atattaaaaa aaattgaccc tatttctggt 240
tggtttctac atctactcgc aataagtggg tgtagggggg ccgaaattca aaaagtaaaa 300
atgcaagata ttactcctct attaaacaaa actggagaaa ctttttacaa tataaaagtt 360
aatatagcta aaaaaagaaa tgtcacttgt attagagaaa ttgtcatcaa atctgaagaa 420
tttgaggcta ttcaaaaagc tcacgaaaat tattttaacg aaaaaaatct tgactcacgg 480
cgtacttatc ttttccaaaa aaccaaacat aaatttaaag ataaccaaat tgatattatc 540
aatatttcta gaaaatttaa aaatcttctc aaaaaatcag gatttcgtgc caataaatct 600
ctccatttat ttagaaattt gtttatttca tatttaaaat ctaatggtta taattctttc 660
caaattaaag aacttatgaa atatcattca acttccgaaa ttgataatat ctacggcctc 720
tctgctgcta ataaaattca cgcttataaa tgcatgaaaa ataaccttaa actttag 777
<210> 17
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 17
atggatacta ttaaattaac cgaacttctt atcaatttaa acgaaattaa acttatagcg 60
gtaatgattt ttgtaacagt gctggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 18
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 18
atggatacta ttaaattaac cgaacttctt atcaatttaa acgaaattaa acttatagcg 60
gtaatgattt ttgtaacagt gctggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 19
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 19
atggatacta ttaaattaac agaacttctt atcaatttaa acgaaattaa acttatagcc 60
gtaatgattt ttgtaacagt gttggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 20
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 20
atggatacta ttaaattaac cgaacttctt atcaatttaa acgaaattaa acttatagcg 60
gtaatgattt ttgtaacagt gctggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 21
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 21
atggatacta ttaaattaac cgaacttctt atcaatttaa acgaaattaa acttatagcg 60
gtaatgattt ttgtaacagt gctggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 22
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 22
atggatacta ttaaattaac cgaacttctt atcaatttaa acgaaattaa acttatagcg 60
gtaatgattt ttgtaacagt gctggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 23
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 23
atggatacta ttaaattaac agaacttctt atcaatttaa atgaaattaa acttatagcc 60
gtaatgattt ttgtaacagt attggtttta ggagtgttaa ttcttctcaa gcctttatta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggcaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 24
<211> 204
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 24
atggatacta ttaaattaac agaacttctt atcaatttaa acgaaattaa acttatagcc 60
gtaatgattt ttgtaacagt gttggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaa 204
<210> 25
<211> 180
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 25
atggatacaa tacttatttt tttgtcaaca attgacaata caaaactaat tatcttagga 60
ggatttattg tgctggtaat aatgccgatg attttggcaa taaaaccaca gtttagagaa 120
aatttaattt tgcttattca aaaactctta aaaaatataa ataaaaagga aaaaaaatga 180
<210> 26
<211> 348
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 26
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgagctgt attctcatta taaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa taaagcactc gctccattaa atgaaaaaat caatcaatgc 240
attgacttag ttaattctga tgaaaaaaat ctcaaaatat ctaatgatct gaaattcaat 300
caggaaggaa aacctatcta taaggaaaga acaaataatg caaaataa 348
<210> 27
<211> 348
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 27
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgagctgt attctcatta taaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa taaagcactc gctccattaa atgaaaaaat caatcaatgc 240
attgacttag ttaattctga tgaaaaaaat ctcaaaatat ctaatgatct gaaattcaat 300
caggaaggaa aacctatcta taaggaaaga acaaataatg caaaataa 348
<210> 28
<211> 348
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 28
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgagctgt attctcatta taaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa taaagcactc gctccattaa atgaaaaaat caatcaatgc 240
attgacttag ttaattctga tgaaaaaaat ctcaaaatat ctaatgatct gaaattcaat 300
caggaaggaa aacctatcta taaggaaaga acaaataatg caaaataa 348
<210> 29
<211> 348
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 29
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgatctgt attctcatta caaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa tgcggcactc gctccattaa atgaaaaaat caatcaatgc 240
attgacttag ttaattctga tgaaaaaaat ctcaaaatat ctaatgatct gaaattcaat 300
caggaaggaa aacctatcta taaggaaaga ataaataatg caaaataa 348
<210> 30
<211> 357
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 30
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgagctgt attctcatta taaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa tgcggcactc gctcctttaa atgcaaaaat caatacggtt 240
attgatttgg ttaattctaa tgataaaaat ctaaaaatat ccaatgatct aaaattcaat 300
aaagacggaa aacctatcta caaagagaga gcaaataatg caaaagaaca ctattga 357
<210> 31
<211> 348
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 31
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgagctgt attctcatta taaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa tgcggcactc gctccattaa atgaaaaaat caatcaatgc 240
attgacttag ttaattctga tgaaaaaaat ctcaaaatat ctaatgatct gaaattcaat 300
caggaaggaa aacctatcta taaggaaaga acaaataatg caaaataa 348
<210> 32
<211> 348
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 32
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgagctgt attctcatta taaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa tgcggcactc gctccattaa atgaaaaaat caatcaatgc 240
attgacttag ttaattctga tgaaaaaaat ctcaaaatat ctaatgatct gaaattcaat 300
caggaaggaa aacctatcta taaggaaaga acaaataatg caaaataa 348
<210> 33
<211> 348
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 33
atgaaattat ccaaagataa tgttgagctt ggacttacgt ctttatcaac ccttattgat 60
atattttcta aatttgaaga tgaatttgat gaaattgcac ataaaggatt ctttttggtt 120
tatgagctgt attctcatta taaattaatc tatacagcaa atatggaaag acttgagagt 180
gcattaaccc cagcaataaa taaagcacta gctccattaa atgaaaaaat caatcaatgc 240
attgacttag ttaattctga tgaaaaaaat ctcaaaatat ctaatgatct gaaattcaat 300
caggaaggaa aacctatcta taaggaaaga acaaataatg caaaataa 348
<210> 34
<211> 426
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 34
atgaacaata aaatgaatat caactctggc attgacgctt taaataattt atatgatttt 60
ctaaaaagta gcgattctcc aacagaagtc aaagtagaaa agggtatata tttggggctt 120
aatctttaca atctaataat gagcatctac aaagacaaaa tcacaaccct tgagaaagaa 180
gaatcattaa aaatattaaa tgaaattaaa aatgttaata ataagattac tcaacttata 240
tcaagcatta atgatgagag agatgcaagc atcattgaac atttaagaga agagagaaat 300
gaactaatgt caatcaaaac tcaagcactc caagaacaaa taaaagagtt tccagaaaat 360
acaagcactg ctaacagcaa agcccaaaac ttaaaagaaa ataaaggaga taaaattgca 420
aattaa 426
<210> 35
<211> 147
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 35
gagtggatag caagcactga ttcaattttt acacaaataa atattactga tgattatgta 60
tttactagcc cgatagcata tttagaccca gcatttagtg ttggcgggga taacactgca 120
ttatgtgtta tggagcgagt tgatgat 147
<210> 36
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 36
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Thr Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Val Lys Leu Thr Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Glu Glu Gln Leu Thr Leu Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Lys Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ser Leu Ile Glu Ile Lys Lys Leu Tyr Ser Ser Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Thr Tyr Lys Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Val Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asp Asp Tyr Val Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Ile Glu Asn Phe Asn Val
325 330 335
His Thr Leu Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Ile Leu Leu Arg Ser Asn Ile Ser Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Thr Leu Ile Thr Pro Phe Thr Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Asn
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Ile Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 37
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 37
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Thr Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Val Lys Leu Thr Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Glu Lys Gln Leu Thr Leu Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Lys Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Phe Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ser Leu Ile Glu Asn Lys Lys Leu Tyr Ser Ser Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Ile Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asp Asp Tyr Val Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Ile Glu Asn Phe Asn Val
325 330 335
His Thr Leu Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Ile Leu Leu Arg Asn Asn Ile Ser Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Thr Leu Ile Thr Pro Phe Thr Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Ser
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Met Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 38
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 38
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Thr Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Val Lys Leu Thr Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Lys Glu Gln Leu Thr Ile Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Gln Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ser Leu Ile Glu Asn Lys Lys Leu Tyr Ser Gly Asp
85 90 95
Ile Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Ile Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asn Asp Tyr Val Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Ile Glu Asn Phe Asn Val
325 330 335
His Thr Leu Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Ile Leu Leu Arg Asn Asn Ile Ser Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Thr Val Ile Thr Pro Phe Thr Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Ser
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Met Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 39
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 39
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Thr Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Val Lys Leu Thr Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Glu Lys Gln Leu Thr Leu Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Lys Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ser Leu Ile Glu Asn Lys Lys Leu Tyr Ser Ser Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Val Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asp Asp Tyr Val Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Ile Glu Asn Phe Asn Val
325 330 335
His Thr Leu Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Ile Leu Leu Arg Ser Asn Ile Ser Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Thr Leu Ile Thr Pro Phe Thr Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Ser
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Met Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 40
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 40
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Thr Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Ile Lys Leu Thr Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Glu Glu Gln Leu Thr Leu Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Lys Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ser Leu Ile Glu Asn Lys Lys Leu Tyr Ser Ser Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Ile Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asn Asp Tyr Val Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Leu Glu Asn Phe Asn Val
325 330 335
His Thr Leu Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Met Leu Leu Arg Asn Asn Met Gly Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Thr Leu Ile Thr Pro Phe Thr Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Asn
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Ile Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 41
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 41
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Thr Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Val Lys Leu Thr Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Glu Glu Gln Leu Thr Leu Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Lys Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ser Leu Ile Glu Asn Lys Lys Leu Tyr Ser Ser Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Thr Tyr Lys Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Val Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asp Asp Tyr Val Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Ile Glu Asn Phe Asn Val
325 330 335
His Thr Leu Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Ile Leu Leu Arg Ser Asn Ile Ser Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Thr Leu Ile Thr Pro Phe Thr Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Asn
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Ile Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 42
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 42
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Thr Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Val Lys Leu Ala Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Glu Lys Gln Leu Thr Leu Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Lys Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ser Leu Ile Glu Asn Lys Lys Leu Tyr Ser Ser Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Ile Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Ile Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asp Asp Tyr Ile Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Ile Glu Asn Phe Asn Val
325 330 335
His Thr Leu Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Ile Leu Leu Arg Asn Asn Ile Ser Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Met Leu Ile Thr Pro Phe Thr Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Ser
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Ile Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 43
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 43
Met Asn Leu Tyr Gln Thr Lys Leu Phe Thr Ile Leu Gln Lys Glu Tyr
1 5 10 15
Lys Asn Lys Tyr Gly Val Asp Ile Ser Gln Phe Val Lys Leu Thr Asn
20 25 30
Ser Ser Ile Asn Phe Asp Lys Phe Glu Glu Glu Gln Leu Thr Leu Lys
35 40 45
Gln Lys Asn Val Ile Lys Ser Ile Lys Lys Asn Asn Glu Lys Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Tyr Leu Ala Cys Tyr
65 70 75 80
Leu Phe Ile Lys Ser Leu Ile Glu Asn Lys Lys Leu Tyr Ser Ser Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Gln Arg Ser Val Glu Val Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Leu Cys Lys Leu Leu Lys Ile Pro Tyr
115 120 125
Ile Pro Arg His Thr Asn Asn Ser Tyr Ile Leu Ile Asp Ser Leu Arg
130 135 140
Ile Asn Leu His Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Phe Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Gln Thr Leu Glu Glu Val Leu Lys Arg Leu Arg Cys Gly Gln Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Ile Ala Thr Phe Lys Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Val Leu Leu Ser Lys Gly Phe Ile Glu Thr Gln Glu
225 230 235 240
Lys Leu Tyr Lys Asp Ile Pro Ser Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Ile Ala Ser Thr Asp Ser Ile Phe Thr Gln Ile Asn Ile Thr
260 265 270
Asn Asp Tyr Val Phe Thr Ser Pro Ile Ala Tyr Leu Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Leu Cys Val Met Glu Arg Val Asp
290 295 300
Asp Lys Tyr Tyr Ala Phe Val Phe Gln Asp Gln Arg Pro Ala Asn Asp
305 310 315 320
Pro Tyr Ile Met Asn Met Val Lys Thr Val Leu Glu Asn Phe Asn Val
325 330 335
His Thr Phe Tyr Leu Glu Asp Arg Asp Asn Thr Lys Gly Ala Gly Gly
340 345 350
Leu Thr Arg Glu Tyr Ile Leu Leu Arg Asn Asn Met Gly Gln Tyr Phe
355 360 365
Arg Ile Val Pro Val Lys Pro Lys Ser Asn Lys Phe Ser Arg Ile Thr
370 375 380
Ala Leu Ile Thr Pro Phe Ile Tyr Lys Lys Leu Tyr Ile Thr Lys Tyr
385 390 395 400
Ser Ser Ser Ser Val Phe Asn Asp Ile Tyr Ser Tyr Lys Gly Asp Asn
405 410 415
Lys Thr His Asp Asp Ala Leu Asp Ala Ile Ser Ala Ala Tyr Leu Met
420 425 430
Leu Ser Leu Gly Tyr Arg Glu Arg Ser Val His Phe Gly Asn Gln Arg
435 440 445
Phe Leu
450
<210> 44
<211> 450
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 44
Met Lys Phe Leu Arg Ser Leu Ala Phe Leu Asn Leu Gln Lys Lys Phe
1 5 10 15
Lys Asn Lys Phe Asn Ile Asn Ile Leu Asp Tyr Ile Lys Pro Lys Pro
20 25 30
Thr Lys Ile Cys Phe Lys Asp Phe Glu Asn Lys Tyr Leu Thr Ala Lys
35 40 45
Gln Lys Glu Val Leu Phe Asp Ile Glu Ser Asn Asn Tyr Ser Lys Val
50 55 60
Ile Phe Ser Gly Gly Ile Ala Ser Gly Lys Thr Phe Leu Ala Ser Tyr
65 70 75 80
Leu Leu Val Lys Lys Leu Ile Glu Asn Lys Ser Phe Tyr Glu Gln Asp
85 90 95
Thr Asn Asn Phe Ile Ile Gly Asn Ser Ile Gly Leu Leu Met Thr Asn
100 105 110
Thr Val Lys Gln Ile Glu Lys Ile Cys Ser Leu Leu Gly Ile Asp Tyr
115 120 125
Glu Lys Lys Lys Ser Gly Gln Ser Phe Cys Lys Ile Ala Gly Leu Lys
130 135 140
Leu Asn Ile Tyr Gly Gly Lys Asn Arg Asp Ala Phe Ser Lys Ile Arg
145 150 155 160
Gly Gly Asn Ser Ala Ile Ile Tyr Val Asn Glu Ala Thr Val Ile His
165 170 175
Arg Glu Thr Leu Leu Glu Val Ile Lys Arg Leu Arg Lys Gly Lys Glu
180 185 190
Ile Ile Ile Phe Asp Thr Asn Pro Glu Ser Pro Ala His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Glu Asn Thr Asp Val Phe Lys Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Pro Leu Asn Ser Ala Asp Phe Ile Gln Thr Gln Glu
225 230 235 240
Lys Leu Tyr Arg Arg Phe Pro Ala Tyr Arg Ala Arg Val Leu Tyr Gly
245 250 255
Glu Trp Ile Leu Asn Glu Ser Thr Leu Phe Asn Glu Met Ile Phe Asn
260 265 270
Gln Asp Tyr Glu Phe Lys Ser Pro Ile Met Tyr Ile Asp Pro Ala Phe
275 280 285
Ser Val Gly Gly Asp Asn Thr Ala Ile Cys Val Leu Glu Arg Thr Phe
290 295 300
Glu Lys Phe Tyr Ala Tyr Ile Tyr Gln Asp Gln Lys Pro Val Ser Asp
305 310 315 320
Ser Leu Met Leu Ala Ser Ile Gln Val Leu Ile Glu Asn Phe Asn Val
325 330 335
Asn Thr Val Tyr Ile Glu Glu Arg Asp Ser Thr Lys Gly Asp Gly Ile
340 345 350
Leu Thr Lys Thr Ile Leu Phe Leu Arg Asn Lys Ser Ser His Tyr Phe
355 360 365
Lys Val Ala Pro Ile Lys Pro Leu Ser Asn Lys Phe Lys Arg Ile Cys
370 375 380
Ala Leu Ile Pro Leu Phe Glu Ser Arg Lys Ile Glu Phe Leu Lys Ile
385 390 395 400
Ile Ser Lys Asn Val Ile Ser Asp Ile Tyr Ser Tyr Lys Gly Asp Gly
405 410 415
Lys Thr Lys Asp Asp Ala Leu Asp Ser Leu Ala Asn Ala Tyr Leu Leu
420 425 430
Leu Thr Leu Asn Tyr Lys Glu Lys Leu Phe His Phe Gly Arg Phe Lys
435 440 445
Tyr Leu
450
<210> 45
<211> 396
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 45
Met Arg Leu Arg Arg Leu Pro Val Tyr Val Asp Ala Tyr Lys Glu Lys
1 5 10 15
Pro Asn Ala Glu Ile Phe Ile Tyr Tyr Ser Ser Arg Gly Thr Gly Lys
20 25 30
Thr Tyr Asp Ile Ala Thr Val Asn Leu Glu Arg Lys Phe Ser Ala Asp
35 40 45
Gly Gly Asp Thr Leu Ala Ile Arg Lys Lys Lys Asn Lys Thr Thr Gln
50 55 60
Ser Ile His Lys Glu Ile Leu Glu Leu Leu Ser Ile Tyr Asn Leu Arg
65 70 75 80
Lys Phe Phe Asn Ile Ser Lys Ala Lys Ile Glu Ser Lys Ser Leu Ile
85 90 95
Phe Gly Lys Lys Arg Ala Phe Val Phe Glu Gly Gly His Asp Thr Arg
100 105 110
Asp Leu Lys Ser Tyr Ala His Phe Lys Asp Leu Trp Leu Glu Glu Ala
115 120 125
Asn Gln Phe Ser Ala Asp Asp Ile Glu Met Leu Val Pro Thr Met Arg
130 135 140
Glu Gln Gly Gly Arg Ile Tyr Met Ser Ser Asn Pro Val Pro Lys Ser
145 150 155 160
His Trp Leu Tyr Lys Arg Tyr Leu Ser Asn Gln Asp Asn Pro Ala Val
165 170 175
Cys Ile Ile Lys Ser Thr Tyr Arg Asp Asn Pro Phe Leu Asn Gly Gly
180 185 190
Asp Val Gln Ala Trp Leu Glu Lys Gln Arg Leu Ala Tyr His Gly Asn
195 200 205
Asp Ile Gly Phe Arg Ile Glu Val Leu Gly Glu Glu Phe Asp Phe Gly
210 215 220
Thr Ala Arg Leu Ile Lys Lys Phe Asn Val Cys Gly Pro Glu Ile Leu
225 230 235 240
Ser Arg Ala Asn Gly Ser Tyr Tyr Thr Gly Ile His Val Lys Gly Asn
245 250 255
Arg Ile Cys Phe Leu Glu Ile Leu Val Gly Arg Ile Ser Tyr Leu Pro
260 265 270
Val Val Ile Ile Thr Asn Ala Cys Ser Lys Val Leu Leu Ser Lys Thr
275 280 285
Asp Tyr Gln Ser Glu Ile Asn Lys Phe Lys Gly Val Phe Val Leu Pro
290 295 300
Thr Ala Arg Glu Glu Leu Lys Tyr Val Phe Ser Arg Phe Gly Arg Gly
305 310 315 320
Thr Leu Leu Ala Lys Lys Arg Asn Leu Tyr Ser Leu Ser Asp Tyr Leu
325 330 335
Ile Pro Ser Asn Leu Asn Val Val Asn Lys Pro Glu Thr Thr Asp Val
340 345 350
Ile Ser Glu Phe Asn Glu Thr Glu Tyr Tyr Tyr Asp Glu Ser Ser Ala
355 360 365
Glu Asp Ser Glu Val Thr Asn Phe Val Met Gln Lys Asp Leu Val Tyr
370 375 380
Ile Pro Ala Phe Leu Asn Ala Ile Ser Val Phe Ser
385 390 395
<210> 46
<211> 407
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 46
Val Cys Asp Leu Arg Lys Thr Lys Leu Ile Asp Lys Ile Ser Ser Leu
1 5 10 15
Glu Leu Tyr Lys Tyr Ser Ile Phe Phe Arg Asn Tyr Ile Glu Asn Val
20 25 30
Ala Glu Asp Cys Leu Lys Asn Gly Leu Ile Leu Glu Ser Ala Ala His
35 40 45
Asn Val Ser Glu Val Glu Leu Ala Arg Leu Lys Val Gln Leu Lys Asn
50 55 60
Ala Leu Leu Asn Cys Ile Ile Ser Tyr Arg Phe His Gly Ile Gly Tyr
65 70 75 80
Val Leu Val Lys Thr Lys Asp Thr Leu Ile Asp Leu Glu Gln Pro Val
85 90 95
Asn Ile Glu Leu Pro Ile Gly Phe Glu Tyr Leu Asp Tyr Glu Tyr Val
100 105 110
Arg Asp Leu Gly Val Asp Phe Asp His Ile Thr Tyr Lys Val Lys Ser
115 120 125
Asn Asn Lys Asn Asn Ser Leu Asp Ala Val Lys Ile His Lys Ser Arg
130 135 140
Leu Ile Ile Tyr Glu Asn Phe Asp Tyr Ile Leu Lys Arg Tyr Val Pro
145 150 155 160
Cys Tyr Thr Glu Ser Phe Leu Leu Asp Ile Tyr Leu Phe Glu Lys Ile
165 170 175
Tyr Val Glu Ile Glu Arg Arg Ile Glu Asn His Asn Phe Leu Phe Tyr
180 185 190
Lys Asp Glu Ser Leu Val Gln Leu Gln Asp Ala Leu Ser Ser Ala Thr
195 200 205
Thr Ser Leu Ser Ala Leu Thr Gln Ser Asn Asn Asp Arg Gly Ser Gly
210 215 220
Ile Leu Ser Ser Phe Leu Arg Lys Gln Asn Ser Asn Asn His Ser Lys
225 230 235 240
Asp Ile Ser Asn Leu Arg Asn Leu Asn Asp Ser Leu Ser Gln Glu Leu
245 250 255
Ala Arg Leu Lys Ser Asn Leu Asn Asn Glu Gly Met Phe Tyr Thr Ala
260 265 270
Thr Pro Ser Ala Ser Leu Glu Val Ile Lys Tyr Asp Leu Ser Tyr Leu
275 280 285
Lys Glu Ala Leu Ala Leu Ile Lys Ala Lys Ile Gly Ala Asp Thr Lys
290 295 300
Glu Pro Leu Thr Arg Ser Phe Asn Glu Gln Ala Lys Gly Leu Gly Asn
305 310 315 320
Asp Gly Lys Gly Asp Arg Ser Asn Tyr Tyr Asp Phe Leu Lys Gly Val
325 330 335
Gln Glu Gln Val Glu Asn Ser Cys Asn Leu Lys Leu Thr Lys Tyr Phe
340 345 350
Gly Leu Asp Met Lys Phe Asn Ser Leu Ile Met Leu Ser Glu Glu Gln
355 360 365
Lys Val Glu Arg Asp Ile Lys Leu Ile Glu Leu Tyr Ser Lys Tyr Asn
370 375 380
Gln Leu Ile Gln Ser Ser Ser Phe Asn Asn Glu Glu Leu Ala Met Leu
385 390 395 400
Lys Glu Lys Leu Phe Ser Phe
405
<210> 47
<211> 407
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 47
Val Cys Asp Leu Arg Lys Thr Lys Leu Ile Asp Lys Ile Ser Ser Leu
1 5 10 15
Glu Leu Tyr Lys Tyr Ser Ile Phe Phe Arg Asn Tyr Ile Glu Asn Val
20 25 30
Ala Glu Asp Cys Leu Lys Asn Gly Leu Ile Leu Glu Ser Ala Ala His
35 40 45
Asn Val Ser Glu Val Glu Leu Ala Arg Leu Lys Val Gln Leu Lys Asn
50 55 60
Ala Leu Leu Asn Cys Ile Ile Ser Tyr Arg Phe His Gly Ile Gly Tyr
65 70 75 80
Val Leu Val Lys Thr Lys Asp Thr Leu Ile Asp Leu Glu Gln Pro Val
85 90 95
Asn Ile Glu Leu Pro Ile Gly Phe Glu Tyr Leu Asp Tyr Glu Tyr Val
100 105 110
Arg Asp Leu Gly Val Asp Phe Asp His Ile Thr Tyr Lys Val Lys Ser
115 120 125
Asn Asn Lys Asn Asn Ser Leu Asp Ala Val Lys Ile His Lys Ser Arg
130 135 140
Leu Ile Ile Tyr Glu Asn Phe Asp Tyr Ile Leu Lys Arg Tyr Val Pro
145 150 155 160
Cys Tyr Thr Glu Ser Phe Leu Leu Asp Ile Tyr Leu Phe Glu Lys Ile
165 170 175
Tyr Val Glu Ile Glu Arg Arg Ile Glu Asn His Asn Phe Leu Phe Tyr
180 185 190
Lys Asp Glu Ser Leu Val Gln Leu Gln Asp Ala Leu Ser Ser Ala Thr
195 200 205
Thr Ser Leu Ser Ala Leu Thr Gln Ser Asn Asn Asp Arg Gly Ser Gly
210 215 220
Ile Leu Ser Ser Phe Leu Arg Lys Gln Asn Ser Asn Asn His Ser Lys
225 230 235 240
Asp Ile Ser Asn Leu Arg Asn Leu Asn Asp Ser Leu Ser Gln Glu Leu
245 250 255
Ala Arg Leu Lys Ser Asn Leu Asn Asn Glu Gly Met Phe Tyr Thr Ala
260 265 270
Thr Pro Ser Ala Ser Leu Glu Val Ile Lys Tyr Asp Leu Ser Tyr Leu
275 280 285
Lys Glu Ala Leu Ala Leu Ile Lys Ala Lys Ile Gly Ala Asp Thr Lys
290 295 300
Glu Pro Leu Thr Arg Ser Phe Asn Glu Gln Ala Lys Gly Leu Gly Asn
305 310 315 320
Asp Gly Lys Gly Asp Arg Ser Asn Tyr Tyr Asp Phe Leu Lys Gly Val
325 330 335
Gln Glu Gln Val Glu Asn Ser Cys Asn Leu Lys Leu Thr Lys Tyr Phe
340 345 350
Gly Leu Asp Met Lys Phe Asn Ser Leu Ile Met Leu Ser Glu Glu Gln
355 360 365
Lys Val Glu Arg Asp Ile Lys Leu Ile Glu Leu Tyr Ser Lys Tyr Asn
370 375 380
Gln Leu Ile Gln Ser Ser Ser Phe Asn Asn Glu Glu Leu Ala Met Leu
385 390 395 400
Lys Glu Lys Leu Phe Ser Phe
405
<210> 48
<211> 407
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 48
Val Cys Asp Leu Arg Lys Thr Lys Leu Ile Asp Lys Ile Ser Ser Leu
1 5 10 15
Glu Leu Tyr Lys Tyr Ser Ile Phe Phe Arg Asn Tyr Ile Glu Asn Val
20 25 30
Ala Glu Asp Cys Leu Lys Asn Gly Leu Ile Leu Glu Ser Ala Ala His
35 40 45
Asn Val Ser Glu Val Glu Leu Ala Arg Leu Lys Val Gln Leu Lys Asn
50 55 60
Ala Leu Leu Asn Cys Ile Ile Ser Tyr Arg Phe His Gly Ile Gly Tyr
65 70 75 80
Val Leu Val Lys Thr Lys Asp Thr Leu Ile Asp Leu Glu Gln Pro Val
85 90 95
Asn Ile Glu Leu Pro Ile Gly Phe Glu Tyr Leu Asp Tyr Glu Tyr Val
100 105 110
Arg Asp Leu Gly Val Asp Phe Asp His Ile Thr Tyr Lys Val Lys Ser
115 120 125
Asn Asn Lys Asn Asn Ser Leu Asp Ala Val Lys Ile His Lys Ser Arg
130 135 140
Leu Ile Ile Tyr Glu Asn Phe Asp Tyr Ile Leu Lys Arg Tyr Val Pro
145 150 155 160
Cys Tyr Thr Glu Ser Phe Leu Leu Asp Ile Tyr Leu Phe Glu Lys Ile
165 170 175
Tyr Val Glu Ile Glu Arg Arg Ile Glu Asn His Asn Phe Leu Phe Tyr
180 185 190
Lys Asp Glu Ser Leu Val Gln Leu Gln Asp Ala Leu Ser Ser Ala Thr
195 200 205
Thr Ser Leu Ser Ala Leu Thr Gln Ser Asn Asn Asp Arg Gly Ser Gly
210 215 220
Ile Leu Ser Ser Phe Leu Arg Lys Gln Asn Ser Asn Asn His Ser Lys
225 230 235 240
Asp Ile Ser Asn Leu Arg Asn Leu Asn Asp Ser Leu Ser Gln Glu Leu
245 250 255
Ala Arg Leu Lys Ser Asn Leu Asn Asn Glu Gly Met Phe Tyr Thr Ala
260 265 270
Thr Pro Ser Ala Ser Leu Glu Val Ile Lys Tyr Asp Leu Ser Tyr Leu
275 280 285
Lys Glu Ala Leu Ala Leu Ile Lys Ala Lys Ile Gly Ala Asp Thr Lys
290 295 300
Glu Pro Leu Thr Arg Ser Phe Asn Glu Gln Ala Lys Gly Leu Gly Asn
305 310 315 320
Asp Gly Lys Gly Asp Arg Ser Asn Tyr Tyr Asp Phe Leu Lys Gly Val
325 330 335
Gln Glu Gln Val Glu Asn Ser Cys Asn Leu Lys Leu Thr Lys Tyr Phe
340 345 350
Gly Leu Asp Met Lys Phe Asn Ser Leu Ile Met Leu Ser Glu Glu Gln
355 360 365
Lys Val Glu Arg Asp Ile Lys Leu Ile Glu Leu Tyr Ser Lys Tyr Asn
370 375 380
Gln Leu Ile Gln Ser Ser Ser Phe Asn Asn Glu Glu Leu Ala Met Leu
385 390 395 400
Lys Glu Lys Leu Phe Ser Phe
405
<210> 49
<211> 407
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 49
Val Cys Asp Leu Arg Lys Thr Lys Leu Ile Asp Lys Ile Ser Ser Leu
1 5 10 15
Glu Leu Tyr Lys Tyr Ser Ile Phe Phe Arg Asn Tyr Ile Glu Asn Val
20 25 30
Ala Glu Asp Cys Leu Lys Asn Gly Leu Ile Leu Glu Ser Ala Ala His
35 40 45
Asn Val Ser Glu Val Glu Leu Ala Arg Leu Lys Val Gln Leu Lys Asn
50 55 60
Ala Leu Leu Asn Cys Ile Ile Ser Tyr Arg Phe His Gly Ile Gly Tyr
65 70 75 80
Val Leu Val Lys Thr Lys Asp Thr Leu Ile Asp Leu Glu Gln Pro Val
85 90 95
Asn Ile Glu Leu Pro Ile Gly Phe Glu Tyr Leu Asp Tyr Glu Tyr Val
100 105 110
Arg Asp Leu Gly Val Asp Phe Asp His Ile Thr Tyr Lys Val Lys Ser
115 120 125
Asn Asn Lys Asn Asn Ser Leu Asp Ala Val Lys Ile His Lys Ser Arg
130 135 140
Leu Ile Ile Tyr Glu Asn Phe Asp Tyr Ile Leu Lys Arg Tyr Val Pro
145 150 155 160
Cys Tyr Thr Glu Ser Phe Leu Leu Asp Ile Tyr Leu Phe Glu Lys Ile
165 170 175
Tyr Val Glu Ile Glu Arg Arg Ile Glu Asn His Asn Phe Leu Phe Tyr
180 185 190
Lys Asp Glu Ser Leu Val Gln Leu Gln Asp Ala Leu Ser Ser Ala Thr
195 200 205
Thr Ser Leu Ser Ala Leu Thr Gln Ser Asn Asn Asp Arg Gly Ser Gly
210 215 220
Ile Leu Ser Ser Phe Leu Arg Lys Gln Asn Ser Asn Asn His Ser Lys
225 230 235 240
Asp Ile Ser Asn Leu Arg Asn Leu Asn Asp Ser Leu Ser Gln Glu Leu
245 250 255
Ala Arg Leu Lys Ser Asn Leu Asn Asn Glu Gly Met Phe Tyr Thr Ala
260 265 270
Thr Pro Ser Ala Ser Leu Glu Val Ile Lys Tyr Asp Leu Ser Tyr Leu
275 280 285
Lys Glu Ala Leu Ala Leu Ile Lys Ala Lys Ile Gly Ala Asp Thr Lys
290 295 300
Glu Pro Leu Thr Arg Ser Phe Asn Glu Gln Ala Lys Gly Leu Gly Asn
305 310 315 320
Asp Gly Lys Gly Asp Arg Ser Asn Tyr Tyr Asp Phe Leu Lys Gly Val
325 330 335
Gln Glu Gln Val Glu Asn Ser Cys Asn Leu Lys Leu Thr Lys Tyr Phe
340 345 350
Gly Leu Asp Met Lys Phe Asn Ser Leu Ile Met Leu Ser Glu Glu Gln
355 360 365
Lys Val Glu Arg Asp Ile Lys Leu Ile Glu Leu Tyr Ser Lys Tyr Asn
370 375 380
Gln Leu Ile Gln Ser Ser Ser Phe Asn Asn Glu Glu Leu Ala Met Leu
385 390 395 400
Lys Glu Lys Leu Phe Ser Phe
405
<210> 50
<211> 408
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 50
Met Ile Asn Glu Phe Lys Met Glu Ile Lys Asn Asn Lys Ser Lys Ile
1 5 10 15
Asn Pro Ile Asp Val Tyr Arg Tyr Ser Ile Phe Phe Arg Asn Tyr Ile
20 25 30
Glu Ser Thr Ala Glu Asp Ala Leu Lys Asn Gly Ile Asn Leu Ser Leu
35 40 45
Leu Glu Lys Ser Cys Leu Lys Glu Ser Ser Lys Ser Leu Leu Gly Leu
50 55 60
Lys Val Gln Leu Lys Glu Ala Leu Leu Glu Ala Met Ile Ser Tyr Arg
65 70 75 80
Phe Asn Gly Ala Gly Tyr Ile Leu Val Lys Pro Lys Ser Glu Asp Glu
85 90 95
Asp Leu Ser Lys Lys Val Asn Ser Glu Leu Pro Thr Gly Phe Lys Tyr
100 105 110
Leu Asp Phe Gln Lys Ile Ile Asn Lys Arg Asp Ser Ser Tyr Val Glu
115 120 125
Tyr Leu Ser Asn Ser Lys Asp Pro Asp Asp Phe Glu Arg Ala Arg Val
130 135 140
Val Lys Ile Asp Lys Ser Arg Val Ile Ile Tyr Glu Asn Tyr Asp Tyr
145 150 155 160
Val Leu Gly Glu Gln Glu Pro Ala Tyr Thr Gln Ser Leu Leu Leu Asn
165 170 175
Ile Cys Leu Leu Glu Gln Ile Tyr Leu Glu Ile Glu Lys Arg Ile Arg
180 185 190
Asn Tyr Asn Phe Leu Phe Tyr Lys Asp Glu His Leu Val Gly Leu Val
195 200 205
Glu Ser Leu Glu Ile Ala Lys Glu Glu Ile Asn Val Leu Ala Asn Ser
210 215 220
Lys Gly Lys Ile Phe Ser Thr Phe Phe Lys Ala Gln Glu Pro Asn Lys
225 230 235 240
Ser Phe Gln Ala Leu Ser Ser Val Ser Asp Glu Leu Ser Arg Glu Leu
245 250 255
Ser Lys Ile Lys Asn Thr Leu Asn Asn Asp Gly Ile Phe Tyr Thr Ala
260 265 270
Ser Glu Asn Ala Arg Leu Glu Val Ile Lys Tyr Asp Leu Glu Phe Leu
275 280 285
Lys Asp Ala Phe Glu Leu Val Lys Ala Lys Ile Gly Ala Asp Thr Lys
290 295 300
Glu Pro Leu Thr Arg Ser Phe Asn Glu Gln Val Lys Gly Leu Gly Ser
305 310 315 320
Ser Gly Lys Gly Asp Lys Ser Asn Tyr Tyr Asp Tyr Leu Lys Gly Val
325 330 335
Gln Glu Ser Val Ala Asn Ala Cys Asn Leu Lys Leu Asn Lys Tyr Tyr
340 345 350
Arg Leu Asn Met Lys Phe Asn Glu Leu Glu Ala Leu Ser Tyr Glu Gln
355 360 365
Arg Leu Gln Lys Asp Asn Leu Leu Leu Asp Val Tyr Phe Lys Tyr Leu
370 375 380
Glu Leu Ile Lys Asn Glu Asn Leu Ser Asp Lys Ala Lys Glu Asn Leu
385 390 395 400
Lys Glu Gln Leu Ser Leu Val Ile
405
<210> 51
<211> 258
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 51
Met Asn Thr Asn Asn Tyr Phe Asn Leu Asn Asn Phe Asn Met Asp Phe
1 5 10 15
Met Leu Lys Leu Phe Gln Asp Tyr Gln Asn Val Leu Asn Glu Asn Lys
20 25 30
Ile Leu Lys Asn Ser Leu Lys Ile Ser Ser Lys Pro Thr Lys Lys Ala
35 40 45
Ser Lys Pro Thr Pro Lys Phe Tyr Leu Thr Pro Lys Ala Ile Lys Ile
50 55 60
Ile Glu Lys Cys Val Lys Ile Leu Lys Lys Ile Asp Pro Ile Ser Gly
65 70 75 80
Trp Phe Leu His Leu Leu Ala Ile Ser Gly Cys Arg Gly Ala Glu Ile
85 90 95
Gln Lys Val Lys Met Gln Asp Ile Thr Pro Leu Leu Asn Lys Thr Gly
100 105 110
Glu Thr Phe Tyr Asn Ile Lys Val Asn Ile Ala Lys Lys Arg Asn Val
115 120 125
Thr Cys Ile Arg Glu Ile Val Ile Lys Ser Glu Glu Phe Glu Ala Ile
130 135 140
Gln Lys Ala His Glu Asn Tyr Phe Asn Glu Lys Asn Leu Asp Ser Arg
145 150 155 160
Arg Thr Tyr Leu Phe Gln Lys Thr Lys His Lys Phe Lys Asp Asn Gln
165 170 175
Ile Asp Ile Ile Asn Ile Ser Arg Lys Phe Lys Asn Leu Leu Lys Lys
180 185 190
Ser Gly Phe Arg Ala Asn Lys Ser Leu His Leu Phe Arg Asn Leu Phe
195 200 205
Ile Ser Tyr Leu Lys Ser Asn Gly Tyr Asn Ser Phe Gln Ile Lys Glu
210 215 220
Leu Met Lys Tyr His Ser Thr Ser Glu Ile Asp Asn Ile Tyr Gly Leu
225 230 235 240
Ser Ala Ala Asn Lys Ile His Ala Tyr Lys Cys Met Lys Asn Asn Leu
245 250 255
Lys Leu
<210> 52
<211> 67
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 52
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg Asp
65
<210> 53
<211> 66
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 53
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg
65
<210> 54
<211> 66
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 54
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg
65
<210> 55
<211> 66
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 55
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg
65
<210> 56
<211> 67
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 56
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg Asp
65
<210> 57
<211> 67
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 57
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg Asp
65
<210> 58
<211> 67
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 58
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg Asp
65
<210> 59
<211> 67
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 59
Met Asp Thr Ile Lys Leu Thr Glu Leu Leu Ile Asn Leu Asn Glu Ile
1 5 10 15
Lys Leu Ile Ala Val Met Ile Phe Val Thr Val Leu Val Leu Gly Val
20 25 30
Leu Ile Leu Leu Lys Pro Leu Leu Lys Asp Ile Leu Thr Ile Val Ile
35 40 45
Gly Lys Ile Phe Lys Asn Gly Asn Gly Asn Gly Lys Asn His Ile Lys
50 55 60
Lys Arg Asp
65
<210> 60
<211> 59
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 60
Met Asp Thr Ile Leu Ile Phe Leu Ser Thr Ile Asp Asn Thr Lys Leu
1 5 10 15
Ile Ile Leu Gly Gly Phe Ile Val Leu Val Ile Met Pro Met Ile Leu
20 25 30
Ala Ile Lys Pro Gln Phe Arg Glu Asn Leu Ile Leu Leu Ile Gln Lys
35 40 45
Leu Leu Lys Asn Ile Asn Lys Lys Glu Lys Lys
50 55
<210> 61
<211> 115
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 61
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Glu Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Lys Ala Leu Ala Pro Leu Asn Glu Lys Ile Asn Gln Cys
65 70 75 80
Ile Asp Leu Val Asn Ser Asp Glu Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Gln Glu Gly Lys Pro Ile Tyr Lys Glu Arg Thr Asn
100 105 110
Asn Ala Lys
115
<210> 62
<211> 115
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 62
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Glu Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Lys Ala Leu Ala Pro Leu Asn Glu Lys Ile Asn Gln Cys
65 70 75 80
Ile Asp Leu Val Asn Ser Asp Glu Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Gln Glu Gly Lys Pro Ile Tyr Lys Glu Arg Thr Asn
100 105 110
Asn Ala Lys
115
<210> 63
<211> 115
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 63
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Glu Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Lys Ala Leu Ala Pro Leu Asn Glu Lys Ile Asn Gln Cys
65 70 75 80
Ile Asp Leu Val Asn Ser Asp Glu Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Gln Glu Gly Lys Pro Ile Tyr Lys Glu Arg Thr Asn
100 105 110
Asn Ala Lys
115
<210> 64
<211> 115
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 64
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Asp Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Ala Ala Leu Ala Pro Leu Asn Glu Lys Ile Asn Gln Cys
65 70 75 80
Ile Asp Leu Val Asn Ser Asp Glu Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Gln Glu Gly Lys Pro Ile Tyr Lys Glu Arg Ile Asn
100 105 110
Asn Ala Lys
115
<210> 65
<211> 118
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 65
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Glu Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Ala Ala Leu Ala Pro Leu Asn Ala Lys Ile Asn Thr Val
65 70 75 80
Ile Asp Leu Val Asn Ser Asn Asp Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Lys Asp Gly Lys Pro Ile Tyr Lys Glu Arg Ala Asn
100 105 110
Asn Ala Lys Glu His Tyr
115
<210> 66
<211> 115
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 66
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Glu Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Ala Ala Leu Ala Pro Leu Asn Glu Lys Ile Asn Gln Cys
65 70 75 80
Ile Asp Leu Val Asn Ser Asp Glu Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Gln Glu Gly Lys Pro Ile Tyr Lys Glu Arg Thr Asn
100 105 110
Asn Ala Lys
115
<210> 67
<211> 115
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 67
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Glu Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Ala Ala Leu Ala Pro Leu Asn Glu Lys Ile Asn Gln Cys
65 70 75 80
Ile Asp Leu Val Asn Ser Asp Glu Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Gln Glu Gly Lys Pro Ile Tyr Lys Glu Arg Thr Asn
100 105 110
Asn Ala Lys
115
<210> 68
<211> 115
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 68
Met Lys Leu Ser Lys Asp Asn Val Glu Leu Gly Leu Thr Ser Leu Ser
1 5 10 15
Thr Leu Ile Asp Ile Phe Ser Lys Phe Glu Asp Glu Phe Asp Glu Ile
20 25 30
Ala His Lys Gly Phe Phe Leu Val Tyr Glu Leu Tyr Ser His Tyr Lys
35 40 45
Leu Ile Tyr Thr Ala Asn Met Glu Arg Leu Glu Ser Ala Leu Thr Pro
50 55 60
Ala Ile Asn Lys Ala Leu Ala Pro Leu Asn Glu Lys Ile Asn Gln Cys
65 70 75 80
Ile Asp Leu Val Asn Ser Asp Glu Lys Asn Leu Lys Ile Ser Asn Asp
85 90 95
Leu Lys Phe Asn Gln Glu Gly Lys Pro Ile Tyr Lys Glu Arg Thr Asn
100 105 110
Asn Ala Lys
115
<210> 69
<211> 141
<212> PRT
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 69
Met Asn Asn Lys Met Asn Ile Asn Ser Gly Ile Asp Ala Leu Asn Asn
1 5 10 15
Leu Tyr Asp Phe Leu Lys Ser Ser Asp Ser Pro Thr Glu Val Lys Val
20 25 30
Glu Lys Gly Ile Tyr Leu Gly Leu Asn Leu Tyr Asn Leu Ile Met Ser
35 40 45
Ile Tyr Lys Asp Lys Ile Thr Thr Leu Glu Lys Glu Glu Ser Leu Lys
50 55 60
Ile Leu Asn Glu Ile Lys Asn Val Asn Asn Lys Ile Thr Gln Leu Ile
65 70 75 80
Ser Ser Ile Asn Asp Glu Arg Asp Ala Ser Ile Ile Glu His Leu Arg
85 90 95
Glu Glu Arg Asn Glu Leu Met Ser Ile Lys Thr Gln Ala Leu Gln Glu
100 105 110
Gln Ile Lys Glu Phe Pro Glu Asn Thr Ser Thr Ala Asn Ser Lys Ala
115 120 125
Gln Asn Leu Lys Glu Asn Lys Gly Asp Lys Ile Ala Asn
130 135 140
<210> 70
<211> 17
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 70
gtgaacttat atcaaac 17
<210> 71
<211> 17
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 71
ataatcttct tttcatt 17
<210> 72
<211> 20
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 72
acagtgctgg ttttaggagt 20
<210> 73
<211> 22
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 73
tgccattacc attaccattc tt 22
<210> 74
<211> 23
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 74
ttgatgaaat tgcacataaa gga 23
<210> 75
<211> 20
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 75
gctggggtta atgcactctc 20
<210> 76
<211> 21
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 76
aacagtgctg gttttaggag t 21
<210> 77
<211> 24
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 77
agataggttt tccttcctga ttga 24
<210> 78
<211> 555
<212> DNA
<213> 来自伯氏疏螺旋体的噬菌体(Phage from Borrelia burgdorferi)
<400> 78
atggatacta ttaaattaac cgaacttctt atcaatttaa acgaaattaa acttatagcg 60
gtaatgattt ttgtaacagt gctggtttta ggagtattaa ttcttctcaa gcctttacta 120
aaagacatat tgactattgt aataggcaag atttttaaga atggtaatgg taatggcaaa 180
aatcacatta aaaaaagaga ttaacttatg aaattatcca aagataatgt tgagcttgga 240
cttacgtctt tatcaaccct tattgatata ttttctaaat ttgaagatga atttgatgaa 300
attgcacata aaggattctt tttggtttat gagctgtatt ctcattataa attaatctat 360
acagcaaata tggaaagact tgagagtgca ttaaccccag caataaataa agcactcgct 420
ccattaaatg aaaaaatcaa tcaatgcatt gacttagtta attctgatga aaaaaatctc 480
aaaatatcta atgatctgaa attcaatcag gaaggaaaac ctatctataa ggaaagaaca 540
aataatgcaa aataa 555
<210> 79
<211> 21
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 79
gagtggatag caagcactga t 21
<210> 80
<211> 22
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 80
atcatcaact cgctccataa ca 22
<210> 81
<211> 26
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 81
tgctgggtct aaatatgcta tcgggc 26
<210> 82
<211> 20
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 82
agactaagat gcgggcaaga 20
<210> 83
<211> 20
<212> DNA
<213> 合成探针(synthetic probe)
<400> 83
ttgcatcaag agcgtcatca 20
<210> 84
<211> 1359
<212> DNA
<213> 宫本疏螺旋体(B. miyamotoi)
<400> 84
atggatatat acaagagcag tttgtttgtt aaatacagaa aaaaatataa acataaatat 60
gggattgata tacaagatta cattaagcct aaaagtttag atgttaactt taaagaattt 120
gagcaaacac atttaacacc aaaacaatta gcagttataa atagtattga agaacataag 180
caaactaaga ttattctttg tggagggatt gccagtggta aaacattctt agcatgttat 240
ctattcttaa aaatactgct tactggtaga catttataca agcaagatac caataatttt 300
attttgggca attcacaaaa atctttagaa cttaatgtat tggggcaatt tgataaaatt 360
gctagtatgc ttaatatact atttgtaccc aagtattcta atacttctta ttttgaagta 420
gattccttaa gagttaattt atatggtgga gataaggcaa gtgactttga acgcttcaga 480
ggctctaatt ctgctatcat ctatataaat gaagcaacta ctcttcataa agaaacatta 540
atagaatgtc ttaaaagact cagagtaggc aagcaaagta tcatctttga taccaatcct 600
gatcatcctg agcattactt taaaactgat tatattgata atactgatat ttatgctaca 660
tataacttta caacatatga taacgcatta atacccttag actttattaa gacacaagaa 720
caactatata aagaccaacc aacatataaa gcaagagtgc tcttaggaga atgggttgca 780
tctaatgagg ctatatttac taatgttaat cttataagta aagaaaaata tgaatttaaa 840
tccccaatag cctacctaga tcctgcttat agtattggtt gtgataacag tgcactttgt 900
gtattagagc gactagataa caagtattat gcatttatat ttcaagacaa acttccagca 960
agtgacccaa gagtattaaa tacaattatg actatactag aaaacttaaa tgtgcataca 1020
ctttatattg aagataggga taatactact gggaaaggta gtattactaa agtgtttatt 1080
aatcttagag cacatatgaa tcatcattat agaattgctc ctattaaacc cataagtaat 1140
aaatttacta gaattgctac tttaataggt cctattaatt catctaattt aagtattcta 1200
gattttagta gtaaatctgc tattgctgat atatacaaat ataaaggtga tgataagact 1260
aatgatgatt caattgatag cctgtcagct ctttatatgt tacttactct gaataagagt 1320
tctttaaaag cacattttac taaaataagg ttcatataa 1359
<210> 85
<211> 452
<212> PRT
<213> 宫本疏螺旋体(B. miyamotoi)
<400> 85
Met Asp Ile Tyr Lys Ser Ser Leu Phe Val Lys Tyr Arg Lys Lys Tyr
1 5 10 15
Lys His Lys Tyr Gly Ile Asp Ile Gln Asp Tyr Ile Lys Pro Lys Ser
20 25 30
Leu Asp Val Asn Phe Lys Glu Phe Glu Gln Thr His Leu Thr Pro Lys
35 40 45
Gln Leu Ala Val Ile Asn Ser Ile Glu Glu His Lys Gln Thr Lys Ile
50 55 60
Ile Leu Cys Gly Gly Ile Ala Ser Gly Lys Thr Phe Leu Ala Cys Tyr
65 70 75 80
Leu Phe Leu Lys Ile Leu Leu Thr Gly Arg His Leu Tyr Lys Gln Asp
85 90 95
Thr Asn Asn Phe Ile Leu Gly Asn Ser Gln Lys Ser Leu Glu Leu Asn
100 105 110
Val Leu Gly Gln Phe Asp Lys Ile Ala Ser Met Leu Asn Ile Leu Phe
115 120 125
Val Pro Lys Tyr Ser Asn Thr Ser Tyr Phe Glu Val Asp Ser Leu Arg
130 135 140
Val Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Ile Ile Tyr Ile Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Glu Thr Leu Ile Glu Cys Leu Lys Arg Leu Arg Val Gly Lys Gln
180 185 190
Ser Ile Ile Phe Asp Thr Asn Pro Asp His Pro Glu His Tyr Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Thr Asp Ile Tyr Ala Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Ala Leu Ile Pro Leu Asp Phe Ile Lys Thr Gln Glu
225 230 235 240
Gln Leu Tyr Lys Asp Gln Pro Thr Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Glu Trp Val Ala Ser Asn Glu Ala Ile Phe Thr Asn Val Asn Leu Ile
260 265 270
Ser Lys Glu Lys Tyr Glu Phe Lys Ser Pro Ile Ala Tyr Leu Asp Pro
275 280 285
Ala Tyr Ser Ile Gly Cys Asp Asn Ser Ala Leu Cys Val Leu Glu Arg
290 295 300
Leu Asp Asn Lys Tyr Tyr Ala Phe Ile Phe Gln Asp Lys Leu Pro Ala
305 310 315 320
Ser Asp Pro Arg Val Leu Asn Thr Ile Met Thr Ile Leu Glu Asn Leu
325 330 335
Asn Val His Thr Leu Tyr Ile Glu Asp Arg Asp Asn Thr Thr Gly Lys
340 345 350
Gly Ser Ile Thr Lys Val Phe Ile Asn Leu Arg Ala His Met Asn His
355 360 365
His Tyr Arg Ile Ala Pro Ile Lys Pro Ile Ser Asn Lys Phe Thr Arg
370 375 380
Ile Ala Thr Leu Ile Gly Pro Ile Asn Ser Ser Asn Leu Ser Ile Leu
385 390 395 400
Asp Phe Ser Ser Lys Ser Ala Ile Ala Asp Ile Tyr Lys Tyr Lys Gly
405 410 415
Asp Asp Lys Thr Asn Asp Asp Ser Ile Asp Ser Leu Ser Ala Leu Tyr
420 425 430
Met Leu Leu Thr Leu Asn Lys Ser Ser Leu Lys Ala His Phe Thr Lys
435 440 445
Ile Arg Phe Ile
450
<210> 86
<211> 810
<212> DNA
<213> 赫姆斯疏螺旋体(B. hermsii)
<400> 86
atggatatat ataaactacc tctttttaag gaaatgcaaa aagaatacaa gcgtgaattt 60
ggtattgata tagcagattt aattaaactg aaagcagctg ttattgattt tagagggttt 120
gaaaagacat atttaactaa gaaacaatgt gaggttttaa aattaattga gaataataat 180
cgaagtaaaa ttatcctgtc aggtggtatt gccagtggta aaacattctt ggcttgctat 240
ttatatctaa agatgctcat taagaataga catttttata agcaggatac caataatttt 300
atattaggta attcccaaaa atcattagag attaatgttt tagggcagtt tgaaaagatt 360
gctagcatgc taagagtacc ctttacgcca aagttttcta atacatcata ttttgaaata 420
gactccttaa gagtcaatct atatggtgga gataaggcaa gtgattttga aaggtttaga 480
ggaagcaatt ccgctcttat ttatgtaaat gaagcaacta cactgcataa ggaaacgcta 540
atagagtgct taaagagact tagagtaggt atggaaacta ttatatttga tacaaatcca 600
gacagtccag aacatttctt taaaactgat tatattgata acaagaaaac ttattctaca 660
tataacttta caacatatga taatgaacta atatcaaaag aatttattaa aacccaagaa 720
gagatttaca gggacatgcc aacatataag gcgagggtac tcttggggag aatgggttgc 780
gtcatacgat tcgatattta ccaatattaa 810
<210> 87
<211> 269
<212> PRT
<213> 赫姆斯疏螺旋体(B. hermsii)
<400> 87
Met Asp Ile Tyr Lys Leu Pro Leu Phe Lys Glu Met Gln Lys Glu Tyr
1 5 10 15
Lys Arg Glu Phe Gly Ile Asp Ile Ala Asp Leu Ile Lys Leu Lys Ala
20 25 30
Ala Val Ile Asp Phe Arg Gly Phe Glu Lys Thr Tyr Leu Thr Lys Lys
35 40 45
Gln Cys Glu Val Leu Lys Leu Ile Glu Asn Asn Asn Arg Ser Lys Ile
50 55 60
Ile Leu Ser Gly Gly Ile Ala Ser Gly Lys Thr Phe Leu Ala Cys Tyr
65 70 75 80
Leu Tyr Leu Lys Met Leu Ile Lys Asn Arg His Phe Tyr Lys Gln Asp
85 90 95
Thr Asn Asn Phe Ile Leu Gly Asn Ser Gln Lys Ser Leu Glu Ile Asn
100 105 110
Val Leu Gly Gln Phe Glu Lys Ile Ala Ser Met Leu Arg Val Pro Phe
115 120 125
Thr Pro Lys Phe Ser Asn Thr Ser Tyr Phe Glu Ile Asp Ser Leu Arg
130 135 140
Val Asn Leu Tyr Gly Gly Asp Lys Ala Ser Asp Phe Glu Arg Phe Arg
145 150 155 160
Gly Ser Asn Ser Ala Leu Ile Tyr Val Asn Glu Ala Thr Thr Leu His
165 170 175
Lys Glu Thr Leu Ile Glu Cys Leu Lys Arg Leu Arg Val Gly Met Glu
180 185 190
Thr Ile Ile Phe Asp Thr Asn Pro Asp Ser Pro Glu His Phe Phe Lys
195 200 205
Thr Asp Tyr Ile Asp Asn Lys Lys Thr Tyr Ser Thr Tyr Asn Phe Thr
210 215 220
Thr Tyr Asp Asn Glu Leu Ile Ser Lys Glu Phe Ile Lys Thr Gln Glu
225 230 235 240
Glu Ile Tyr Arg Asp Met Pro Thr Tyr Lys Ala Arg Val Leu Leu Gly
245 250 255
Arg Met Gly Cys Val Ile Arg Phe Asp Ile Tyr Gln Tyr
260 265
<210> 88
<211> 26
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 88
agtgcacttt gtgtgcttga aatggt 26
<210> 89
<211> 22
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 89
agcctaccta gatcctgctt at 22
<210> 90
<211> 20
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 90
gggtcacttg ctggtagttt 20
<210> 91
<211> 23
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 91
acgcttcaga ggctctaatt ctg 23
<210> 92
<211> 19
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 92
gtggagataa ggcaagtga 19
<210> 93
<211> 22
<212> DNA
<213> 合成探针(Synthetic probe)
<400> 93
ctttatgaag agtagttgct tc 22
<210> 94
<211> 29
<212> DNA
<213> 合成探针(synthetic probe)
<400> 94
aggcaccaat agcatattta gatcctgca 29
<210> 95
<211> 20
<212> DNA
<213> 合成探针(synthetic probe)
<400> 95
ggagaatggg ttgcgtcata 20
<210> 96
<211> 23
<212> DNA
<213> 合成探针(synthetic probe)
<400> 96
gcgcagtatt atcacctcca ata 23
<210> 97
<211> 201
<212> DNA
<213> 帕克疏螺旋体(Borrelia parkeri)
<400> 97
ctttatctgt gtctttacct tgttttttat cttactagct aaaatagtta gtatatcttt 60
gattgctggc ttgaagatca gtccaagact taagataaag gcaccaataa taactaactt 120
cacttcattg atgttaatca gttgaagtaa aaaatttaat acattattct caacttcatt 180
caactttatg tctcctctca t 201
<210> 98
<211> 213
<212> DNA
<213> 墨西哥疏螺旋体(Borrelia turicatae)
<400> 98
atgaaaggag acataaagtt gaatgaactt gatagtaatt tattaaaatt tttacttcaa 60
ctaattaaca tcaatgaaat gaagttagtt attattggtg cctttatctt aagtcttggt 120
ctgattttca agccagcaat caaagatata ctaactattt tagtaagtaa gataaaaaca 180
cgggttaaag acacagataa aggggaggat tta 213
<210> 99
<211> 192
<212> DNA
<213> 赫姆斯疏螺旋体(Borrelia hermsii)
<400> 99
atcccccttt tctttgtctc tgtcatgtat ttttatctta cttaataaga tacttagtat 60
atctttgatt actggtttga aaatcaaacc caaacttaat ataaaaatac taataatgat 120
taactttact tcattgatgt taattagttg aagtaaaaaa tctaatatgt tattaatgct 180
taattcattc aa 192
<210> 100
<211> 210
<212> DNA
<213> 革质疏螺旋体(Borrelia coriaceae)
<400> 100
atgcgcggcg ataacctgaa cgaagtgaac aacaacctgc tggattttct gctgcagctg 60
attagcgtga acgaagtgaa actgattatt attggcattt ttattctggg cattggcctg 120
atttttaaac cgaccattaa agatatgctg aacattattg cgaacaaaat taaaaccaac 180
gataaagata aaaacgataa agaaaacaaa 210
<210> 101
<211> 321
<212> DNA
<213> 赫姆斯疏螺旋体(Borrelia hermsii)
<400> 101
atgatacttg atcgtaattc tttagatact gctttaaatg caatcgtaac attatttgac 60
acactatcta attttgaaga tgggtcgttt aatgaaaatg ctcataaaac attcatgcta 120
ctaaacgaca tttacacaga atatcagatt atctatacac aaaatatgga aaggttagag 180
aatgccttaa ctccgcaaat aagagaaaca cttgaaccta tccaaactaa aatcaaaaaa 240
tttatagaaa aggttaatag caatccagat aatatgcaat taccattaga aatctcatca 300
attgaaaagg aggtcaaatg a 321
<210> 102
<211> 1212
<212> DNA
<213> 墨西哥疏螺旋体(Borrelia turicatae)
<400> 102
atgatttcaa atagcaatat caacgcaaga gagttatata agtattcaat attttttaga 60
aactatattt caaatgtagc agaggacact cttaagaatg gaattacctt aaatagtatt 120
gatactgctt ttaatgttaa tgatgcttta gaagccttaa aaatagagtt aaaagaagca 180
ttattgcagt gtttaattag ttaccgtttt aatggtgttg gatacatttt agttaaaact 240
gctgacttac ttgaagattt acatttaagc gtgaacctag aacttcctat tgggtttatg 300
tatcttgact ataacaatgt tcgtgatgag gggcctgact ttaattatat aacatacatt 360
ttcaaagtaa atacagatga gaagatatct tatagagagt tgaagattca taagaataga 420
gtaatcatac actctaatta tgattatata cttaaagctt acagtccatg ttatacgcaa 480
agctttttgc ttaatatata tctttttgaa caaatatata aagaaataga gaggagaata 540
gagcaacata actttttatt ttacaaggat gaatcattag taacattaca agatgcctta 600
agtgatgcta cgacttcatt agagatttta actaagggcg ttaatgataa gccacgtata 660
ttctctaatc tatttaaaag aaatgtagat gaaaatcata taagcacatt taagagtgta 720
aatagagatt tagagcgaga gcttacgcgg cttaaatcta acttaaataa taatggtatt 780
ttttacagtg gaacacctga tgcgtcactg gaggttatta agtatgactt aacctattta 840
aaggaagcat tagccttagt aaaggcaaag ataggtgctg atacaaaaga gccattaact 900
agaagtttta atgagcaaac taaggggctt ggaaatgatg ggaaagggga taggtctaat 960
tattatgact ttttaaaaag tgttcaagaa caaattgaag ttactattaa tagtaaactt 1020
gttaagtatt tcaatcttaa gatgcatttt aattcacttt atgtgttaag tgaagaagaa 1080
aaaatagagc gagatatgag attacttgaa atgtatgaca agtatgtagc tttaatgcta 1140
aatcctgcct taagtaaagt tgataaggca aatttacaag acagattatt tatgaaaata 1200
aaaggagatt aa 1212
<210> 103
<211> 759
<212> DNA
<213> 墨西哥疏螺旋体(Borrelia turicatae)
<400> 103
atgaaagaga cacatttaat taactgcaat cttaacttaa aaattaaaga attagagctg 60
caaaatgaac gcttaagtga agaattaact ctacttaaaa gcaatagcaa aactaaaaat 120
actaaaaaat tatcccctcc tgtaagattt tatctaaatg acaggacaat taaactagta 180
aaacgttcta tagagagact taaagaacaa gacccaatct ctggatggtt tgtacattta 240
ctctcaatta ctggttgtag gggtgttgaa atgcaaaatg taaaacttac tgatatatat 300
aaagagacaa gcagtaatgg tgaagtattt tattctattc gtgttaatgt agctaaaaag 360
cgtagcaata tctgtataag agaagtagtt attagtaagc ttgaatttga ttctattatg 420
agggcacatc aagaatattt caactctaga gataaagaca gtaggcgtac ttatctattt 480
caaaaaagta aacttaaatt ccgtgacaac aaaattaaca taactgaaat ttcaaaacag 540
tttaaggaat tactaattaa gggaggattt aaacatcgca aatctctaca tatactccgt 600
aatatattta tagcatcact taaagctaaa ggatataatt catttgaaat taaagagctt 660
atgaaatact catctacttc ggagattgat aatgtttatg gactctcaag tgcaagtaaa 720
atacaggctt acaaagatat caaaactagc ttgaaataa 759
<210> 104
<211> 70
<212> PRT
<213> 帕克疏螺旋体(Borrelia parkeri)
<400> 104
Met Asn Glu Val Glu Asn Asn Val Leu Asn Phe Leu Leu Gln Leu Ile
1 5 10 15
Asn Ile Asn Glu Val Lys Leu Val Ile Ile Gly Ala Phe Ile Leu Ser
20 25 30
Leu Gly Leu Ile Phe Lys Pro Ala Ile Lys Asp Ile Leu Thr Ile Leu
35 40 45
Ala Ser Lys Ile Lys Asn Lys Val Lys Thr Gln Ile Lys Glu Arg Thr
50 55 60
Tyr Glu Ile Lys His Cys
65 70
<210> 105
<211> 71
<212> PRT
<213> 墨西哥疏螺旋体(Borrelia turicatae)
<400> 105
Met Lys Gly Asp Ile Lys Leu Asn Glu Leu Asp Ser Asn Leu Leu Lys
1 5 10 15
Phe Leu Leu Gln Leu Ile Asn Ile Asn Glu Met Lys Leu Val Ile Ile
20 25 30
Gly Ala Phe Ile Leu Ser Leu Gly Leu Ile Phe Lys Pro Ala Ile Lys
35 40 45
Asp Ile Leu Thr Ile Leu Val Ser Lys Ile Lys Thr Arg Val Lys Asp
50 55 60
Thr Asp Lys Gly Glu Asp Leu
65 70
<210> 106
<211> 65
<212> PRT
<213> 赫姆斯疏螺旋体(Borrelia hermsii)
<400> 106
Met Asn Glu Leu Ser Ile Asn Asn Ile Leu Asp Phe Leu Leu Gln Leu
1 5 10 15
Ile Asn Ile Asn Glu Val Lys Leu Ile Ile Ile Ser Ile Phe Ile Leu
20 25 30
Ser Leu Gly Leu Ile Phe Lys Pro Val Ile Lys Asp Ile Leu Ser Ile
35 40 45
Leu Leu Ser Lys Ile Lys Ile His Asp Arg Asp Lys Glu Lys Gly Asp
50 55 60
Leu
65
<210> 107
<211> 70
<212> PRT
<213> 革质疏螺旋体(Borrelia coriaceae)
<400> 107
Met Arg Gly Asp Asn Leu Asn Glu Val Asn Asn Asn Leu Leu Asp Phe
1 5 10 15
Leu Leu Gln Leu Ile Ser Val Asn Glu Val Lys Leu Ile Ile Ile Gly
20 25 30
Ile Phe Ile Leu Gly Ile Gly Leu Ile Phe Lys Pro Thr Ile Lys Asp
35 40 45
Met Leu Asn Ile Ile Ala Asn Lys Ile Lys Thr Asn Asp Lys Asp Lys
50 55 60
Asn Asp Lys Glu Asn Lys
65 70
<210> 108
<211> 106
<212> PRT
<213> 赫姆斯疏螺旋体(Borrelia hermsii)
<400> 108
Met Ile Leu Asp Arg Asn Ser Leu Asp Thr Ala Leu Asn Ala Ile Val
1 5 10 15
Thr Leu Phe Asp Thr Leu Ser Asn Phe Glu Asp Gly Ser Phe Asn Glu
20 25 30
Asn Ala His Lys Thr Phe Met Leu Leu Asn Asp Ile Tyr Thr Glu Tyr
35 40 45
Gln Ile Ile Tyr Thr Gln Asn Met Glu Arg Leu Glu Asn Ala Leu Thr
50 55 60
Pro Gln Ile Arg Glu Thr Leu Glu Pro Ile Gln Thr Lys Ile Lys Lys
65 70 75 80
Phe Ile Glu Lys Val Asn Ser Asn Pro Asp Asn Met Gln Leu Pro Leu
85 90 95
Glu Ile Ser Ser Ile Glu Lys Glu Val Lys
100 105
<210> 109
<211> 403
<212> PRT
<213> 墨西哥疏螺旋体(Borrelia turicatae)
<400> 109
Met Ile Ser Asn Ser Asn Ile Asn Ala Arg Glu Leu Tyr Lys Tyr Ser
1 5 10 15
Ile Phe Phe Arg Asn Tyr Ile Ser Asn Val Ala Glu Asp Thr Leu Lys
20 25 30
Asn Gly Ile Thr Leu Asn Ser Ile Asp Thr Ala Phe Asn Val Asn Asp
35 40 45
Ala Leu Glu Ala Leu Lys Ile Glu Leu Lys Glu Ala Leu Leu Gln Cys
50 55 60
Leu Ile Ser Tyr Arg Phe Asn Gly Val Gly Tyr Ile Leu Val Lys Thr
65 70 75 80
Ala Asp Leu Leu Glu Asp Leu His Leu Ser Val Asn Leu Glu Leu Pro
85 90 95
Ile Gly Phe Met Tyr Leu Asp Tyr Asn Asn Val Arg Asp Glu Gly Pro
100 105 110
Asp Phe Asn Tyr Ile Thr Tyr Ile Phe Lys Val Asn Thr Asp Glu Lys
115 120 125
Ile Ser Tyr Arg Glu Leu Lys Ile His Lys Asn Arg Val Ile Ile His
130 135 140
Ser Asn Tyr Asp Tyr Ile Leu Lys Ala Tyr Ser Pro Cys Tyr Thr Gln
145 150 155 160
Ser Phe Leu Leu Asn Ile Tyr Leu Phe Glu Gln Ile Tyr Lys Glu Ile
165 170 175
Glu Arg Arg Ile Glu Gln His Asn Phe Leu Phe Tyr Lys Asp Glu Ser
180 185 190
Leu Val Thr Leu Gln Asp Ala Leu Ser Asp Ala Thr Thr Ser Leu Glu
195 200 205
Ile Leu Thr Lys Gly Val Asn Asp Lys Pro Arg Ile Phe Ser Asn Leu
210 215 220
Phe Lys Arg Asn Val Asp Glu Asn His Ile Ser Thr Phe Lys Ser Val
225 230 235 240
Asn Arg Asp Leu Glu Arg Glu Leu Thr Arg Leu Lys Ser Asn Leu Asn
245 250 255
Asn Asn Gly Ile Phe Tyr Ser Gly Thr Pro Asp Ala Ser Leu Glu Val
260 265 270
Ile Lys Tyr Asp Leu Thr Tyr Leu Lys Glu Ala Leu Ala Leu Val Lys
275 280 285
Ala Lys Ile Gly Ala Asp Thr Lys Glu Pro Leu Thr Arg Ser Phe Asn
290 295 300
Glu Gln Thr Lys Gly Leu Gly Asn Asp Gly Lys Gly Asp Arg Ser Asn
305 310 315 320
Tyr Tyr Asp Phe Leu Lys Ser Val Gln Glu Gln Ile Glu Val Thr Ile
325 330 335
Asn Ser Lys Leu Val Lys Tyr Phe Asn Leu Lys Met His Phe Asn Ser
340 345 350
Leu Tyr Val Leu Ser Glu Glu Glu Lys Ile Glu Arg Asp Met Arg Leu
355 360 365
Leu Glu Met Tyr Asp Lys Tyr Val Ala Leu Met Leu Asn Pro Ala Leu
370 375 380
Ser Lys Val Asp Lys Ala Asn Leu Gln Asp Arg Leu Phe Met Lys Ile
385 390 395 400
Lys Gly Asp
<210> 110
<211> 252
<212> PRT
<213> 墨西哥疏螺旋体(Borrelia turicatae)
<400> 110
Met Lys Glu Thr His Leu Ile Asn Cys Asn Leu Asn Leu Lys Ile Lys
1 5 10 15
Glu Leu Glu Leu Gln Asn Glu Arg Leu Ser Glu Glu Leu Thr Leu Leu
20 25 30
Lys Ser Asn Ser Lys Thr Lys Asn Thr Lys Lys Leu Ser Pro Pro Val
35 40 45
Arg Phe Tyr Leu Asn Asp Arg Thr Ile Lys Leu Val Lys Arg Ser Ile
50 55 60
Glu Arg Leu Lys Glu Gln Asp Pro Ile Ser Gly Trp Phe Val His Leu
65 70 75 80
Leu Ser Ile Thr Gly Cys Arg Gly Val Glu Met Gln Asn Val Lys Leu
85 90 95
Thr Asp Ile Tyr Lys Glu Thr Ser Ser Asn Gly Glu Val Phe Tyr Ser
100 105 110
Ile Arg Val Asn Val Ala Lys Lys Arg Ser Asn Ile Cys Ile Arg Glu
115 120 125
Val Val Ile Ser Lys Leu Glu Phe Asp Ser Ile Met Arg Ala His Gln
130 135 140
Glu Tyr Phe Asn Ser Arg Asp Lys Asp Ser Arg Arg Thr Tyr Leu Phe
145 150 155 160
Gln Lys Ser Lys Leu Lys Phe Arg Asp Asn Lys Ile Asn Ile Thr Glu
165 170 175
Ile Ser Lys Gln Phe Lys Glu Leu Leu Ile Lys Gly Gly Phe Lys His
180 185 190
Arg Lys Ser Leu His Ile Leu Arg Asn Ile Phe Ile Ala Ser Leu Lys
195 200 205
Ala Lys Gly Tyr Asn Ser Phe Glu Ile Lys Glu Leu Met Lys Tyr Ser
210 215 220
Ser Thr Ser Glu Ile Asp Asn Val Tyr Gly Leu Ser Ser Ala Ser Lys
225 230 235 240
Ile Gln Ala Tyr Lys Asp Ile Lys Thr Ser Leu Lys
245 250
Claims (36)
1.一种确定样品中存在或不存在广义伯氏疏螺旋体(Borrelia burgdorferi sensulato)或回归热疏螺旋体属(Borrelia)的方法,所述方法包括以下步骤:
a)检测所述样品中存在或不存在对广义伯氏疏螺旋体或回归热疏螺旋体属特异性的噬菌体;和
b)在检测到所述噬菌体的基础上确定所述样品中存在广义伯氏疏螺旋体或回归热疏螺旋体属,或在没有检测到所述噬菌体的基础上确定所述样品中不存在广义伯氏疏螺旋体或回归热疏螺旋体属。
2.根据权利要求1所述的方法,其中检测所述噬菌体包括检测噬菌体基因或基因片段。
3.根据权利要求1或2所述的方法,其中步骤a)之前是从所述样品分离核酸的步骤,并且步骤a)是对所分离的核酸实施的。
4.根据权利要求2或3所述的方法,其中所述噬菌体基因或基因片段编码末端酶、穴蛋白、内溶菌素、整合酶、门蛋白或衣壳蛋白或其组合。
5.根据权利要求4所述的方法,其中所述噬菌体基因或基因片段编码末端酶蛋白。
6.根据权利要求2至5中任一项所述的方法,其中所述噬菌体基因包含根据以下序列的核酸:
a)SEQ ID NO.1-34;或与SEQ ID NO.1-34具有大于或等于70%-99.5%序列同源性的核酸;或其片段;或其中所述噬菌体基因能够编码根据SEQ ID NO.36-69的任何一种的蛋白质或其片段;或
b)SEQ ID NO.84、SEQ ID NO.86或SEQ ID NO.97-103;或与SEQ ID NO.84、SEQ IDNO.86或SEQ ID NO.97-103具有大于或等于70%-99.5%序列同源性的核酸;或其片段;或其中所述噬菌体基因能够编码根据SEQ ID NO.85、SEQ ID NO.87或SEQ ID NO.104-110的任何一种的蛋白质或其片段。
7.根据权利要求6所述的方法,其中所检测的噬菌体基因片段包含SEQ ID NO.35;或与SEQ ID NO.35具有大于或等于70%-99.5%序列同源性的核酸。
8.根据权利要求1所述的方法,其中检测所述噬菌体包括检测噬菌体蛋白质或其片段。
9.根据权利要求8所述的方法,其中所述噬菌体蛋白质包含以下氨基酸序列:
a)SEQ ID NO.36-69或由根据SEQ ID NO.1-34的基因编码;或与SEQ ID NO.36-69中的任一个具有70%-99.5%序列同源性的氨基酸序列或由与SEQ ID NO.1-34中的任一个具有70%-99.5%序列同源性的基因编码;或由根据SEQ ID NO.35的基因片段编码;或
b)SEQ ID NO.85、SEQ ID NO.87或SEQ ID NO.104-110,或由根据SEQ ID NO.84、SEQID NO.86或SEQ ID NO.97-103的基因编码;或与SEQ ID NO.85、SEQ ID NO.87或SEQ IDNO.104-110中的任一个具有70%-99.5%序列同源性的氨基酸序列;或由与SEQ ID NO.84、SEQ ID NO.86或SEQ ID NO.97-103具有70%-99.5%序列同源性的基因编码。
10.根据权利要求1所述的方法,其中检测所述噬菌体包括检测噬菌体RNA或RNA片段。
11.根据前述权利要求中任一项所述的方法,其中所述样品是血浆、全血、脑脊液、尿液、粪便物质、皮肤、脑组织、神经胶质细胞、淋巴、汗液、羊膜液或其任何组合。
12.根据前述权利要求中任一项所述的方法,其中所述样品是已经在广义伯氏疏螺旋体或回归热疏螺旋体属感染的早期或晚期获得的样品。
13.根据前述权利要求中任一项所述的方法,其中所述广义伯氏疏螺旋体可以是以下中的任一种或其任何组合:阿氏疏螺旋体(Borrelia afzelii)、斯氏疏螺旋体(Borreliaspielmanii)、法雷斯疏螺旋体(Borrelia valaisiana)、伽氏疏螺旋体(Borreliagarinii)、芬兰疏螺旋体(Borrelia finlandensis)、狭义伯氏疏螺旋体(Borreliabugdorferi sensu stricto)、比塞蒂疏螺旋体(Borrelia bissettii)、巴伐利亚疏螺旋体(Borrelia bavariensis)、日本疏螺旋体(Borrelia japonica)、卢西塔尼亚疏螺旋体(Borrelia lusitaniae)、西尼伽疏螺旋体(Borrelia sinica)、斯氏疏螺旋体(Borreliaspielmanii)、塔卢基疏螺旋体(Borrelia tanukii)、土德疏螺旋体(Borrelia turdi)、法雷斯疏螺旋体(Borrelia valaisiana)、Borrelia Yangtze、梅奥疏螺旋体(Borreliamayonii)、Borrelia carolinensis和安德森疏螺旋体(Borrelia andersonii)、孤星疏螺旋体(Borrelia lonestari)和美洲疏螺旋体(Borrelia Americana)。
14.根据前述权利要求中任一项所述的方法,其中所述回归热疏螺旋体属可以是以下中的任一种或其任何组合:宫本疏螺旋体(Borrelia miyamotoi)、赫姆斯疏螺旋体(Borrelia hermsii)、回归热疏螺旋体(Borrelia recurrentis)、麝鼩疏螺旋体(Borreliacrocidurae)、中非疏螺旋体(Borrelia duttoni)、西班牙疏螺旋体(Borreliahispanica)、帕克疏螺旋体(Borrelia parkeri)和墨西哥疏螺旋体(Borreliaturicatae)。
15.根据前述权利要求中任一项所述的方法,其中所述方法能够确定所述样品中存在或不存在广义伯氏疏螺旋体或回归热疏螺旋体属的物种,所述方法包括以下步骤:
a)检测所述样品中存在或不存在所述广义伯氏疏螺旋体或回归热疏螺旋体属物种特异性噬菌体;
b)在检测到所述物种特异性噬菌体的基础上确定所述样品中存在所述广义伯氏疏螺旋体或回归热疏螺旋体属的物种,或在没有检测到所述物种特异性噬菌体的基础上确定所述样品中不存在所述广义伯氏疏螺旋体或回归热疏螺旋体属的物种。
16.一种监测来自广义伯氏疏螺旋体或回归热疏螺旋体属的感染的进展的方法,所述方法包括以下步骤:
a)确定在从受试者获得的第一样品中的噬菌体的量;
b)检测在第二时间点从所述受试者获得的第二样品中的噬菌体的量;
c)比较在步骤a)中鉴定的所述第一样品中的噬菌体的量与在步骤b)中鉴定的所述第二样品中的噬菌体的量。
17.根据权利要求16所述的方法,其中确定所述噬菌体的量包括根据权利要求1至15中任一项所述的方法。
18.根据权利要求1至17中任一项所述的方法,其中,所述方法另外包括治疗患者的莱姆病,所述患者的样品已经测试为广义伯氏疏螺旋体阳性。
19.根据权利要求1至17中任一项所述的方法,其中所述方法另外包括治疗患者的回归热,所述患者的样品已经测试为回归热疏螺旋体属阳性。
20.根据权利要求18或19所述的方法,其中治疗包括施用至少一种抗生素。
21.一种用于检测样品中对广义伯氏疏螺旋体和/或回归热疏螺旋体属特异性的噬菌体的试剂盒,所述试剂盒包含对广义伯氏疏螺旋体和/或回归热疏螺旋体属特异性的一种或更多种检测分子。
22.根据权利要求21所述的试剂盒,其中所述一种或更多种检测分子特异性地结合:a)编码所述末端酶、穴蛋白、内溶菌素、整合酶、门蛋白或衣壳蛋白或其组合的噬菌体基因或基因片段;或b)噬菌体末端酶、穴蛋白、内溶菌素、整合酶、门蛋白或衣壳蛋白或其组合。
23.根据权利要求22所述的试剂盒,其中所述检测分子特异性地结合:a)SEQ ID NO.1-35、SEQ ID NO.84、SEQ ID NO.86或SEQ ID NO.97-103;或b)SEQ ID NO.36-69、SEQ IDNO.85、SEQ ID NO.87或SEQ ID NO.104-110。
24.根据权利要求22或23所述的试剂盒,其中所述试剂盒包含一种或更多种以下的引物:a)SEQ ID NO.70-77和/或SEQ ID NO.79-83;和/或b)SEQ ID NO.88-96。
25.根据权利要求22或23所述的试剂盒,其中所述检测分子是对噬菌体末端酶、穴蛋白、内溶菌素、整合酶、门蛋白或衣壳蛋白特异性的抗体,优选地是对根据SEQ ID NO.36-69、SEQ ID NO.85、SEQ ID NO.87或SEQ ID NO.104-110的蛋白质特异性的抗体。
26.对广义伯氏疏螺旋体特异性的噬菌体作为莱姆病的诊断标志物或作为广义伯氏疏螺旋体感染的诊断标志物的用途。
27.根据权利要求26所述的用途,其中对广义伯氏疏螺旋体特异性的所述噬菌体包含根据SEQ ID NO.1-34中任一个的核酸或其片段或SEQ ID NO.35或根据SEQ ID NO.36-69中任一个的蛋白质或其片段。
28.对回归热疏螺旋体属特异性的噬菌体作为回归热的诊断标志物的用途。
29.根据权利要求28所述的用途,其中对回归热疏螺旋体属特异性的所述噬菌体包含根据SEQ ID NO.84、SEQ ID NO.86或SEQ ID NO.97-103中任一个的核酸或其片段;或根据SEQ ID NO.85、SEQ ID NO.87或SEQ ID NO.104-110中任一个的蛋白质或其片段。
30.一种疏螺旋体属噬菌体核酸,包含:a)SEQ ID NO.1-34或其片段;或与SEQ IDNO.1-34具有等于或大于70%-99.5%同源性的噬菌体核酸序列;或b)SEQ ID NO.84、SEQID NO.86或SEQ ID NO.97-103或与SEQ ID NO.84、SEQ ID NO.86或SEQ ID NO.97-103具有等于或大于70%-99.5%同源性的噬菌体核酸序列。
31.一种疏螺旋体属噬菌体核酸片段,所述疏螺旋体属噬菌体核酸片段包含SEQ IDNO.35或与SEQ ID NO.35具有等于或大于70%-99.5%同源性的噬菌体核酸序列。
32.一种DNA引物,所述DNA引物是对根据权利要求30-31任一项所述的噬菌体核酸特异性的。
33.根据权利要求32所述的DNA引物,所述DNA引物包含以下任一个或更多个:a)SEQ IDNO.70-77和/或SEQ ID NO.79-83;或b)SEQ ID NO.88-96。
34.一种疏螺旋体属噬菌体蛋白质,包含:a)SEQ ID NO.36-69或其片段;或与SEQ IDNO.36-69具有等于或大于70%-99.5%同源性的噬菌体蛋白质;或b)SEQ ID NO.85、SEQ IDNO.87或SEQ ID NO.104-110或其片段;或与SEQ ID NO.85、SEQ ID NO.87或SEQ IDNO.104-110具有等于或大于70%-99.5%同源性的噬菌体蛋白质。
35.一种抗体或其他结合分子,所述抗体或其他结合分子是对权利要求34所述的噬菌体蛋白质或蛋白质片段特异性的。
36.一种从疏螺旋体属提取噬菌体DNA的方法,所述方法包括:a)在氢氧化铵中孵育所述疏螺旋体属;和b)将苯酚-氯仿添加至所述疏螺旋体属和氢氧化铵混合物。
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