本发明涉及一种诊断疾病的方法,所述方法包括使供体组织切片与能够从所述供体组织切片提取抗体的液体接触,并使所述液体与包含抗原的受体材料接触,然后检测包括所述抗体和所述抗原的复合物,以及包含供体组织切片和包含抗原的受体材料的诊断上有用的载体。
在大多数人类中,默认地存在所有身体成分的耐受性,即它们不会触发针对其自身组织抗原的抗体的产生。相比之下,发生称为自身免疫疾病的病症,其中宿主对他自己的组织有明显的免疫反应,常常导致组织损伤。
无论怎么强调早期可靠诊断自身免疫疾病的重要性都不过分。许多自身免疫疾病不能治愈,但可以使用可用于显著改善其症状的治疗,通常使患者能够正常生活。诊断越早,利用例如免疫抑制药物的施用或严格的无免疫原性抗原饮食来给患者提供全部益处的一系列疗法的机会越多。
自身免疫疾病的诊断通常基于在包含抗体的液体样品中检测自身抗体的方法的结果(output)。最常使用从患者常规获得的血清。在其他情况下,使用如脑脊髓液(csf)、特别是用于检测与神经症状相关的自身抗体的其它液体。
乳糜泻(腹部疾病)(coeliacdisease,cd)是自身免疫疾病的一个特例。它是由麸质(gluten)引发的,其在遗传易感受试者中激活针对cd自身抗原(即组织转谷氨酰胺酶(ttg))的免疫反应。在发现针对麦醇溶蛋白和/或ttg的抗体的存在与cd相关联之后,可以确定cd不是罕见的,而是在全世界人群内预期患病率高于1%的常见病症。存在与麸质敏感性相关的一系列疾病,包括以摄取含麸质食物相关肠症状为特征的非腹腔麸质敏感性(noncoeliacglutensensitivity,ncgs),而在许多患者的情况下,仍然难以彻底区分它们。
需要具有重要经验的病理学家来检查由活检样品制成的组织切片作为可靠诊断的基础。在许多情况下,这样的检查是不确定的,因为不能观察到特征性特征。然而,病理学家可能不具有由他处理的来自患者的血清样品以补充组织学分析,或者包含外周血的血清样品中抗体的浓度可能不足。
在这种情况下,很可能会误诊患者。假阴性结果可能导致患者被拒绝适当治疗,导致继发性疾病的风险增加,其中包括例如肠淋巴瘤的癌症,以及生活质量差。
因此,本发明内在的一个问题是提供一种诊断自身免疫疾病的方法,该方法可用于证实患病组织的不确定的组织学检查。另一个问题是提供具有增加的灵敏度并降低假阴性结果的可能性的改进的测试。
本发明内在的至少一个问题在于提供一种可以在不存在血清样品的情况下进行的测定,并允许病理学家基于不确定的组织学检查证实诊断。
本发明内在的问题由所附独立和从属权利要求的主题来解决。
在第一方面,本发明内在的问题通过诊断疾病的方法来解决,该方法包括使供体组织切片与能够从所述供体组织切片提取抗体的液体接触,并使所述液体与包含抗原(任选两种或更多种抗原)的受体材料接触,随后检测包含所述抗体和所述抗原的复合物。
在优选实施方案中,将所述供体组织切片和所述包含抗原的受体材料在所述液体中共孵育。
在第二方面,该问题通过包括供体组织切片和包含抗原的受体材料的诊断上有用的载体来解决,其中所述载体被构造成使得所述供体组织切片和所述包含抗原的受体材料可以在能够从所述供体组织切片提取抗体并将所述抗体转运到所述受体材料的液体中共孵育。
在优选实施方案中,所述供体组织切片和所述包含抗原的受体材料包被于所述载体的表面上,并且优选在空间上是分开的。
在优选实施方案中,供体组织切片和包含抗原的受体材料在空间上分开的生物芯片上。
在优选实施方案中,所述载体被构造成使得能够从所述供体组织切片提取抗体的液体可以置于载体的表面上,使得可以从所述供体组织切片中提取抗体并且抗体通过所述液体扩散到所述包含抗原的受体材料。
在优选实施方案中,所述载体包含具有用所述供体组织切片包被的表面的第一部分和具有用所述受体材料包被的表面的第二部分,
其中所述第一部分和第二部分是分开的,并且所述载体被构造成使得所述第一部分和第二部分可以接触,优选组装,使得所述第一部分或所述第二部分的表面面朝下地位于所述第一部分和所述第二部分中另一个的表面上,使得一滴液体可以置于第一部分和第二部分的表面之间,以允许任何来自所述供体组织切片的抗体扩散到所述受体材料。
在优选实施方案中,已经从待诊断的患者获得供体组织切片。
在优选实施方案中,包含抗原的受体材料是包含优选天然多肽形式的抗原的组织样品,产生所述抗原的细胞或分离的多肽。
在优选实施方案中,供体组织切片是冷冻组织切片。
在优选实施方案中,疾病是胃肠疾病,优选炎性或自身免疫性胃肠疾病,更优选乳糜泻,供体组织切片是胃肠道组织,优选十二指肠组织,抗原是组织转谷氨酰胺酶或脱酰氨麦醇溶蛋白或其变体,优选脱酰氨麦醇溶蛋白或其变体的寡聚物。
在一个优选实施方案中,该疾病是天疱疮和/或类天疱疮,优选大疱性类天疱疮,供体组织切片为患病的皮肤组织,受体材料包含来自dsg1、dsg3、nc16a、bp180、bp230、lama3(seqidno14),层粘连蛋白332的一种或多种抗原,其优选是包含包括lama3(seqidno14)、lamb3(seqidno15)和lamc2(seqidno16)和β4整联蛋白(seqidno17)和vii型胶原及其变体的多肽的组合物,任选以组织的形式,优选选自灵长类动物食管、人盐裂皮肤(humansaltsplitskin)和大鼠膀胱。
在优选实施方案中,所述疾病是肺出血-肾炎综合征或sle,供体组织切片是患病的肾组织,并且包含抗原的受体材料是或来源于选自抗肾小球基底膜、dsdns、人上皮细胞(hep-2)、pla2r和thsd7a及其变体的材料。
在优选实施方案中,该疾病是克罗恩病,供体组织切片是患病的肠组织,并且包含抗原的受体材料选自健康胰腺组织、cuzd1和gp2及其变体,后两者优选为表达cuzd1和/或gp2或其变体的细胞的形式。
在优选实施方案中,该疾病是格雷夫斯病和/或桥本病,供体组织切片来源于甲状腺,并且包含抗原的受体材料是或来源于健康的甲状腺组织。
在优选实施方案中,疾病是肌炎,供体组织切片是肌肉或皮肤,优选肌肉组织,并且包含抗原的受体材料是mup-44或其变体。
本发明基于本发明人出乎意料的发现:可以在组织切片中检测自身抗体。例如,这适用于与cd相关的抗体。
不希望受任何理论约束,本发明人假设自身抗体积累在病变组织和器官中,并且其转移到患者的血液是不完全的,不足以用于基于血清学的可靠分析。
根据本发明,提供供体组织切片。这是从患有或怀疑患有待诊断的疾病的患者获得的一块组织。在优选实施方案中,将供体组织切片冷冻,优选浸入液氮或融化异戊烷。组织切片的厚度优选为1-100μm,优选为4-50μm。供体组织切片优选不用任何试剂处理或暴露于基本上改变供体组织切片中存在的抗体的条件。
根据本发明,供体组织切片与能够从所述供体组织切片提取抗体的液体接触。这是任何液体,其在暴露于供体组织切片时可能接收供体组织切片中存在的抗体,保存其化学组成、结构或结合性质,例如特异性结合抗原的能力,并允许其递送到包含抗原的受体材料。优选地,液体是包含生理缓冲液的水性液体,更优选在ph5至9,更优选6至8,最优选6.2至7.8。在最优选实施方案中,液体为pbsph7.4。
在优选实施方案中,本文所用的术语“结合特异性”是指比以下特征结合反应更强的结合,其特征在于解离常数为1×10-5m,更优选1×10-7m,更优选1×10-8m,更优选1×10-9m,更优选1×10-10m,更优选1×10-11m,更优选1×10-12m,如通过使用biacore设备在25℃,ph7.4的pbs缓冲液中的表面等离子体共振所测定的。
能够提取抗体的液体优选包含有助于从组织中解离任何抗体同时保存其化学组成、结构或结合性质,特别是特异性结合抗原的能力的洗涤剂,并且优选为吐温。在优选实施方案中,液体包含0.1至10%,优选0.5至5%,更优选1至4%,最优选1.5至2.5%的洗涤剂。
根据本发明,供体组织切片与能够从所述供体组织切片提取抗体的液体接触,并使所述液体与包含抗原的受体材料接触。受体材料包含允许抗体特异性结合的状态和构象的抗原。受体材料可以选自含有抗原的组织,优选天然多肽形式,更优选内源性天然多肽,产生抗原的细胞,优选过表达抗原的重组细胞和分离的多肽。细胞可以是完整的或裂解的细胞。
在优选实施方案中,将供体组织切片和包含抗原的受体材料在液体中共孵育,即供体组织切片和受体材料二者都与液体接触,同时将抗体从供体组织切片提取并运送到受体材料。该共孵育步骤进行至少10、20、30、60分钟、2、3、4、6、8、10或12小时。
或者,可以首先将供体组织切片与液体接触,然后分离供体组织切片和液体,并使液体与包含抗原的受体材料接触。在液体与包含抗原的受体材料接触之前,可以将液体储存、冷冻和/或浓缩。两个接触步骤可以进行至少10、20、30、60分钟、2、3、4、6、8、10或12小时。
随后,将受体材料与能够提取抗体的液体和/或供体组织切片分离,例如通过分离载体的第一部分或第二部分,或通过例如通过抽吸方式除去液体。受体材料可以使用洗涤缓冲液洗涤至少一次。能够提取抗体的新鲜液体可以用作洗涤缓冲液,优选包含较小浓度的洗涤剂,例如在孵育步骤中使用的浓度的10%,或根本不含洗涤剂。
随后,可以检测到包含抗体和抗原的任何复合物。在优选实施方案中,使用选自包括免疫扩散技术、免疫电泳技术、光散射免疫测定、凝集技术、标记的免疫测定法、例如包括放射性标记的免疫测定法、例如elisa的免疫测定法、化学发光免疫测定法和免疫荧光技术。本领域技术人员熟悉这些方法。
本发明涉及一种诊断上有用的载体,其优选是用于使供体组织切片和/或优选和包含抗原的受体材料与能够从供体组织切片提取抗体的液体接触的固体人造载体,所述供体组织切片和/或受体材料包被于所述载体表面上。优选地,供体组织切片和受体材料被疏水表面包围以固定能够提取抗体的液体的位置。在优选实施方案中,固体载体包含两个或多个分开的部分,一个包括供体组织切片,一个包含受体材料,每个分开的部分优选选自珠、微量滴定板、玻璃表面、生物芯片和膜,最优选生物芯片。
在优选实施方案中,本文所用的术语“生物芯片”是指厚度为0.01至1mm,优选为0.02至0.5mm,更优选为0.05至0.4mm的平面薄载片。它们优选由玻璃或塑料制成。它们用生物制剂如供体组织切片或包含抗原的受体材料包被。
在优选实施方案中,诊断上有用的载体可以用供体组织切片包被并包被有包含抗原的受体材料。供体组织切片和受体材料(优选二者均在生物芯片上)可以设定尺寸和可以非常接近地放置,使得能够提取抗体的一滴液体(优选体积为10至80μl,优选为20至60μl)可以同时与供体组织切片和受体材料接触以进行孵育步骤。
优选地,在诊断上有用的载体上包被的供体组织切片和包被的包含抗原的受体材料在空间上分开,特别是使得可以独立于另一个从诊断上有用的载体添加或除去。
在优选实施方案中,接触步骤可以如下进行:将一滴液体放置在表面上,并且使诊断载体与所述一滴液体接触,使得供体组织切片和受体材料(二者在诊断上有用的载体的同一侧上)在接触位于载体上的液滴时面朝下。由于其亲水性,表面、供体组织切片和受体材料粘附并固定液滴以用于整体的共孵育。抗体通过液体从供体组织切片向受体材料的提取和递送,可以通过轻轻摇动诊断上有用的载体与表面来得到支持。us2010/0124750中描述了用于进行孵育的装置。
图1示出了实施根据本发明的方法的这种方式:诊断上相关的载体(1),其包含包括抗体(3)的供体组织切片(2)和非常接近的包含抗原(5)的受体材料(4),所述诊断上相关的载体(1)放置在亲水表面(6)上方,能够提取抗体(7)的一滴液体位于所述亲水表面(6)上。诊断相关的载体(1)放置在亲水表面(6)上方,使得抗体(3)可以通过液体从供体组织切片(2)递送到抗原(5)并形成复合物。随后,分离亲水表面和诊断上有用的载体。可以检测包含抗原和抗体的复合物(如果存在)。
在另一个优选实施方案中,载体包括具有用所述供体组织切片包被的表面的第一部分和具有用所述包含抗原的受体材料包被的表面的第二部分,其中第一部分和第二部分是分开的并且载体被构造成使得第一部分和第二部分可以接触,优选组装,使得所述第一部分或所述第二部分的表面面朝下地位于所述第一部分和所述第二部分中另一个的表面上,使得一滴液体可以置于第一部分和第二部分的表面之间,以允许任何来自所述供体组织切片的抗体扩散到所述受体材料。图2示出了进行根据本发明的方法的这种方式。
在另一个优选实施方案中,载体的第一部分是用包含抗原的受体材料包被的珠,并且与供体组织切片接触。后者优选包被于载体的第二部分的表面上,但也可以是非固定的,例如漂浮在溶液中而不是包被。
受体材料可以包括多肽抗原或由其组成,例如在组织、细胞中或以纯化形式由本申请中明确提及的确切序列表示,例如通过功能、名称、序列或登录号,或隐含提及,而且还包括这些多肽的变体。
在优选实施方案中,本文所用的术语“变体”可以指所提到的全长序列的至少一个片段,更具体地相对于全长序列,在一个或两个末端被截短了一个或多个氨基酸的一个或多个氨基酸。这样的片段包含或编码具有原始序列或其变体的肽的至少6、7、8、10、12、15、20、25、50、75、100、150或200个连续氨基酸。变体的总长度可以是至少6、7、8、9、10、11、12、20、25、30、40、50、60、70、80、90、100个或更多个氨基酸。变体可以包括与提到的参考氨基酸序列或所述参考氨基酸序列的相应片段至少40、50、60、70、75、80、85、90、92、94、95、96、97、98或99%相同的全长序列或片段。
此外,其任何变体还可包含化学修饰,例如同位素标记或共价修饰,例如糖基化、磷酸化、乙酰化、脱羧、瓜氨酸化、甲基化、羟基化等。
优选包含抗原的多肽的变体具有生物活性。在优选实施方案中,这样的生物学活性是特异性结合抗体的能力,优选在患有相应疾病的患者中发现的感兴趣的自身抗体。例如,抗原如脱酰氨麦醇溶蛋白的变体的生物活性可以是特异性结合患者抗体的脱酰氨麦醇溶蛋白的能力,其存在表明患者患有疾病,例如在脱酰氨麦醇溶蛋白变体的情况下患者患有cd。
多肽或其变体可以以任何形式和以任何纯化的程度来提供,包括,以内源形式包含所述多肽的液体样品、组织或细胞(更优选过表达多肽的细胞,此类细胞的粗产物或富集裂解物),以及包括纯化和/或分离的多肽,其任选地基本上是纯的。在优选实施方案中,多肽是天然多肽,其中如本文所用的术语“天然多肽”是指折叠的多肽,更优选从组织或细胞、更优选哺乳动物细胞或组织、任选地来自非重组组织或细胞纯化的折叠多肽。在另一个优选实施方案中,多肽是重组蛋白,其中如本文所用的术语“重组蛋白”是指在生产过程的任何阶段使用遗传工程方法产生的多肽。在优选实施方案中,如果通过sdspage然后进行考马斯蓝染色和目视检查判断的相应样品中至少60、70、80、90、95或99%的多肽由所述多肽组成,则多肽是纯的。重组多肽可以包含两个或更多个抗原拷贝,优选彼此融合。
如果以组织形式提供抗原,则组织优选为哺乳动物组织,例如人、大鼠、灵长类动物、驴、小鼠、山羊、马、绵羊、猪或牛。
根据本发明,提供了一种诊断疾病的方法。在优选实施方案中,本文所用的术语“诊断”是指旨在获得有用信息的任何种类的方法,所述信息有助于评估患者是否患有或可能或更可能比普通受试者或比较受试者(后者优选具有相似的症状)在过去、在诊断时或在将来患有某种疾病或病症,以了解疾病进展情况或将来可能进展或评估患者对某种治疗例如免疫抑制药物的施用的应答情况。换句话说,术语“诊断”不仅包括疾病或病症的诊断,而且包括预测和/或监测疾病或病症的过程。
在许多情况下,仅仅检测,换句话说,确定样品中是否存在可检测的抗体水平,对于诊断是有用的,因为它表明患者患有疾病的可能性增加。在优选实施方案中,可以测定血清中抗体相对于普通健康受试者中可能发现的水平的相对浓度。尽管在许多情况下,测定样品中自身抗体是否存在或可检测可能是足够的,但为了获得诊断有用的信息而进行的方法可以包括测定浓度是否是比普通健康受试者中发现浓度高至少0.1、优选0.2、0.5、1、2、5、10、20、25、50、100、200、500、1000、10000或100000倍。
本领域技术人员将理解,临床医师通常不会仅仅基于单个诊断参数而断定患者是否患有或可能患有疾病、病况或病症,而是还基于其他方面,例如其他自身抗体、标志物的存在、血液参数、患者症状的临床评估或医学成像或其他非侵入性方法如多导睡眠监测仪的结果,以得出确定的诊断。(参见baenklerh.w.(2012),generalaspectsofautoimmunediagnostics,inrenz,h.,autoimmunediagnostics,2012,degruyter,第3页)。诊断剂或方法的价值也可能是属于这样的可能性,排除一种疾病,从而允许间接诊断另一种疾病。
术语“诊断”还可以指用于区分与相似或相同症状相关联的两种或多种病况的方法或药剂。术语“诊断”也可以指用于选择对于患者最有希望的治疗方案的方法或药剂。换句话说,该方法或药剂可涉及选择受试者的治疗方案。
在优选实施方案中,待检测的抗体是自身抗体。在另一个优选实施方案中,待检测的抗体是igg或iga类抗体,优选iga类抗体。
证实存在或不存在包含抗体和本发明多肽的复合物的任何数据可以与参考数据相关联。例如,所述复合物的检测表示提供所分析的样本的患者已经患有、正在患有或者可能在将来遭受疾病。如果先前已经诊断出患者并且再次运行获得诊断相关信息的方法,则在两次运行中检测到的复合物的量可能相关,以了解疾病的进展和/或治疗的成功。例如,如果发现复合物的量增加,这表明该疾病正在进展,可能在未来显现和/或表明任何尝试的治疗是不成功的。
本发明的教导提供了一种试剂盒,优选用于诊断疾病的试剂盒,其包含根据本发明的诊断上有用的载体。诊断上有用的载体可以最初包含仅包含抗原的受体材料,并且被构造成使得最终用户可以在实施本发明的方法之前添加供体组织切片。另外,所述试剂盒可以包括详细说明如何使用试剂盒的说明书和用于使本发明多肽与来自受试者、优选人受试者的体液样品接触的手段。此外,试剂盒可以包含阳性对照(例如已知结合本发明多肽的一批抗体或重组抗体)和阴性对照(例如对本发明多肽无可检测亲和力的蛋白质如牛血清白蛋白)。最后,这样的试剂盒可以包含用于制备校准曲线的抗体或抗原的标准溶液。
在优选实施方案中,试剂盒包括用于检测与在受体材料中抗原结合的抗体的手段,优选通过检测包含本发明多肽和与本发明多肽结合的抗体的复合物。此类手段优选是结合所述复合物并修饰复合物或携带使得复合物可检测到的标记的试剂。例如,所述手段可以是在除了由第一抗体识别的结合位点结合所述多肽或结合第一抗体的恒定区的标记抗体。或者,所述手段可以是与抗体的恒定区结合的第二抗体,优选特异性针对人抗体的第二抗体。
在优选实施方案中,待诊断的疾病是胃肠疾病,优选炎性或自身免疫性胃肠疾病,更优选乳糜泻,供体组织切片是胃肠道组织,优选十二指肠组织,并且抗原选自组织转谷氨酰胺酶(uniprot数据库:p21980,参考优先权数据在线版本,作为本文引用的所有数据库编号)或其变体或选自包含seqidno1、seqidno3、seqidno4、seqidno5及其变体的麦醇溶蛋白变体,优选其融合的多于一个拷贝,例如seqidno2。方法或载体可用于诊断ncgs,优选区分与麸质敏感性相关的胃肠病,更优选cd和ncgs。待检测的抗体优选为ttg或脱酰氨麦醇溶蛋白、更优选为脱酰氨麦醇溶蛋白的iga类抗体。
在另一个优选实施方案中,待诊断的疾病是天疱疮和/或类天疱疮,优选大疱性类天疱疮,供体组织切片为患病皮肤组织,抗原选自dsg1(uniprot数据库编号q02413)dsg3(uniprot数据库编号p32926)、nc16a(seqidno6),任选以包含seqidno7的两个或更多个拷贝的融合体的形式、bp180(uniprot数据库编号q9umd9)、bp230(seqidno8)和vii型胶原(uniprot数据库编号q02388)或其变体。
在另一个优选实施方案中,待诊断的疾病是肺出血-肾炎综合征或sle,供体组织切片是患病的肾组织,并且包含抗原的受体材料选自抗肾小球基底膜、dsdns、人上皮细胞(hep-2)、pla2r(seqidno9)和thsd7a(seqidno10)或其变体。
在另一个优选实施方案中,待诊断的疾病是克罗恩病,供体组织切片是患病的肠组织,抗原选自cuzd1(seqidno11)和gp2(seqidno12)或其变体。
在另一个优选实施方案中,待诊断的疾病是格雷夫斯病和/或桥本病,供体组织切片来源于甲状腺,并且抗原是或来源于健康的甲状腺组织。
在另一个优选实施方案中,待诊断的疾病是肌炎,供体组织切片是肌肉或皮肤,优选肌肉组织,并且抗原是或来源于mup-44(seqidno13)或其变体。
通过非限制性附加附图和以下非限制性示例进一步说明本发明,从中可以得到本发明的其它特征、实施方案、方面和优点。
图3示出了本发明中使用的包含biochip马赛克的载片。
图4显示了皮肤活检样品以及各种受体材料的位置,更具体地分别显示表达桥粒黏蛋白1、bp230gc(seqidno18)、桥粒黏蛋白3和bp180-nc16a的hek293细胞。
图5显示了阳性活检样品的免疫荧光分析,其中可检测到bp230的自身抗体。
图6显示了阳性活检样品的免疫荧光分析,其中可检测到桥粒黏蛋白3的自身抗体。
实施例1:
制造根据本发明的诊断上有用的载体及其用途
在显微镜载片上的一个反应场内将用作为组织供体切片的活检样品(冷冻于液氮中)的冷冻切片(4μm)包被的生物芯片放置在用作为包含抗原的受体材料的纯化的重组抗原gaf-3x(europlus)(seqidno2)的点包被的生物芯片旁边。从其他患者获得的所有活检样品都重复了这一步骤。
在4℃下将包含两个生物芯片的显微镜载片和一滴与活检样品和抗原二者放置接触的pbst缓冲液(含有2%(v/v)吐温-20的pbs,ph7.4)孵育过夜。这样,从组织洗脱的抗体可以扩散到缓冲液中,结合脱酰氨麦醇溶蛋白的抗体可以结合相邻的gaf-3x抗原点并被检测到。第二天,使用pbst(含有0.2%(v/v)吐温-20的pbs,ph7.4)洗涤生物芯片5分钟。随后,将生物芯片与fitc缀合的抗人iga抗体(euroimmunag)孵育30分钟,并再次洗涤5分钟。最后,使用荧光显微镜(eurostar)分析载片。
样品
经分析的队列包括37名患有乳糜泻的患者和35名健康对照人员,所述患者经根据有经验的临床医师对活检样品进行组织学检查而得到诊断。获得来自十二指肠球部(bulbusduodeni)的十二指肠活检样品(36名cd患者,34名对照个体)。所有个体的血清样品可供使用。将样品于暗处(通过盲式)孵育和显微镜分析,并对数据进行最终评估。
使用市售抗gaf-3x-iift(iga)和抗gaf-3xelisa(iga)根据制造商的说明书(euroimmunag,germany,产品号分别是fv3011-#a和ev3011-9601a)另外测试血清样品。
81份血清样品的血清学分析(不是根据本发明)
使用免疫荧光试验,37个测试cd患者中28个样品为iga抗gaf-3x抗体阳性,灵敏度为76%。在35个对照样品中,抗gaf-3x-iift(iga)中没有阳性。因此,iift(iga)达到100%的特异性。
使用根据本发明的方法获得的iga检测的增强的灵敏度
在根据本发明的方法中使用十二指肠球部(bulbusduodeni)活检样品,36名cd患者(其样品可获得)中有32名对于iga抗gaf-3x抗体检测为阳性。在32名患者中,5名在elisa和iift二者中均检测为阴性。进一步测试对照个体的34例十二指肠球部(bulbus)活检样品,其中仅一例显示iga类抗gaf-3x抗体。
总之,对于iga抗gaf-3x,十二指肠球部(bulbusduodeni)活检样品共孵育测试灵敏度为89%,从而达到97%的特异性。
结果表明,与脱酰氨麦醇溶蛋白结合的抗体存在于小肠粘膜内,并且使用本发明的方法在来自患者血清样品为阴性的组织切片中可以检测到。结果,可减少假阴性结果的数量。
实施例2:
大疱性类天疱疮(bp)和寻常型天疱疮(pv)与针对bp180、bp230和桥粒黏蛋白(dsg1、dsg3)的循环自身抗体相关。
针对bp180和/或bp230的抗体产生大疱性类天疱疮的血清学指征。它也可以是很少发现的扁平苔藓类天疱疮或类似的不常见的粘膜类天疱疮(仅bp180)或瘢痕性类天疱疮(仅bp180),其主要发生在老年人中。在孕妇中,应考虑妊娠类天疱疮。
抗桥粒黏蛋白1的抗体指示落叶型天疱疮病,而抗桥粒黏蛋白3的抗体(有时另外还包括抗桥粒黏蛋白1的抗体)出现在寻常型天疱疮中。
活检样品的直接免疫荧光(dif)显示天疱疮病中的桥粒和类天疱疮病中的表皮基底膜的染色。为此,制备患者活检材料的组织切片,用荧光染料标记的抗人单克隆抗体孵育,然后使用荧光显微镜进行评价。
然后,血清学鉴别必须用单特异性测试(例如通过与重组hek细胞(表达特异性抗原)的间接免疫荧光(iif))和抗原制剂
本研究的目的是通过活检样品和重组蛋白的细胞制剂或抗原点在iif(与使用血清样品进行的常规iift相反)中的共孵育单特异性测定从组织洗脱的抗体。这样就可以一步进行搜索测试(通常是dif)和确认测试(通常是iift)。
将一例pv和两例bp患者的活检样品的冷冻切片分别在具有用桥粒黏蛋白1(euroimmun,底物fd1495-50;dsg1)、桥粒黏蛋白3(euroimmun,底物fd1496-50;dsg3)、bp230(euroimmun底物fd1502-56;bp230gc(seqidno18))或抗bp180-nc16a-4x(euroimmun底物fd1502-52;bp180-nc16a-4x)转染的hek293细胞的一个载片上组合成biochiptm-马赛克(图1和2)。将组织切片、丙酮或甲醛固定的重组细胞制剂或抗原制剂
将个体马赛克与30μlpbs-吐温20(2%)在4℃孵育15小时。通过fitc标记的抗人igg抗体(euroimmun,产品号af102,fitc标记的抗人igg(山羊))显现结合的抗体。室温(20-25℃)孵育30分钟。两个孵育步骤后将载片用pbs-吐温20(0.2%)洗涤5分钟。然后覆盖它们并在荧光显微镜(axioscopea1,zeiss,jena,germany,第490035-9100-000号)下进行评估。
pv活检样品的共孵育显示与桥粒和dsg3的平行反应性(参见图6的示例性图像),而两例bp活检样品的共孵育导致表皮基底膜和bp180和bp230抗原的染色(参见图5的示例性图像)。与在患者血清中检测到抗体的常规iift相反,使用共孵育技术检测到存在于活检样品内体液残留物中的抗体。
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leugluprogluthrpheglnasnpheaspglyasphisalacysser
195200205
valargaspaspgluphelyspheglnglyleuarghisthrvalthr
210215220
alaargglnleuvalglualalysleuleuaspmetargthrileglu
225230235240
glnleuargleuglyleulysthrvalglugluvalglnlysthrleu
245250255
asnlyspheleuthrlysalathrserilealaglyleutyrleuglu
260265270
serthrlysglulysileserphealaseralaalagluargileile
275280285
ileasplysmetvalalaleualapheleuglualaglnalaalathr
290295300
glypheileileaspproileserglyglnthrtyrservalgluasp
305310315320
alavalleulysglyvalvalaspproglupheargileargleuleu
325330335
glualaglulysalaalavalglytyrsertyrserserlysthrleu
340345350
servalpheglnalametgluasnargmetleuaspargglnlysgly
355360365
lyshisileleuglualaglnilealaserglyglyvalileasppro
370375380
valargglyileargvalproprogluilealaleuglnglnglyleu
385390395400
leuasnasnalaileleuglnpheleuhisgluproserserasnthr
405410415
argvalpheproasnproasnasnlysglnalaleutyrtyrserglu
420425430
leuleuargmetcysvalpheaspvalgluserglncyspheleuphe
435440445
propheglygluargasnileserasnleuasnvallyslysthrhis
450455460
argileservalvalaspthrlysthrglysergluleuthrvaltyr
465470475480
glualapheglnargasnleuileglulysseriletyrleugluleu
485490495
serglyglnglntyrglntrplysglualametphepheglusertyr
500505510
glyhisserserhismetleuthraspthrlysthrglyleuhisphe
515520525
asnileasnglualailegluglnglythrileasplysalaleuval
530535540
lyslystyrglngluglyleuilethrleuthrgluleualaaspser
545550555560
leuleuserargleuvalprolyslysaspleuhisserprovalala
565570575
glytyrtrpleuthralaserglygluargileservalleulysala
580585590
serargargasnleuvalaspargilethralaleuargcysleuglu
595600605
alaglnvalserthrglyglyileileaspproleuthrglylyslys
610615620
tyrargvalalaglualaleuhisargglyleuvalaspgluglyphe
625630635640
alaglnglnleuargglncysgluleuvalilethrglyileglyhis
645650655
proilethrasnlysmetmetservalvalglualavalasnalaasn
660665670
ileileasnlysglumetglyileargcysleuglupheglntyrleu
675680685
thrglyglyleuilegluproglnvalhisserargleuserileglu
690695700
glualaleuglnvalglyileileaspvalleuilealathrlysleu
705710715720
lysaspglnlyssertyrvalargasnileilecysproglnthrlys
725730735
arglysleuthrtyrlysglualaleuglulysalaasppheaspphe
740745750
histhrglyleulysleuleugluvalsergluproleumetthrgly
755760765
ileserserleutyrtyrserserleuleuglu
770775
<210>9
<211>1463
<212>prt
<213>人工序列
<220>
<223>seqidno9人工抗原
<400>9
metleuleuserproserleuleuleuleuleuleuleuglyalapro
151015
argglycysalagluglyvalalaalaalaleuthrprogluargleu
202530
leuglutrpglnasplysglyilephevalileglnsergluserleu
354045
lyslyscysileglnalaglylysservalleuthrleugluasncys
505560
lysglnalaasnlyshismetleutrplystrpvalserasnhisgly
65707580
leupheasnileglyglyserglycysleuglyleuasnpheserala
859095
progluglnproleuserleutyrglucysaspserthrleuvalser
100105110
leuargtrpargcysasnarglysmetilethrglyproleuglntyr
115120125
servalglnvalalahisaspasnthrvalvalalaserarglystyr
130135140
ilehislystrpilesertyrglyserglyglyglyaspilecysglu
145150155160
tyrleuhislysaspleuhisthrilelysglyasnthrhisglymet
165170175
procysmetphepropheglntyrasnhisglntrphishisglucys
180185190
thrarggluglyarggluaspaspleuleutrpcysalathrthrser
195200205
argtyrgluargaspglulystrpglyphecysproaspprothrser
210215220
alagluvalglycysaspthriletrpglulysaspleuasnserhis
225230235240
ilecystyrglnpheasnleuleuserserleusertrpsergluala
245250255
hissersercysglnmetglnglyglythrleuleuserilethrasp
260265270
gluthrglugluasnpheilearggluhismetserserlysthrval
275280285
gluvaltrpmetglyleuasnglnleuaspgluhisalaglytrpgln
290295300
trpseraspglythrproleuasntyrleuasntrpserprogluval
305310315320
asnphegluprophevalgluasphiscysglythrpheserserphe
325330335
metproseralatrpargserargaspcysgluserthrleuprotyr
340345350
ilecyslyslystyrleuasnhisileasphisgluilevalglulys
355360365
aspalatrplystyrtyralathrhiscysgluproglytrpasnpro
370375380
tyrasnargasncystyrlysleuglnlysgluglulysthrtrphis
385390395400
glualaleuargsercysglnalaaspasnseralaleuileaspile
405410415
thrserleualagluvalglupheleuvalthrleuleuglyaspglu
420425430
asnalasergluthrtrpileglyleuserserasnlysileproval
435440445
serpheglutrpserasnaspserservalilephethrasntrphis
450455460
thrleugluprohisilepheproasnargserglnleucysvalser
465470475480
alagluglnsergluglyhistrplysvallysasncysglugluarg
485490495
leuphetyrilecyslyslysalaglyhisvalleuseraspalaglu
500505510
serglycysglngluglytrpgluarghisglyglyphecystyrlys
515520525
ileaspthrvalleuargserpheaspglnalaserserglytyrtyr
530535540
cysproproalaleuvalthrilethrasnargphegluglnalaphe
545550555560
ilethrserleuileserservalvallysmetlysaspsertyrphe
565570575
trpilealaleuglnaspglnasnaspthrglyglutyrthrtrplys
580585590
provalglyglnlysprogluprovalglntyrthrhistrpasnthr
595600605
hisglnproargtyrserglyglycysvalalametargglyarghis
610615620
proleuglyargtrpgluvallyshiscysarghisphelysalamet
625630635640
serleucyslysglnprovalgluasnglnglulysalaglutyrglu
645650655
gluargtrpprophehisprocystyrleuasptrpgluserglupro
660665670
glyleualasercysphelysvalphehisserglulysvalleumet
675680685
lysargthrtrpargglualaglualaphecysgluglupheglyala
690695700
hisleualaserphealahisilegluglugluasnphevalasnglu
705710715720
leuleuhisserlyspheasntrpthrglugluargglnphetrpile
725730735
glypheasnlysargasnproleuasnalaglysertrpglutrpser
740745750
aspargthrprovalvalserserpheleuaspasnthrtyrphegly
755760765
gluaspalaargasncysalavaltyrlysalaasnlysthrleuleu
770775780
proleuhiscysglyserlysargglutrpilecyslysileproarg
785790795800
aspvallysprolysileprophetrptyrglntyraspvalprotrp
805810815
leuphetyrglnaspalaglutyrleuphehisthrphealaserglu
820825830
trpleuasnphegluphevalcyssertrpleuhisseraspleuleu
835840845
thrilehisseralahisgluglnglupheilehisserlysilelys
850855860
alaleuserlystyrglyalasertrptrpileglyleuglngluglu
865870875880
argalaasnaspglupheargtrpargaspglythrprovaliletyr
885890895
glnasntrpaspthrglyarggluargthrvalasnasnglnsergln
900905910
argcysglypheileserserilethrglyleutrpglysergluglu
915920925
cysservalsermetproserilecyslysarglyslysvaltrpleu
930935940
ileglulyslyslysaspthrprolysglnhisglythrcysprolys
945950955960
glytrpleutyrpheasntyrlyscysleuleuleuasnileprolys
965970975
aspprosersertrplysasntrpthrhisalaglnhisphecysala
980985990
glugluglyglythrleuvalalaileglusergluvalgluglnala
99510001005
pheilethrmetasnleupheglyglnthrthrservaltrpilegly
101010151020
leuglnasnaspasptyrgluthrtrpleuasnglylysprovalval
1025103010351040
tyrserasntrpserpropheaspileileasnileproserhisasn
104510501055
thrthrgluvalglnlyshisileproleucysalaleuleuserser
106010651070
asnproasnphehisphethrglylystrptyrphegluaspcysgly
107510801085
lysgluglytyrglyphevalcysglulysmetglnaspthrsergly
109010951100
hisglyvalasnthrseraspmettyrprometproasnthrleuglu
1105111011151120
tyrglyasnargthrtyrlysileileasnalaasnmetthrtrptyr
112511301135
alaalailelysthrcysleumethislysalaglnleuvalserile
114011451150
thraspglntyrhisglnserpheleuthrvalvalleuasnargleu
115511601165
glytyralahistrpileglyleuphethrthraspasnglyleuasn
117011751180
pheasptrpseraspglythrlysserserphethrphetrplysasp
1185119011951200
glugluserserleuleuglyaspcysvalphealaaspserasngly
120512101215
argtrphisserthralacysgluserpheleuglnglyalailecys
122012251230
hisvalproprogluthrargglnsergluhisprogluleucysser
123512401245
gluthrserileprotrpilelysphelysserasncystyrserphe
125012551260
serthrvalleuaspsermetserpheglualaalahisgluphecys
1265127012751280
lyslysgluglyserasnleuleuthrilelysaspglualagluasn
128512901295
alapheleuleuglugluleuphealapheglyserservalglnmet
130013051310
valtrpleuasnalaglnpheaspglyasnasngluthrilelystrp
131513201325
pheaspglythrprothraspglnserasntrpglyilearglyspro
133013351340
aspthrasptyrphelysprohishiscysvalalaleuargilepro
1345135013551360
gluglyleutrpglnleuserprocysglnglulyslysglypheile
136513701375
cyslysmetglualaaspilehisthralaglualaleuproglulys
138013851390
glyproserhisserileileproleualavalvalleuthrleuile
139514001405
valilevalalailecysthrleuserphecysiletyrlyshisasn
141014151420
glyglyphepheargargleualaglypheargasnprotyrtyrpro
1425143014351440
alathrasnpheserthrvaltyrleuglugluasnileleuileser
144514501455
aspleuglulysseraspgln
1460
<210>10
<211>1657
<212>prt
<213>人工序列
<220>
<223>seqidno10人工抗原
<400>10
metglyleuglnalaargargtrpalaserglyserargglyalaala
151015
glyproargargglyvalleuglnleuleuproleuproleuproleu
202530
proleuleuleuleuleuleuleuargproglyalaglyargalaala
354045
alaglnglyglualaglualaprothrleutyrleutrplysthrgly
505560
protrpglyargcysmetglyaspglucysglyproglyglyilegln
65707580
thrargalavaltrpcysalahisvalgluglytrpthrthrleuhis
859095
thrasncyslysglnalagluargproasnasnglnglnasncysphe
100105110
lysvalcysasptrphislysgluleutyrasptrpargleuglypro
115120125
trpasnglncysglnprovalileserlysserleuglulysproleu
130135140
glucysilelysglyglugluglyileglnvalarggluilealacys
145150155160
ileglnlysasplysaspileproalagluaspileilecysglutyr
165170175
phegluprolysproleuleugluglnalacysleuileprocysgln
180185190
glnaspcysilevalserglupheseralatrpserglucysserlys
195200205
thrcysglyserglyleuglnhisargthrarghisvalvalalapro
210215220
proglnpheglyglyserglycysproasnleuthrglupheglnval
225230235240
cysglnserserprocysglualaglugluleuargtyrserleuhis
245250255
valglyprotrpserthrcyssermetprohisserargglnvalarg
260265270
glnalaargargargglylysasnlysgluargglulysaspargser
275280285
lysglyvallysaspproglualaarggluleuilelyslyslysarg
290295300
asnargasnargglnasnargglngluasnlystyrtrpaspilegln
305310315320
ileglytyrglnthrarggluvalmetcysileasnlysthrglylys
325330335
alaalaaspleuserphecysglnglnglulysleuprometthrphe
340345350
glnsercysvalilethrlysglucysglnvalserglutrpserglu
355360365
trpserprocysserlysthrcyshisaspmetvalserproalagly
370375380
thrargvalargthrargthrileargglnpheproileglyserglu
385390395400
lysglucysprogluphegluglulysgluprocysleuserglngly
405410415
aspglyvalvalprocysalathrtyrglytrpargthrthrglutrp
420425430
thrglucysargvalaspproleuleuserglnglnasplysargarg
435440445
glyasnglnthralaleucysglyglyglyileglnthrarggluval
450455460
tyrcysvalglnalaasngluasnleuleuserglnleuserthrhis
465470475480
lysasnlysglualaserlysprometaspleulysleucysthrgly
485490495
proileproasnthrthrglnleucyshisileprocysprothrglu
500505510
cysgluvalserprotrpseralatrpglyprocysthrtyrgluasn
515520525
cysasnaspglnglnglylyslysglyphelysleuarglysargarg
530535540
ilethrasngluprothrglyglyserglyvalthrglyasncyspro
545550555560
hisleuleuglualaileprocysglugluproalacystyrasptrp
565570575
lysalavalargleuglyasncysgluproaspasnglylysglucys
580585590
glyproglythrglnvalglngluvalvalcysileasnseraspgly
595600605
glugluvalaspargglnleucysargaspalailepheproilepro
610615620
valalacysaspalaprocysprolysaspcysvalleuserthrtrp
625630635640
serthrtrpsersercysserhisthrcysserglylysthrthrglu
645650655
glylysglnileargalaargserileleualatyralaglygluglu
660665670
glyglyileargcysproasnserseralaleuglngluvalargser
675680685
cysasngluhisprocysthrvaltyrhistrpglnthrglyprotrp
690695700
glyglncysilegluaspthrservalserserpheasnthrthrthr
705710715720
thrtrpasnglyglualasercysservalglymetglnthrarglys
725730735
valilecysvalargvalasnvalglyglnvalglyprolyslyscys
740745750
progluserleuargprogluthrvalargprocysleuleuprocys
755760765
lyslysaspcysilevalthrprotyrserasptrpthrsercyspro
770775780
sersercyslysgluglyaspserserilearglysglnserarghis
785790795800
argvalileileglnleuproalaasnglyglyargaspcysthrasp
805810815
proleutyrgluglulysalacysglualaproglnalacysglnser
820825830
tyrargtrplysthrhislystrpargargcysglnleuvalprotrp
835840845
servalglnglnaspserproglyalaglngluglycysglyprogly
850855860
argglnalaargalailethrcysarglysglnaspglyglyglnala
865870875880
glyilehisglucysleuglntyralaglyprovalproalaleuthr
885890895
glnalacysglnileprocysglnaspaspcysglnleuthrsertrp
900905910
serlysphesersercysasnglyaspcysglyalavalargthrarg
915920925
lysargthrleuvalglylysserlyslyslysglulyscyslysasn
930935940
serhisleutyrproleuilegluthrglntyrcysprocysasplys
945950955960
tyrasnalaglnprovalglyasntrpseraspcysileleuproglu
965970975
glylysvalgluvalleuleuglymetlysvalglnglyaspilelys
980985990
glucysglyglnglytyrargtyrglnalametalacystyraspgln
99510001005
asnglyargleuvalgluthrserargcysasnserhisglytyrile
101010151020
gluglualacysileileprocysproseraspcyslysleuserglu
1025103010351040
trpserasntrpserargcysserlyssercysglyserglyvallys
104510501055
valargserlystrpleuargglulysprotyrasnglyglyargpro
106010651070
cysprolysleuasphisvalasnglnalaglnvaltyrgluvalval
107510801085
procyshisseraspcysasnglntyrleutrpvalthrgluprotrp
109010951100
serilecyslysvalthrphevalasnmetarggluasncysglyglu
1105111011151120
glyvalglnthrarglysvalargcysmetglnasnthralaaspgly
112511301135
prosergluhisvalgluasptyrleucysaspprogluglumetpro
114011451150
leuglyserargvalcyslysleuprocysprogluaspcysvalile
115511601165
serglutrpglyprotrpthrglncysvalleuprocysasnglnser
117011751180
serpheargglnargseralaaspproileargglnproalaaspglu
1185119011951200
glyargsercysproasnalavalglulysgluprocysasnleuasn
120512101215
lysasncystyrhistyrasptyrasnvalthrasptrpserthrcys
122012251230
glnleuserglulysalavalcysglyasnglyilelysthrargmet
123512401245
leuaspcysvalargseraspglylysservalaspleulystyrcys
125012551260
glualaleuglyleuglulysasntrpglnmetasnthrsercysmet
1265127012751280
valglucysprovalasncysglnleuserasptrpserprotrpser
128512901295
glucysserglnthrcysglyleuthrglylysmetileargargarg
130013051310
thrvalthrglnpropheglnglyaspglyargprocysproserleu
131513201325
metaspglnserlysprocysprovallysprocystyrargtrpgln
133013351340
tyrglyglntrpserprocysglnvalglnglualaglncysglyglu
1345135013551360
glythrargthrargasnilesercysvalvalseraspglyserala
136513701375
aspasppheserlysvalvalaspglugluphecysalaaspileglu
138013851390
leuileileaspglyasnlysasnmetvalleugluglusercysser
139514001405
glnprocysproglyaspcystyrleulysasptrpsersertrpser
141014151420
leucysglnleuthrcysvalasnglygluaspleuglypheglygly
1425143014351440
ileglnvalargserargprovalileileglngluleugluasngln
144514501455
hisleucysprogluglnmetleugluthrlyssercystyraspgly
146014651470
glncystyrglutyrlystrpmetalaseralatrplysglyserser
147514801485
argthrvaltrpcysglnargseraspglyileasnvalthrglygly
149014951500
cysleuvalmetserglnproaspalaaspargsercysasnpropro
1505151015151520
cysserglnprohissertyrcyssergluthrlysthrcyshiscys
152515301535
glugluglytyrthrgluvalmetserserasnserthrleuglugln
154015451550
cysthrleuileprovalvalvalleuprothrmetgluasplysarg
155515601565
glyaspvallysthrserargalavalhisprothrglnproserser
157015751580
asnproalaglyargglyargthrtrppheleuglnpropheglypro
1585159015951600
aspglyargleulysthrtrpvaltyrglyvalalaalaglyalaphe
160516101615
valleuleuilepheilevalsermetiletyrleualacyslyslys
162016251630
prolyslysproglnargargglnasnasnargleulysproleuthr
163516401645
leualatyraspglyaspalaaspmet
16501655
<210>11
<211>607
<212>prt
<213>人工序列
<220>
<223>seqidno11人工抗原
<400>11
metgluleuvalargargleumetproleuthrleuleuileleuser
151015
cysleualagluleuthrmetalaglualagluglyasnalasercys
202530
thrvalserleuglyglyalaasnmetalagluthrhislysalamet
354045
ileleuglnleuasnprosergluasncysthrtrpthrilegluarg
505560
progluasnlysserileargileilephesertyrvalglnleuasp
65707580
proaspglysercysglusergluasnilelysvalpheaspglythr
859095
serserasnglyproleuleuglyglnvalcysserlysasnasptyr
100105110
valprovalphegluserserserserthrleuthrpheglnileval
115120125
thraspseralaargileglnargthrvalphevalphetyrtyrphe
130135140
pheserproasnileserileproasncysglyglytyrleuaspthr
145150155160
leugluglyserphethrserproasntyrprolysprohisproglu
165170175
leualatyrcysvaltrphisileglnvalglulysasptyrlysile
180185190
lysleuasnphelysgluilepheleugluileasplysglncyslys
195200205
pheasppheleualailetyraspglyproserthrasnserglyleu
210215220
ileglyglnvalcysglyargvalthrprothrphegluserserser
225230235240
asnserleuthrvalvalleuserthrasptyralaasnsertyrarg
245250255
glypheseralasertyrthrseriletyralagluasnileasnthr
260265270
thrserleuthrcysserseraspargmetargvalileileserlys
275280285
sertyrleuglualapheasnserasnglyasnasnleuglnleulys
290295300
aspprothrcysargprolysleuserasnvalvalglupheserval
305310315320
proleuasnglycysglythrilearglysvalgluaspglnserile
325330335
thrtyrthrasnileilethrpheseralaserserthrsergluval
340345350
ilethrargglnlysglnleuglnileilevallyscysglumetgly
355360365
hisasnserthrvalgluileiletyrilethrgluaspaspvalile
370375380
glnserglnasnalaleuglylystyrasnthrsermetalaleuphe
385390395400
gluserasnserpheglulysthrileleugluserprotyrtyrval
405410415
aspleuasnglnthrleuphevalglnvalserleuhisthrserasp
420425430
proasnleuvalvalpheleuaspthrcysargalaserprothrser
435440445
aspphealaserprothrtyraspleuilelysserglycysserarg
450455460
aspgluthrcyslysvaltyrproleupheglyhistyrglyargphe
465470475480
glnpheasnalaphelyspheleuargsermetserservaltyrleu
485490495
glncyslysvalleuilecysaspserserasphisglnserargcys
500505510
asnglnglycysvalserargserlysargaspilesersertyrlys
515520525
trplysthraspserileileglyproileargleulysargasparg
530535540
seralaserglyasnserglypheglnhisgluthrhisalagluglu
545550555560
thrproasnglnpropheasnservalhisleupheserphemetval
565570575
leualaleuasnvalvalthrvalalathrilethrvalarghisphe
580585590
valasnglnargalaasptyrlystyrglnlysleuglnasntyr
595600605
<210>12
<211>530
<212>prt
<213>人工序列
<220>
<223>seqidno12人工抗原
<400>12
metgluargmetvalglyserglyleuleutrpleualaleuvalser
151015
cysileleuthrglnalaseralavalglnargglytyrglyasnpro
202530
ileglualasersertyrglyleuaspleuaspcysglyalaprogly
354045
thrproglualahisvalcyspheaspprocysglnasntyrthrleu
505560
leuaspglupropheargserthrgluasnseralaglyserglngly
65707580
cysasplysasnmetserglytrptyrargphevalglygluglygly
859095
valargmetsergluthrcysvalglnvalhisargcysglnthrasp
100105110
alapromettrpleuasnglythrhisproalaleuglyaspglyile
115120125
thrasnhisthralacysalahistrpserglyasncyscysphetrp
130135140
lysthrgluvalleuvallysalacysproglyglytyrhisvaltyr
145150155160
argleugluglythrprotrpcysasnleuargtyrcysthrasppro
165170175
serthrvalgluasplyscysglulysalacysargproglugluglu
180185190
cysleualaleuasnserthrtrpglycysphecysargglnaspleu
195200205
asnserseraspvalhisserleuglnproglnleuaspcysglypro
210215220
arggluilelysvallysvalasplyscysleuleuglyglyleugly
225230235240
leuglyglugluvalilealatyrleuargaspproasncysserser
245250255
ileleuglnthrglugluargasntrpvalservalthrserproval
260265270
glnalaseralacysargasnileleugluargasnglnthrhisala
275280285
iletyrlysasnthrleuserleuvalasnasppheileileargasp
290295300
thrileleuasnileasnpheglncysalatyrproleuaspmetlys
305310315320
valserleuglnalaalaleuglnproilevalserserleuasnval
325330335
servalaspglyasnglyglupheilevalargmetalaleuphegln
340345350
aspglnasntyrthrasnprotyrgluglyaspalavalgluleuser
355360365
valgluservalleutyrvalglyalaileleugluglnglyaspthr
370375380
serargpheasnleuvalleuargasncystyralathrprothrglu
385390395400
asplysalaaspleuvallystyrpheileileargasnsercysser
405410415
asnglnargaspserthrilehisvalglugluasnglyglnserser
420425430
gluserargpheservalglnmetphemetphealaglyhistyrasp
435440445
leuvalpheleuhiscysgluilehisleucysaspserleuasnglu
450455460
glncysglnprosercysserargserglnvalargsergluvalpro
465470475480
alaileaspleualaargvalleuaspleuglyproilethrargarg
485490495
glyalaglnserproglyvalmetasnglythrproserthralagly
500505510
pheleuvalalatrpprometvalleuleuthrvalleuleualatrp
515520525
leuphe
530
<210>13
<211>368
<212>prt
<213>人工序列
<220>
<223>seqidno13人工
<400>13
metgluproglyglnproarggluproglngluproarggluprogly
151015
proglyalagluthralaalaalaprovaltrpgluglualalysile
202530
phetyraspasnleualaprolyslyslysprolysserprolyspro
354045
glnasnalavalthrilealavalserserargalaleupheargmet
505560
aspglugluglnglniletyrthrgluglnglyvalgluglutyrval
65707580
argtyrglnleugluhisgluasnglupropheserproglyproala
859095
pheprophevallysalaleuglualavalasnargargleuargglu
100105110
leutyrproaspsergluaspvalpheaspilevalleumetthrasn
115120125
asnhisalaglnvalglyvalargleuileasnserileasnhistyr
130135140
aspleupheilegluargphecysmetthrglyglyasnserproile
145150155160
cystyrleulysalatyrhisthrasnleutyrleuseralaaspala
165170175
glulysvalargglualaileaspgluglyilealaalaalathrile
180185190
pheserproserargaspvalvalvalserglnserglnleuargval
195200205
alapheaspglyaspalavalleupheseraspglusergluargile
210215220
vallysalahisglyleuaspargphephegluhisglulysalahis
225230235240
gluasnlysproleualaglnglyproleulysglypheleugluala
245250255
leuglyargleuglnlyslysphetyrserlysglyleuargleuglu
260265270
cysproileargthrtyrleuvalthralaargseralaalaserser
275280285
glyalaargalaleulysthrleuargsertrpglyleugluthrasp
290295300
glualaleupheleualaglyalaprolysglyproleuleuglulys
305310315320
ileargprohisilephepheaspaspglnmetphehisvalalagly
325330335
alaglnglumetglythrvalalaalahisvalprotyrglyvalala
340345350
glnthrproargargthralaproalalysglnalaproseralagln
355360365
<210>14
<211>1724
<212>prt
<213>人工序列
<220>
<223>seqidno14lama3
<400>14
metproproalavalargargseralacyssermetglytrpleutrp
151015
ilepheglyalaalaleuglyglncysleuglytyrserserglngln
202530
glnargvalpropheleuglnproproglyglnserglnleuglnala
354045
sertyrvalglupheargproserglnglycysserproglytyrtyr
505560
argasphislysglyleutyrthrglyargcysvalprocysasncys
65707580
asnglyhisserasnglncysglnaspglyserglyilecysvalasn
859095
cysglnhisasnthralaglygluhiscysgluargcysglnglugly
100105110
tyrtyrglyasnalavalhisglysercysargalacysprocyspro
115120125
histhrasnserphealathrglycysvalvalasnglyglyaspval
130135140
argcyssercyslysalaglytyrthrglythrglncysgluargcys
145150155160
alaproglytyrpheglyasnproglnlyspheglyglysercysgln
165170175
procyssercysasnserasnglyglnleuglysercyshisproleu
180185190
thrglyaspcysileasnglngluprolysaspserserproalaglu
195200205
glucysaspaspcysaspsercysvalmetthrleuleuasnaspleu
210215220
alathrmetglygluglnleuargleuvallysserglnleuglngly
225230235240
leuseralaseralaglyleuleugluglnmetarghismetgluthr
245250255
glnalalysaspleuargasnglnleuleuasntyrargseralaile
260265270
serasnhisglyserlysilegluglyleugluarggluleuthrasp
275280285
leuasnglngluphegluthrleuglnglulysalaglnvalasnser
290295300
arglysalaglnthrleuasnasnasnvalasnargalathrglnser
305310315320
alalysgluleuaspvallysilelysasnvalileargasnvalhis
325330335
ileleuleulysglnileserglythraspglygluglyasnasnval
340345350
proserglyasppheserargglutrpalaglualaglnargmetmet
355360365
arggluleuargasnargasnpheglylyshisleuargglualaglu
370375380
alaasplysarggluserglnleuleuleuasnargileargthrtrp
385390395400
glnlysthrhisglnglygluasnasnglyleualaasnserilearg
405410415
aspserleuasnglutyrglualalysleuseraspleuargalaarg
420425430
leuglnglualaalaalaglnalalysglnalaasnglyleuasngln
435440445
gluasngluargalaleuglyalaileglnargglnvallysgluile
450455460
asnserleuglnseraspphethrlystyrleuthrthralaaspser
465470475480
serleuleuglnthrasnilealaleuglnleumetglulyssergln
485490495
lysglutyrglulysleualaalaserleuasnglualaargglnglu
500505510
leuserasplysvalarggluleuserargseralaglylysthrser
515520525
leuvalgluglualaglulyshisalaargserleuglngluleuala
530535540
lysglnleuglugluilelysargasnalaserglyaspgluleuval
545550555560
argcysalavalaspalaalathralatyrgluasnileleuasnala
565570575
ilelysalaalagluaspalaalaasnargalaalaseralaserglu
580585590
seralaleuglnthrvalilelysgluaspleuproarglysalalys
595600605
thrleuserserasnserasplysleuleuasnglualalysmetthr
610615620
glnlyslysleulysglngluvalserproalaleuasnasnleugln
625630635640
glnthrleuasnilevalthrvalglnlysgluvalileaspthrasn
645650655
leuthrthrleuargaspglyleuhisglyileglnargglyaspile
660665670
aspalametileserseralalyssermetvalarglysalaasnasp
675680685
ilethraspgluvalleuaspglyleuasnproileglnthraspval
690695700
gluargilelysaspthrtyrglyargthrglnasngluaspphelys
705710715720
lysalaleuthraspalaaspasnservalasnlysleuthrasnlys
725730735
leuproaspleutrparglysilegluserileasnglnglnleuleu
740745750
proleuglyasnileseraspasnmetaspargilearggluleuile
755760765
glnglnalaargaspalaalaserlysvalalavalprometargphe
770775780
asnglylysserglyvalgluvalargleuproasnaspleugluasp
785790795800
leulysglytyrthrserleuserleupheleuglnargproasnser
805810815
arggluasnglyglythrgluasnmetphevalmettyrleuglyasn
820825830
lysaspalaserargasptyrileglymetalavalvalaspglygln
835840845
leuthrcysvaltyrasnleuglyaspargglualagluleuglnval
850855860
aspglnileleuthrlyssergluthrlysglualavalmetasparg
865870875880
vallyspheglnargiletyrglnphealaargleuasntyrthrlys
885890895
glyalathrserserlysprogluthrproglyvaltyraspmetasp
900905910
glyargasnserasnthrleuleuasnleuaspprogluasnvalval
915920925
phetyrvalglyglytyrproproaspphelysleuproserargleu
930935940
serpheproprotyrlysglycysilegluleuaspaspleuasnglu
945950955960
asnvalleuserleutyrasnphelyslysthrpheasnleuasnthr
965970975
thrgluvalgluprocysargargarglysglugluserasplysasn
980985990
tyrphegluglythrglytyralaargvalprothrglnprohisala
99510001005
proileprothrpheglyglnthrileglnthrthrvalasparggly
101010151020
leuleuphephealagluasnglyaspargpheileserleuasnile
1025103010351040
gluaspglylysleumetvalargtyrlysleuasnsergluleupro
104510501055
lysgluargglyvalglyaspalaileasnasnglyargasphisser
106010651070
ileglnilelysileglylysleuglnlysargmettrpileasnval
107510801085
aspvalglnasnthrileileaspglygluvalpheasppheserthr
109010951100
tyrtyrleuglyglyileproilealailearggluargpheasnile
1105111011151120
serthrproalapheargglycysmetlysasnleulyslysthrser
112511301135
glyvalvalargleuasnaspthrvalglyvalthrlyslyscysser
114011451150
gluasptrplysleuvalargseralaserpheserargglyglygln
115511601165
leuserphethraspleuglyleuproprothrasphisleuglnala
117011751180
serpheglypheglnthrpheglnproserglyileleuleuasphis
1185119011951200
glnthrtrpthrargasnleuglnvalthrleugluaspglytyrile
120512101215
gluleuserthrseraspserglyserproilephelysserprogln
122012251230
thrtyrmetaspglyleuleuhistyrvalservalileseraspasn
123512401245
serglyleuargleuleuileaspaspglnleuleuargasnserlys
125012551260
argleulyshisileserserserargglnserleuargleuglygly
1265127012751280
serasnphegluglycysileserasnvalphevalglnargleuser
128512901295
leuserprogluvalleuaspleuthrserasnserleulysargasp
130013051310
valserleuglyglycysserleuasnlyspropropheleumetleu
131513201325
leulysglyserthrargpheasnlysthrlysthrpheargileasn
133013351340
glnleuleuglnaspthrprovalalaserproargservallysval
1345135013551360
trpglnaspalacysserproleuprolysthrglnalaasnhisgly
136513701375
alaleuglnpheglyaspileprothrserhisleuleuphelysleu
138013851390
proglngluleuleulysproargserglnphealavalaspmetgln
139514001405
thrthrserserargglyleuvalphehisthrglythrlysasnser
141014151420
phemetalaleutyrleuserlysglyargleuvalphealaleugly
1425143014351440
thraspglylyslysleuargilelysserlysglulyscysasnasp
144514501455
glylystrphisthrvalvalpheglyhisaspglyglulysglyarg
146014651470
leuvalvalaspglyleuargalaarggluglyserleuproglyasn
147514801485
serthrileserileargalaprovaltyrleuglyserproproser
149014951500
glylysprolysserleuprothrasnserphevalglycysleulys
1505151015151520
asnpheglnleuaspserlysproleutyrthrproserserserphe
152515301535
glyvalsersercysleuglyglyproleuglulysglyiletyrphe
154015451550
serglugluglyglyhisvalvalleualahisservalleuleugly
155515601565
progluphelysleuvalpheserileargproargserleuthrgly
157015751580
ileleuilehisileglyserglnproglylyshisleucysvaltyr
1585159015951600
leuglualaglylysvalthralasermetaspserglyalaglygly
160516101615
thrserthrservalthrprolysglnserleucysaspglyglntrp
162016251630
hisservalalavalthrilelysglnhisileleuhisleugluleu
163516401645
aspthraspsersertyrthralaglyglnilepropheproproala
165016551660
serthrglngluproleuhisleuglyglyalaproalaasnleuthr
1665167016751680
thrleuargileprovaltrplysserphepheglycysleuargasn
168516901695
ilehisvalasnhisileprovalprovalthrglualaleugluval
170017051710
glnglyprovalserleuasnglycysproaspgln
17151720
<210>15
<211>1172
<212>prt
<213>人工序列
<220>
<223>seqidno15lamb3
<400>15
metargprophepheleuleucysphealaleuproglyleuleuhis
151015
alaglnglnalacysserargglyalacystyrproprovalglyasp
202530
leuleuvalglyargthrargpheleuargalaserserthrcysgly
354045
leuthrlysprogluthrtyrcysthrglntyrglyglutrpglnmet
505560
lyscyscyslyscysaspserargglnprohisasntyrtyrserhis
65707580
argvalgluasnvalalaserserserglyprometargtrptrpgln
859095
serglnasnaspvalasnprovalserleuglnleuaspleuasparg
100105110
argpheglnleuglngluvalmetmetglupheglnglyprometpro
115120125
alaglymetleuilegluargserserasppheglylysthrtrparg
130135140
valtyrglntyrleualaalaaspcysthrserthrpheproargval
145150155160
argglnglyargproglnsertrpglnaspvalargcysglnserleu
165170175
proglnargproasnalaargleuasnglyglylysvalglnleuasn
180185190
leumetaspleuvalserglyileproalathrglnserglnlysile
195200205
glngluvalglygluilethrasnleuargvalasnphethrargleu
210215220
alaprovalproglnargglytyrhisproproseralatyrtyrala
225230235240
valserglnleuargleuglnglysercysphecyshisglyhisala
245250255
aspargcysalaprolysproglyalaseralaglyproserthrala
260265270
valglnvalhisaspvalcysvalcysglnhisasnthralaglypro
275280285
asncysgluargcysalaprophetyrasnasnargprotrpargpro
290295300
alagluglyglnaspalahisglucysglnargcysaspcysasngly
305310315320
hissergluthrcyshispheaspproalavalphealaalasergln
325330335
glyalatyrglyglyvalcysaspasncysargasphisthrglugly
340345350
lysasncysgluargcysglnleuhistyrpheargasnargargpro
355360365
glyalaserileglngluthrcysilesercysglucysaspproasp
370375380
glyalavalproglyalaprocysaspprovalthrglyglncysval
385390395400
cyslysgluhisvalglnglygluargcysaspleucyslysprogly
405410415
phethrglyleuthrtyralaasnproglnglycyshisargcysasp
420425430
cysasnileleuglyserargargaspmetprocysaspglugluser
435440445
glyargcysleucysleuproasnvalvalglyprolyscysaspgln
450455460
cysalaprotyrhistrplysleualaserglyglnglycysglupro
465470475480
cysalacysaspprohisasnserleuserproglncysasnglnphe
485490495
thrglyglncysprocysarggluglypheglyglyleumetcysser
500505510
alaalaalaileargglncysproaspargthrtyrglyaspvalala
515520525
thrglycysargalacysaspcysasppheargglythrgluglypro
530535540
glycysasplysalaserglyargcysleucysargproglyleuthr
545550555560
glyproargcysaspglncysglnargglytyrcysasnargtyrpro
565570575
valcysvalalacyshisprocyspheglnthrtyraspalaaspleu
580585590
arggluglnalaleuargpheglyargleuargasnalathralaser
595600605
leutrpserglyproglyleugluaspargglyleualaserargile
610615620
leuaspalalysserlysilegluglnileargalavalleuserser
625630635640
proalavalthrgluglngluvalalaglnvalalaseralaileleu
645650655
serleuargargthrleuglnglyleuglnleuaspleuproleuglu
660665670
glugluthrleuserleuproargaspleugluserleuaspargser
675680685
pheasnglyleuleuthrmettyrglnarglysarggluglnpheglu
690695700
lysileserseralaaspproserglyalapheargmetleuserthr
705710715720
alatyrgluglnseralaglnalaalaglnglnvalseraspserser
725730735
argleuleuaspglnleuargaspserargargglualagluargleu
740745750
valargglnalaglyglyglyglyglythrglyserprolysleuval
755760765
alaleuargleuglumetserserleuproaspleuthrprothrphe
770775780
asnlysleucysglyasnserargglnmetalacysthrproileser
785790795800
cysproglygluleucysproglnaspasnglythralacysglyser
805810815
argcysargglyvalleuproargalaglyglyalapheleumetala
820825830
glyglnvalalagluglnleuargglypheasnalaglnleuglnarg
835840845
thrargglnmetileargalaalaglugluseralaserglnilegln
850855860
serseralaglnargleugluthrglnvalseralaserargsergln
865870875880
metglugluaspvalargargthrargleuleuileglnglnvalarg
885890895
asppheleuthraspproaspthraspalaalathrileglngluval
900905910
serglualavalleualaleutrpleuprothraspseralathrval
915920925
leuglnlysmetasngluileglnalailealaalaargleuproasn
930935940
valaspleuvalleuserglnthrlysglnaspilealaargalaarg
945950955960
argleuglnalaglualagluglualaargserargalahisalaval
965970975
gluglyglnvalgluaspvalvalglyasnleuargglnglythrval
980985990
alaleuglnglualaglnaspthrmetglnglythrserargserleu
99510001005
argleuileglnaspargvalalagluvalglnglnvalleuargpro
101010151020
alaglulysleuvalthrsermetthrlysglnleuglyaspphetrp
1025103010351040
thrargmetglugluleuarghisglnalaargglnglnglyalaglu
104510501055
alavalglnalaglnglnleualagluglyalasergluglnalaleu
106010651070
seralaglngluglyphegluargilelysglnlystyralagluleu
107510801085
lysaspargleuglyglnsersermetleuglygluglnglyalaarg
109010951100
ileglnservallysthrglualaglugluleupheglygluthrmet
1105111011151120
glumetmetaspargmetlysaspmetgluleugluleuleuarggly
112511301135
serglnalailemetleuargseralaaspleuthrglyleuglulys
114011451150
argvalgluglnileargasphisileasnglyargvalleutyrtyr
115511601165
alathrcyslys
1170
<210>16
<211>1111
<212>prt
<213>人工序列
<220>
<223>seqidno16lamc2-短
<400>16
metproalaleutrpleuglycyscysleucyspheserleuleuleu
151015
proalaalaargalathrserargarggluvalcysaspcysasngly
202530
lysserargglncysilepheasparggluleuhisargglnthrgly
354045
asnglypheargcysleuasncysasnaspasnthraspglyilehis
505560
cysglulyscyslysasnglyphetyrarghisarggluargasparg
65707580
cysleuprocysasncysasnserlysglyserleuseralaargcys
859095
aspasnserglyargcyssercyslysproglyvalthrglyalaarg
100105110
cysaspargcysleuproglyphehismetleuthraspalaglycys
115120125
thrglnaspglnargleuleuaspserlyscysaspcysaspproala
130135140
glyilealaglyprocysaspalaglyargcysvalcyslysproala
145150155160
valthrglygluargcysaspargcysargserglytyrtyrasnleu
165170175
aspglyglyasnprogluglycysthrglncysphecystyrglyhis
180185190
seralasercysargserseralaglutyrservalhislysilethr
195200205
serthrphehisglnaspvalaspglytrplysalavalglnargasn
210215220
glyserproalalysleuglntrpserglnarghisglnaspvalphe
225230235240
serseralaglnargleuaspprovaltyrphevalalaproalalys
245250255
pheleuglyasnglnglnvalsertyrglyglnserleuserpheasp
260265270
tyrargvalaspargglyglyarghisproseralahisaspvalile
275280285
leugluglyalaglyleuargilethralaproleumetproleugly
290295300
lysthrleuprocysglyleuthrlysthrtyrthrpheargleuasn
305310315320
gluhisproserasnasntrpserproglnleusertyrpheglutyr
325330335
argargleuleuargasnleuthralaleuargileargalathrtyr
340345350
glyglutyrserthrglytyrileaspasnvalthrleuileserala
355360365
argprovalserglyalaproalaprotrpvalgluglncysilecys
370375380
provalglytyrlysglyglnphecysglnaspcysalaserglytyr
385390395400
lysargaspseralaargleuglypropheglythrcysileprocys
405410415
asncysglnglyglyglyalacysaspproaspthrglyaspcystyr
420425430
serglyaspgluasnproaspileglucysalaaspcysproilegly
435440445
phetyrasnaspprohisaspproargsercyslysprocysprocys
450455460
hisasnglyphesercysservalmetprogluthrglugluvalval
465470475480
cysasnasncysproproglyvalthrglyalaargcysgluleucys
485490495
alaaspglytyrpheglyasppropheglygluhisglyprovalarg
500505510
procysglnprocysglncysasnasnasnvalaspproseralaser
515520525
glyasncysaspargleuthrglyargcysleulyscysilehisasn
530535540
thralaglyiletyrcysaspglncyslysalaglytyrpheglyasp
545550555560
proleualaproasnproalaasplyscysargalacysasncysasn
565570575
prometglysergluprovalglycysargseraspglythrcysval
580585590
cyslysproglypheglyglyproasncysgluhisglyalapheser
595600605
cysproalacystyrasnglnvallysileglnmetaspglnphemet
610615620
glnglnleuglnargmetglualaleuileserlysalaglnglygly
625630635640
aspglyvalvalproaspthrgluleugluglyargmetglnglnala
645650655
gluglnalaleuglnaspileleuargaspalaglnileserglugly
660665670
alaserargserleuglyleuglnleualalysvalargserglnglu
675680685
asnsertyrglnserargleuaspaspleulysmetthrvalgluarg
690695700
valargalaleuglyserglntyrglnasnargvalargaspthrhis
705710715720
argleuilethrglnmetglnleuserleualagluserglualaser
725730735
leuglyasnthrasnileproalaserasphistyrvalglyproasn
740745750
glyphelysserleualaglnglualathrargleualagluserhis
755760765
valgluseralaserasnmetgluglnleuthrarggluthrgluasp
770775780
tyrserlysglnalaleuserleuvalarglysalaleuhisglugly
785790795800
valglyserglyserglyserproaspglyalavalvalglnglyleu
805810815
valglulysleuglulysthrlysserleualaglnglnleuthrarg
820825830
glualathrglnalagluileglualaaspargsertyrglnhisser
835840845
leuargleuleuaspservalserargleuglnglyvalseraspgln
850855860
serpheglnvalgluglualalysargilelysglnlysalaaspser
865870875880
leuserserleuvalthrarghismetaspgluphelysargthrgln
885890895
lysasnleuglyasntrplysgluglualaglnglnleuleuglnasn
900905910
glylysserglyargglulysseraspglnleuleuserargalaasn
915920925
leualalysserargalaglnglualaleusermetglyasnalathr
930935940
phetyrgluvalgluserileleulysasnleuargglupheaspleu
945950955960
glnvalaspasnarglysalaglualagluglualametlysargleu
965970975
sertyrileserglnlysvalseraspalaserasplysthrglngln
980985990
alagluargalaleuglyseralaalaalaaspalaglnargalalys
99510001005
asnglyalaglyglualaleugluilesersergluilegluglnglu
101010151020
ileglyserleuasnleuglualaasnvalthralaaspglyalaleu
1025103010351040
alametglulysglyleualaserleulysserglumetarggluval
104510501055
gluglygluleugluarglysgluleuglupheaspthrasnmetasp
106010651070
alavalglnmetvalilethrglualaglnlysvalaspthrargala
107510801085
lysasnalaglyvalthrileglnaspthrleuasnthrleuaspgly
109010951100
leuleuhisleumetglymet
11051110
<210>17
<211>1800
<212>prt
<213>人工序列
<220>
<223>seqidno17b4-整联蛋白-his
<400>17
metalaglyproargproserprotrpalaargleuleuleualaala
151015
leuileservalserleuserglythrleualaasnargcyslyslys
202530
alaprovallyssercysthrglucysvalargvalasplysaspcys
354045
alatyrcysthraspglumetpheargaspargargcysasnthrgln
505560
alagluleuleualaalaglycysglnarggluserilevalvalmet
65707580
gluserserpheglnilethrglugluthrglnileaspthrthrleu
859095
argargserglnmetserproglnglyleuargvalargleuargpro
100105110
glyglugluarghisphegluleugluvalphegluproleugluser
115120125
provalaspleutyrileleumetasppheserasnsermetserasp
130135140
aspleuaspasnleulyslysmetglyglnasnleualaargvalleu
145150155160
serglnleuthrserasptyrthrileglypheglylysphevalasp
165170175
lysvalservalproglnthraspmetargproglulysleulysglu
180185190
protrpproasnserasppropropheserphelysasnvalileser
195200205
leuthrgluaspvalaspglupheargasnlysleuglnglygluarg
210215220
ileserglyasnleuaspalaprogluglyglypheaspalaileleu
225230235240
glnthralavalcysthrargaspileglytrpargproaspserthr
245250255
hisleuleuvalpheserthrgluseralaphehistyrglualaasp
260265270
glyalaasnvalleualaglyilemetserargasnaspgluargcys
275280285
hisleuaspthrthrglythrtyrthrglntyrargthrglnasptyr
290295300
proservalprothrleuvalargleuleualalyshisasnileile
305310315320
proilephealavalthrasntyrsertyrsertyrtyrglulysleu
325330335
histhrtyrpheprovalserserleuglyvalleuglngluaspser
340345350
serasnilevalgluleuleugluglualapheasnargileargser
355360365
asnleuaspileargalaleuaspserproargglyleuargthrglu
370375380
valthrserlysmetpheglnlysthrargthrglyserphehisile
385390395400
argargglygluvalglyiletyrglnvalglnleuargalaleuglu
405410415
hisvalaspglythrhisvalcysglnleuprogluaspglnlysgly
420425430
asnilehisleulysproserpheseraspglyleulysmetaspala
435440445
glyileilecysaspvalcysthrcysgluleuglnlysgluvalarg
450455460
seralaargcysserpheasnglyaspphevalcysglyglncysval
465470475480
cyssergluglytrpserglyglnthrcysasncysserthrglyser
485490495
leuseraspileglnprocysleuarggluglygluasplysprocys
500505510
serglyargglyglucysglncysglyhiscysvalcystyrglyglu
515520525
glyargtyrgluglyglnphecysglutyraspasnpheglncyspro
530535540
argthrserglypheleucysasnaspargglyargcyssermetgly
545550555560
glncysvalcysgluproglytrpthrglyprosercysaspcyspro
565570575
leuserasnalathrcysileaspserasnglyglyilecysasngly
580585590
argglyhiscysglucysglyargcyshiscyshisglnglnserleu
595600605
tyrthraspthrilecysgluileasntyrseralailehisprogly
610615620
leucysgluaspleuargsercysvalglncysglnalatrpglythr
625630635640
glyglulyslysglyargthrcysgluglucysasnphelysvallys
645650655
metvalaspgluleulysargalaglugluvalvalvalargcysser
660665670
pheargaspgluaspaspaspcysthrtyrsertyrthrmetglugly
675680685
aspglyalaproglyproasnserthrvalleuvalhislyslyslys
690695700
aspcysproproglyserphetrptrpleuileproleuleuleuleu
705710715720
leuleuproleuleualaleuleuleuleuleucystrplystyrcys
725730735
alacyscyslysalacysleualaleuleuprocyscysasnarggly
740745750
hismetvalglyphelysgluasphistyrmetleuarggluasnleu
755760765
metalaserasphisleuaspthrprometleuargserglyasnleu
770775780
lysglyargaspvalvalargtrplysvalthrasnasnmetglnarg
785790795800
proglyphealathrhisalaalaserileasnprothrgluleuval
805810815
protyrglyleuserleuargleualaargleucysthrgluasnleu
820825830
leulysproaspthrargglucysalaglnleuargglngluvalglu
835840845
gluasnleuasngluvaltyrargglnileserglyvalhislysleu
850855860
glnglnthrlyspheargglnglnproasnalaglylyslysglnasp
865870875880
histhrilevalaspthrvalleumetalaproargseralalyspro
885890895
alaleuleulysleuthrglulysglnvalgluglnargalaphehis
900905910
aspleulysvalalaproglytyrtyrthrleuthralaaspglnasp
915920925
alaargglymetvalglupheglngluglyvalgluleuvalaspval
930935940
argvalproleupheileargprogluaspaspaspglulysglnleu
945950955960
leuvalglualaileaspvalproalaglythralathrleuglyarg
965970975
argleuvalasnilethrileilelysgluglnalaargaspvalval
980985990
serphegluglnproglupheservalserargglyaspglnvalala
99510001005
argileprovalileargargvalleuaspglyglylysserglnval
101010151020
sertyrargthrglnaspglythralaglnglyasnargasptyrile
1025103010351040
provalgluglygluleuleupheglnproglyglualatrplysglu
104510501055
leuglnvallysleuleugluleuglngluvalaspserleuleuarg
106010651070
glyargglnvalargargphehisvalglnleuserasnprolysphe
107510801085
glyalahisleuglyglnprohisserthrthrileileileargasp
109010951100
proaspgluleuaspargserphethrserglnmetleusersergln
1105111011151120
proproprohisglyaspleuglyalaproglnasnproasnalalys
112511301135
alaalaglyserarglysilehispheasntrpleuproprosergly
114011451150
lysprometglytyrargvallystyrtrpileglnglyaspserglu
115511601165
serglualahisleuleuaspserlysvalproservalgluleuthr
117011751180
asnleutyrprotyrcysasptyrglumetlysvalcysalatyrgly
1185119011951200
alaglnglygluglyprotyrserserleuvalsercysargthrhis
120512101215
glngluvalprosergluproglyargleualapheasnvalvalser
122012251230
serthrvalthrglnleusertrpalagluproalagluthrasngly
123512401245
gluilethralatyrgluvalcystyrglyleuvalasnaspaspasn
125012551260
argproileglyprometlyslysvalleuvalaspasnprolysasn
1265127012751280
argmetleuleuilegluasnleuarggluserglnprotyrargtyr
128512901295
thrvallysalaargasnglyalaglytrpglyprogluarggluala
130013051310
ileileasnleualathrglnprolysargprometserileproile
131513201325
ileproaspileproilevalaspalaglnserglygluasptyrasp
133013351340
serpheleumettyrseraspaspvalleuargserproserglyser
1345135013551360
glnargproservalseraspaspthrglycysglytrplyspheglu
136513701375
proleuleuglyglugluleuaspleuargargvalthrtrpargleu
138013851390
proprogluleuileproargleuseralaserserglyargserser
139514001405
aspalaglualaprohisglyproproaspaspglyglyalaglygly
141014151420
lysglyglyserleuproargseralathrproglyproproglyglu
1425143014351440
hisleuvalasnglyargmetaspphealapheproglyserthrasn
144514501455
serleuhisargmetthrthrthrseralaalaalatyrglythrhis
146014651470
leuserprohisvalprohisargvalleuserthrserserthrleu
147514801485
thrargasptyrasnserleuthrargsergluhisserhisserthr
149014951500
thrleuproargasptyrserthrleuthrservalserserhisasp
1505151015151520
serargleuthralaglyvalproaspthrprothrargleuvalphe
152515301535
seralaleuglyprothrserleuargvalsertrpglngluproarg
154015451550
cysgluargproleuglnglytyrservalglutyrglnleuleuasn
155515601565
glyglygluleuhisargleuasnileproasnproalaglnthrser
157015751580
valvalvalgluaspleuleuproasnhissertyrvalpheargval
1585159015951600
argalaglnserglngluglytrpglyarggluarggluglyvalile
160516101615
thrilegluserglnvalhisproglnserproleucysproleupro
162016251630
glyseralaphethrleuserthrproseralaproglyproleuval
163516401645
phethralaleuserproaspserleuglnleusertrpgluargpro
165016551660
argargproasnglyaspilevalglytyrleuvalthrcysglumet
1665167016751680
alaglnglyglyglyproalathralapheargvalaspglyaspser
168516901695
progluserargleuthrvalproglyleusergluasnvalprotyr
170017051710
lysphelysvalglnalaargthrthrgluglypheglyprogluarg
171517201725
gluglyileilethrilegluserglnaspglyglypropheprogln
173017351740
leuglyserargalaglyleupheglnhisproleuglnserglutyr
1745175017551760
serserilethrthrthrhisthrseralathrglupropheleuval
176517701775
aspglyprothrleuglyalaglnhisleuglualaglyglyleuglu
178017851790
hishishishishishishishis
17951800
<210>18
<211>787
<212>prt
<213>人工序列
<220>
<223>seqidno18bp230-g-c-his
<400>18
metaspcysthrphelysproasppheglumetthrvallysglucys
151015
glnhisserglygluleuserserargasnthrglyhisleuhispro
202530
thrproargserproleuleuargtrpthrglngluproglnproleu
354045
gluglulystrpglnhisargvalvalgluglnileprolysgluval
505560
glnpheglnproproglyalaproleuglulysglulysserglngln
65707580
cystyrserglutyrpheserglnthrserthrgluleuglnilethr
859095
pheaspgluthrasnproilethrargleusergluileglulysile
100105110
argaspglnalaleuasnasnserargproprovalargtyrglnasp
115120125
asnalacysglumetgluleuvallysvalleuthrproleugluile
130135140
alalysasnlysglntyraspmethisthrgluvalthrthrleulys
145150155160
glnglulysasnprovalproseralagluglutrpmetleuglugly
165170175
cysargalaserglyglyleulyslysglyasppheleulyslysgly
180185190
leugluprogluthrpheglnasnpheaspglyasphisalacysser
195200205
valargaspaspgluphelyspheglnglyleuarghisthrvalthr
210215220
alaargglnleuvalglualalysleuleuaspmetargthrileglu
225230235240
glnleuargleuglyleulysthrvalglugluvalglnlysthrleu
245250255
asnlyspheleuthrlysalathrserilealaglyleutyrleuglu
260265270
serthrlysglulysileserphealaseralaalagluargileile
275280285
ileasplysmetvalalaleualapheleuglualaglnalaalathr
290295300
glypheileileaspproileserglyglnthrtyrservalgluasp
305310315320
alavalleulysglyvalvalaspproglupheargileargleuleu
325330335
glualaglulysalaalavalglytyrsertyrserserlysthrleu
340345350
servalpheglnalametgluasnargmetleuaspargglnlysgly
355360365
lyshisileleuglualaglnilealaserglyglyvalileasppro
370375380
valargglyileargvalproprogluilealaleuglnglnglyleu
385390395400
leuasnasnalaileleuglnpheleuhisgluproserserasnthr
405410415
argvalpheproasnproasnasnlysglnalaleutyrtyrserglu
420425430
leuleuargmetcysvalpheaspvalgluserglncyspheleuphe
435440445
propheglygluargasnileserasnleuasnvallyslysthrhis
450455460
argileservalvalaspthrlysthrglysergluleuthrvaltyr
465470475480
glualapheglnargasnleuileglulysseriletyrleugluleu
485490495
serglyglnglntyrglntrplysglualametphepheglusertyr
500505510
glyhisserserhismetleuthraspthrlysthrglyleuhisphe
515520525
asnileasnglualailegluglnglythrileasplysalaleuval
530535540
lyslystyrglngluglyleuilethrleuthrgluleualaaspser
545550555560
leuleuserargleuvalprolyslysaspleuhisserprovalala
565570575
glytyrtrpleuthralaserglygluargileservalleulysala
580585590
serargargasnleuvalaspargilethralaleuargcysleuglu
595600605
alaglnvalserthrglyglyileileaspproleuthrglylyslys
610615620
tyrargvalalaglualaleuhisargglyleuvalaspgluglyphe
625630635640
alaglnglnleuargglncysgluleuvalilethrglyileglyhis
645650655
proilethrasnlysmetmetservalvalglualavalasnalaasn
660665670
ileileasnlysglumetglyileargcysleuglupheglntyrleu
675680685
thrglyglyleuilegluproglnvalhisserargleuserileglu
690695700
glualaleuglnvalglyileileaspvalleuilealathrlysleu
705710715720
lysaspglnlyssertyrvalargasnileilecysproglnthrlys
725730735
arglysleuthrtyrlysglualaleuglulysalaasppheaspphe
740745750
histhrglyleulysleuleugluvalsergluproleumetthrgly
755760765
ileserserleutyrtyrserserleuleugluhishishishishis
770775780
hishishis
785