CN111471096B - 用于阿尔茨海默症自身抗体检测的含有adarb1蛋白片段组合物 - Google Patents

用于阿尔茨海默症自身抗体检测的含有adarb1蛋白片段组合物 Download PDF

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CN111471096B
CN111471096B CN202010304884.5A CN202010304884A CN111471096B CN 111471096 B CN111471096 B CN 111471096B CN 202010304884 A CN202010304884 A CN 202010304884A CN 111471096 B CN111471096 B CN 111471096B
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朱树勇
邹永红
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Abstract

本发明属于生物检测领域，具体涉及一种用于阿尔茨海默症自身抗体检测的含有ADARB1蛋白的蛋白片段组合物。所采用的技术方案为：一种阿尔茨海默症检测用抗原，所述抗原为蛋白片段组合物。所述蛋白片段组合物包括：ADARB1蛋白的蛋白片段、HSP60蛋白的蛋白片段、ASXL1蛋白的蛋白片段；所述ADARB1蛋白的蛋白片段序列如SEQ ID NO.2所示，所述HSP60蛋白的蛋白片段序列如SEQ ID NO.3所示，所述ASXL1蛋白的蛋白片段序列如SEQ ID NO.8所示。基于上述抗原，可以制备用于诊断、尤其是早期诊断阿尔茨海默症或其相关风险的试剂盒。使用本发明提供的抗原，可针对性地快速、准确诊断出早期阿尔茨海默症，具有重要的现实意义。

Description

用于阿尔茨海默症自身抗体检测的含有ADARB1蛋白片段组 合物
技术领域
本发明属于生物检测领域，具体涉及一种用于阿尔茨海默症自身抗体检测的含有ADARB1蛋白片段组合物。
背景技术
阿尔茨海默症(Alzheimer＇s disease，简称AD)是以记忆和认知功能障碍为主要特征的进行性神经变性疾病，多发于老年群体，病程缓慢且不可逆。按照认知功能障碍的发展，可以将阿尔茨海默症分成早、中、晚期。在早期，患者没有明显的症状，多数只表现出健忘、焦虑，因此难以察觉或确诊；中期则会出现混乱性症状，性格、脾性都会发生改变，记忆力也出现混乱；到后期，患者完全表现为痴呆，没有自理的生活能力，最终多继发多种疾病而死亡。
目前对阿尔茨海默症的诊断方式包括如下几种：1、出现早期和显著的情景记忆障碍；2、颞中回萎缩；3、异常的脑脊液生物标记，包括β淀粉样蛋白1-42(Aβ1-42)浓度降低，总Tau蛋白浓度升高，磷酸化Tau蛋白浓度升高，或此三者的组合；4、PET功能神经影像的特异性成像，双侧颞、顶叶葡萄糖代谢率减低；5、直系亲属中有明确的AD相关的常染色体显性突变。然而，由于早期症状不明显、中期与其他疾病难以区分等问题，阿尔茨海默症的诊断尚存在较为显著的漏诊、误诊问题，因此急需AD诊断或分期的有效工具。
自身抗体是由个体免疫系统产生的、针对个体自身蛋白抗原的抗体。正常情况下，免疫系统响应体内的外源性蛋白或物质而产生抗体，但有时也会识别机体的一种或多种内源性组分，导致产生自身抗体。已经有大量证据证实，血清中存在多种自身抗体参与神经性疾病和综合症。就阿尔茨海默症而言，有许多文献报道了患者具有高滴度的针对非脑和脑相关靶的自身抗体，包括结合神经元的自身抗体。
血清中抗体的检测已经是一种很成熟的技术，这使得阿尔茨海默症自身抗体的精确检测成为可能，进而可以以血清中阿尔茨海默症自身抗体作为检测靶标，实现诊断目的。例如，中国专利申请公布号CN103154736A公开了一系列蛋白，该文献提出，这些蛋白潜在地可用作结合阿尔茨海默症自身抗体的抗原，因此可用作阿尔茨海默症的诊断性指示剂。
然而，CN103154736A中公开的蛋白抗原的数量极其庞杂，仍需要从中选择出可以组合使用的蛋白，作为蛋白抗原组合以高特异性和灵敏度检测受试者血清中可能存在的阿尔茨海默症自身抗体，以提供切实可行的阿尔茨海默症诊断或分期手段。况且，即使就单个蛋白抗原而言，蛋白全长与可能存在的对应的阿尔茨海默症自身抗体的结合作用可能很弱，导致检测效果差，进而检测失效、甚至误诊。因此，无论是单独使用还是组合使用，筛选出特定蛋白抗原组合、或者筛选出蛋白中的特定区域或片段以有效结合、检测对应的自身抗体，是至关重要的。
发明内容
本发明的目的是提供一种用于检测人血清样本中阿尔茨海默症自身抗体的蛋白抗原组合及其应用。
为实现上述发明目的，本发明所采用的技术方案是：一种抗原组合，所述抗原组合至少包括三种下述蛋白的全蛋白和/或蛋白片段：MAPT、ADARB1、HSP60、P21、DAG、DNAJC8、RAGE、ASXL1、JMJD2D。
优选的，所述抗原组合中，所述蛋白片段中至少包括9个氨基酸。
优选的，选用蛋白片段或选用蛋白片段与全蛋白组合时，所述蛋白片段序列选自如下序列之一或其组合：
所述MAPT蛋白片段序列如SEQ ID NO.1所示；
所述ADARB1蛋白片段序列如SEQ ID NO.2所示；
所述HSP60蛋白片段序列如SEQ ID NO.3所示；
所述P21蛋白片段序列如SEQ ID NO.4所示；
所述DAG蛋白片段序列如SEQ ID NO.5所示；
所述DNAJC8蛋白片段序列如SEQ ID NO.6所示；
所述RAGE蛋白片段序列如SEQ ID NO.7所示；
所述ASXL1蛋白片段序列如SEQ ID NO.8所示；
所述JMJD2D蛋白片段序列如SEQ ID NO.9所示。
优选的，同时包括MAPT蛋白片段、ADARB1蛋白片段、HSP60蛋白片段。
优选的，同时包括DNAJC8蛋白片段、RAGE蛋白片段、ASXL1蛋白片段。
优选的，同时包括JMJD2D蛋白片段、P21蛋白片段、DAG蛋白片段。
优选的，同时包括MAPT蛋白片段、HSP60蛋白片段、DNAJC8蛋白片段；或；
同时包括ADARB1蛋白片段、RAGE蛋白片段、ASXL1蛋白片段；或；
同时包括HSP60蛋白片段、RAGE蛋白片段、P21蛋白片段。
相应的，所述抗原组合在检测阿尔茨海默症中的应用。
相应的，利用所述抗原组合制备的检测阿尔茨海默症的试剂盒。
优选的，所述试剂盒所用样本为全血或血清。
本发明具有以下有益效果：
本发明提供了一种用于检测阿尔茨海默症自身抗体的蛋白抗原组合物，其中所涉及的蛋白抗原或其片段可利用人工合成的方法制得。例如，合成蛋白抗原或其片段的编码DNA，以合成DNA为模板，设计引物，通过PCR、酶切、连接等分子克隆手段，将所述蛋白抗原或其片段的基因片段克隆到表达质粒上。同时，还可选择性地在蛋白抗原或其片段的N-端增加HIS、c-myc等标签。
本发明提供的抗原组合具有下述用途：
(1)测试来自受试者的生物学样品中针对上述抗原组合的自身抗体的存在或存在水平，从而判断所述受试者是否患有阿尔兹海默症；预测所述受试者是否具有罹患阿尔兹海默症的风险；评估所述受试者所患有的阿尔兹海默症的进展；或者判断所述受试者是否具有阿尔兹海默症的复发风险。其中，所述生物学样品可以是血清、血浆、全血、唾液、口腔粘膜拭子、尿液、淋巴液、脑脊液等等。根据具体情况，所述生物学样品可经提取、稀释、富集等手段进行预处理。使用方法多样、简便易操作。
在将本发明提供的抗原组合用于上述测试时，通过使所述抗原组合中的蛋白或其片段与可能存在的对应自身抗体发生结合或相互作用，来测试该自身抗体的存在或存在水平。
(2)本发明提供的蛋白抗原组合还可用于制备阿尔茨海默症自身抗体检测试剂或阿尔茨海默症诊断试剂。应当理解的，所述蛋白抗原组合还可用于制备阿尔茨海默症自身抗体检测试剂盒，所述试剂盒可以参照本发明实施例中使用所述蛋白抗原组合进行阿尔茨海默症自身抗体检测时的方法和试剂制备，也可根据需要进行相应调整。
综上，本发明提供了一种蛋白抗原组合，其可用于阿尔兹海默症的检测或诊断，尤其适用于早期检测或诊断；还可用于阿尔兹海默症的患病风险预测；并可根据需要进一步制备为相关试剂或试剂盒。
具体实施方式
下面结合具体实施例对本申请进行进一步阐释。如无特殊说明，下述实施例中所使用的实验方法均为常规方法；所用的材料、试剂等均可从商业途径得到；所获得的数据均为进行至少3次重复后获得的平均值，且各重复获得的均为有效数据。
实施例一：抗原的重组载体构建、表达和纯化
1、抗原的选择。选择115种与阿尔茨海默症高度相关的抗原蛋白进行构建、表达和纯化。为节省文章篇幅，本文中只体现其中具有代表性的其中50，其分别的数据库ID具体如表1所示。
表1待测蛋白的数据库ID对应表
Figure GDA0003299800950000051
2、抗原重组载体的构建和表达。从表1的50种蛋白中筛选出目标蛋白抗原。以人类cDNA文库(购自Invitrogen公司)或者全基因合成DNA为模板，分别设计引物，通过PCR、酶切、连接等分子克隆手段，将所述蛋白的全长基因克隆到pET28质粒上。同时，在蛋白N-端增加HIS、c-myc等标签，形成融合蛋白。将得到的重组表达载体通过DNA测序鉴定，确认包含正确的蛋白基因片段。需要说明的是，添加的标签只是方便识别和提取蛋白，并未对蛋白作为抗原时的功能产生决定性影响，使用时，不添加标签或根据需要添加其它标签均可。
将上述包含蛋白基因片段的重组质粒转化至大肠杆菌BL21(DE3)感受态细胞中，挑取克隆接种至LB培养基中，37℃摇床培养。当菌体密度达到OD600约为0.8时，降温至16℃，在每个LB培养基中加入0.1mM异丙基硫代-β-D-半乳糖苷(IPTG)，诱导表达过夜，获得菌体。
3、抗原的纯化。离心收集诱导表达的所述菌体，用PBS漂洗两遍。用裂解液(每g菌体加5～10ml裂解液)重悬并分散菌体，冰浴，超声破碎菌体(超声功率200W，破碎5S，休息5S)。破碎后13000rpm，10℃，离心20分钟，取上清，经Ni柱亲和层析和分子筛层析两步纯化后，采用SDS-PAGE电泳分析，确认蛋白的分子量、纯度，用Bradford法测定浓度后，保存在-80℃备用。即获得纯化后的待测蛋白。
实施例二：从待测蛋白中筛选获得候选抗原
1、本实施例中使用的溶液和试剂如下：
(1)包被缓冲液为PBS缓冲液，pH＝7.4。其制备方法为：准确称取3.58g Na2HPO4·12H2O、0.23g KH2PO4·2H2O、0.2g KCl、8.0g NaCl，溶解于水中，清水定容至1L。
(2)封闭液/样本稀释液/抗体稀释液：将10g BSA(牛血清白蛋白)溶解于包被缓冲液中，清水定容至1L。
(3)洗涤液：现配现用。使用前在包被缓冲液中加入0.5％Tween20(V/V)，pH＝7.4。
(4)TMB显色剂，购自KPL公司。
(5)终止液：1M盐酸。
2、固相包被待测蛋白。用包被缓冲液将实施例一获得的纯化后的待测蛋白稀释至5μg/ml，加至96孔板，每孔50μl，4℃包被过夜。次日倒掉溶液，甩干，用洗涤液洗三次，每次每孔200μl。然后每孔加入200μl封闭液，室温孵育1h后，将封闭液倒掉，甩干，再用洗涤液洗三次，每次每孔200μl，并再次甩干；获得位于96孔板中的固相包被的抗原。
3、加入待测样本。将待测人血清用样本稀释液稀释100倍后，加入所述含待测蛋白的96孔板，每孔加稀释后的待测样本50μl。随后将96孔板置于微孔板振荡仪上，室温振荡孵育1h；甩干，用洗涤液洗三次，每次每孔200μl，并再次甩干。
4、加入酶标二抗。将1.0mg/ml辣根过氧化物酶标记的重组羊抗人免疫球蛋白G抗体(购自Jackson ImmunoResearch Inc.)用抗体稀释液稀释20000倍后，加入步骤3处理后的96孔板中，每孔加50μl。随后将96孔板置于微孔板振荡仪上，室温振荡孵育0.5h，将板甩干，用洗涤液洗三次，每次每孔200μl，并再次甩干。
5、显色反应及光密度值读数。在步骤4处理后的96孔板中，每孔加50μl TMB显色剂，振荡15s，室温下避光反应15min，再加入50μl终止液；然后用酶标仪读取450nm波长的吸收值，获得每个待测样本的检测信号(S)。
6、敏感性和特异性分析。分别取180例阳性样本(确诊为阿尔兹海默症患者的血清)和180例阴性样本(健康受试者血清)，按上述方法(450nm波长的吸收值)测定每个样本的检测信号(S)。以阴性样本为阴性参考样本，计算所有阴性参考样本检测信号(S)的平均值(M)及标准差(SD)，以M+3SD为Cut Off值。将检测信号(S)≥Cut Off值的样本(S≥M+3SD)定为阳性；将检测信号(S)＜Cut Off值的样本(S＜M+3SD)定为阴性。
基于样本阳性和阴性结果计算特异性和敏感性。其中，特异性是指健康受试者样本被正确地判定为阴性的比例，即在阴性样本中被正确地判定为阴性的数量除以阴性样本总数。敏感性是指阿尔兹海默症患者样本被正确地判定为阳性的比例，即在阳性样本中被判定为阳性的数量除以阳性样本总数。通过计算得出使用每个被测蛋白作为抗原进行样本检测时的敏感性和特异性，结果如表2所示。
表2各被测蛋白作为抗原的敏感性和特异性展示表
Figure GDA0003299800950000071
Figure GDA0003299800950000081
7、从上述各被测蛋白中选择筛选出敏感性≥20％且特异性≥75％的蛋白作为候选抗原。筛选结果如表3所示。
表3从被测蛋白中筛选出的候选抗原展示表
Figure GDA0003299800950000082
Figure GDA0003299800950000091
实施例三：候选抗原的活性位点筛选
1、对实施例二获得的所述候选抗原的氨基酸序列及其结构进行分析，经大量前期试验后，从各所述候选抗原中选择不同的序列片段和抗原表位。所选择的序列片段或全长如表4所示。
表4各候选抗原上截取的序列片段对应表
Figure GDA0003299800950000092
Figure GDA0003299800950000101
2、按照实施例一的方法，对表4中各蛋白片段进行表达载体构建、表达和纯化，得到构建后的蛋白片段，再按实施例二的方法检测各构建后的蛋白片段作为抗原时的敏感性和特异性。与实施例一相同的是，此处虽然也在各蛋白片段中添加了一些标签，但添加标签的目的主要在于便于提取和识别，并未对蛋白片段作为抗原的功能产生实质性影响。使用添加标签前后的蛋白均可实现发明目的，实际使用时，本领域技术人员可以根据需要选择是否添加标签、及添加标签的种类。因此，本发明全文只提供了添加标签前的蛋白序列。测试结果如表5所示。
表5各候选抗原截取片段优化后的敏感性和特异性对照表
Figure GDA0003299800950000111
Figure GDA0003299800950000121
3、根据表5结果可知，对于某些蛋白而言，与使用全蛋白相比，截取特定氨基酸序列区域并进行相应优化后获得的蛋白片段具有更优的检测效果；而对于某些抗原而言，全蛋白的检测效果优于其片段。这可能是因为，在与阿尔兹海默症相关位点结合或识别过程中，某些全蛋白中的部分蛋白片段发挥了抑制作用。
根据表5的结果，进一步筛选出9种蛋白全长或其片段，入选的蛋白全长或其片段的敏感性在33％以上，且特异性在88％以上；作为区分阿尔兹海默症患者和健康受试者的候选抗原。筛选结果如表6所示。
表6各候选抗原的敏感性和特异性对照表
Figure GDA0003299800950000122
Figure GDA0003299800950000131
实施例四：候选抗原的检测效果展示
1、根据表6的筛选结果，从所述候选蛋白抗原中选择形成不同的抗原组合。并基于候选组1～8的候选蛋白片段对应的全蛋白，设置对应的全蛋白组合，分别为对照组1～8；根据专利CN 109738653 A中表5的组合5、6分别设置对照组9、10。具体如表7所示。需要说明的是，发明人并非只进行了表7的组合试验；发明人经过大量前期试验后才获得表7中所示的各抗原组合，因篇幅限制，只选取了部分效果较优的组合。
表7各抗原组合对照表
组别 组合
侯选组1 MAPT[502-758],ADARB1[678-741],HSP60[1-573]
侯选组2 DNAJC8[114-253],RAGE[23-54],ASXL1[1-84]
侯选组3 JMJD2D[1-160],P21[2-164],DAG[1-9]
侯选组4 MAPT[502-758],DNAJC8[114-253],JMJD2D[1-160]
侯选组5 MAPT[502-758],HSP60[1-573],DNAJC8[114-253]
侯选组6 ADARB1[678-741],RAGE[23-54],ASXL1[1-84]
侯选组7 HSP60[1-573],RAGE[23-54],P21[2-164]
侯选组8 ADARB1[678-741],HSP60[1-573],ASXL1[1-84]
侯选组9 MAPT[502-758],RAGE[23-54],JMJD2D[1-160]
侯选组10 ADARB1[678-741],ASXL1[1-84],DAG[1-9]
对照组1 MAPT,ADARB1,HSP60
对照组2 DNAJC8,RAGE,ASXL1
对照组3 JMJD2D,P21,DAG
对照组4 MAPT,DNAJC8,JMJD2D
对照组5 MAPT,HSP60,DNAJC8
对照组6 ADARB1,RAGE,ASXL1
对照组7 HSP60,RAGE,P21
对照组8 ADARB1,HSP60,ASXL1
对照组9 RAGE,DNAJC8,HSP60,MRPL34,ADARB1,CCL2,MAPT,ASXL1,GDF11
对照组10 RAGE,DNAJC8,HSP60,MRPL34,ADARB1,CCL2,MAPT,MBP,GDF11
2、按简易精神状态检查表(Mini-Mental State Examination，MMSE)-Folstein版，对阿尔兹海默症患者和健康受试者进行评分，分数27～30的测试者为正常人，分数21～26的测试者为轻度(早期)患者，分数10～20的测试者为中度(中期)患者，分数0～9的测试者为重度(晚期)患者。
根据MMSE评分，取阿尔兹海默症患者早期180例，中期90例，晚期90例作为检测样本；取180例MMSE评分正常的健康受试者作为健康受试者的样本。参照实施例二的检测方法，分别使用表7中各组抗原组合对上述各样本进行敏感性和特异性检测(需要说明的是，对照组中所用各蛋白为未经切割的全蛋白)。本实施例中的敏感性和特异性具体为总体特异性、总体敏感性、早期敏感性、中期敏感性和晚期敏感性。
其中，早期敏感性、中期敏感性和晚期敏感性对阳性和阴性的定义方法参照实施例二。
早期敏感性是指180例早期阿尔兹海默症患者样本被该组合正确地判定为阳性的比例，即在早期阿尔兹海默症患者样本中被判定为阳性的数量除以早期阿尔兹海默症患者样本总数；中期敏感性是指90例中期阿尔兹海默症患者样本被正确地判定为阳性的比例，即在中期阿尔兹海默症患者样本中被判定为阳性的数量除以中期阿尔兹海默症患者样本总数；晚期敏感性是指90例晚期阿尔兹海默症患者样本被正确地判定为阳性的比例，即在晚期阿尔兹海默症患者样本中被判定为阳性的数量除以晚期阿尔兹海默症患者样本总数。总体敏感性是指在180例早期、90例中期、90例晚期阿尔兹海默症患者样本总共被正确地判定为阳性的比例，即在所有阿尔兹海默症患者样本中被判定为阳性的数量除以所有阿尔兹海默症患者样本总数；总体特异性是指180例健康受试者样本被正确地判定为阴性的比例，即在180例健康受试者样本中，健康受试者样本中被判定为阴性的数量除以健康受试者样本总数。
各组抗原组合检测获得结果如表8所示。
表8各组抗原组合检测结果对照表
组别 早期敏感性 中期敏感性 晚期敏感性 总体敏感性 总体特异性
候选组1 94.44％ 88.89％ 93.33％ 92.78％ 90.00％
候选组2 93.33％ 88.89％ 88.89％ 91.11％ 88.89％
候选组3 94.44％ 84.44％ 91.11％ 91.11％ 88.33％
候选组4 90.00％ 86.67％ 86.67％ 88.33％ 88.33％
候选组5 92.22％ 88.89％ 91.11％ 91.11％ 90.00％
候选组6 94.44％ 84.44％ 91.11％ 91.11％ 88.89％
候选组7 93.33％ 91.11％ 80.00％ 89.44％ 90.00％
候选组8 93.33％ 73.33％ 91.11％ 87.78％ 88.89％
候选组9 74.44％ 88.89％ 91.11％ 82.22％ 88.33％
候选组10 66.67％ 84.44％ 93.33％ 77.78％ 88.89％
对照组1 77.78％ 66.67％ 80.00％ 75.56％ 83.33％
对照组2 76.67％ 73.33％ 77.78％ 76.11％ 80.56％
对照组3 80.00％ 62.22％ 80.00％ 75.56％ 77.78％
对照组4 73.33％ 64.44％ 71.11％ 70.56％ 79.44％
对照组5 75.56％ 66.67％ 75.56％ 73.33％ 80.00％
对照组6 77.78％ 68.89％ 80.00％ 76.11％ 78.89％
对照组7 78.89％ 68.89％ 77.78％ 76.11％ 77.78％
对照组8 76.67％ 64.44％ 80.00％ 74.44％ 75.56％
对照组9 83.33％ 77.78％ 82.22％ 81.67％ 87.22％
对照组10 85.56％ 84.44％ 75.56％ 82.78％ 87.78％
从表8可以看出，候选组1～8具有更高的敏感性和特异性；总体敏感性都大于85％，总体特异性都大于88％。同时，与中期和晚期样本相比，使用本发明提供的抗原组合在检测早期样本时具有更高的敏感性。因此，本发明为阿尔茨海默病的诊断、尤其是早期诊断提供了更为精确的检测方法。
在后期应用时，还可根据需要，从本发明提供的候选抗原或抗原组合中选择一种或多种，制作成AD诊断试剂盒。
序 列 表
<110> 上海众启生物科技有限公司
<120> 用于阿尔兹海默症自身抗体检测的蛋白抗原组合及其应用
<160> 9
<210> 1
<211> 257
<212> PRT
<213> MAPT[502-758]
<400> 1
Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro
1 5 10 15
Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro
20 25 30
Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro
35 40 45
Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val
50 55 60
Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu
65 70 75 80
Asn Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys
85 90 95
Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn
100 105 110
Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro
115 120 125
Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile
130 135 140
His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu
145 150 155 160
Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile
165 170 175
Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu
180 185 190
Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile
195 200 205
Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu
210 215 220
Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln
225 230 235 240
Leu Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly
245 250 255
Leu
<210> 2
<211> 64
<212> PRT
<213> ADARB1[678-741]
<400> 2
Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys Ile Thr
1 5 10 15
Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys Glu Tyr Gln
20 25 30
Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys Ala Gly Leu Gly
35 40 45
Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe Ser Leu Thr Pro
50 55 60
<210> 3
<211> 573
<212> PRT
<213> HSP60[1-573]
<400> 3
Met Leu Arg Leu Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg
1 5 10 15
Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe
20 25 30
Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu Ala
35 40 45
Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr Val Ile Ile
50 55 60
Glu Gln Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val
65 70 75 80
Ala Lys Ser Ile Asp Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys
85 90 95
Leu Val Gln Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly
100 105 110
Thr Thr Thr Ala Thr Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe
115 120 125
Glu Lys Ile Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val
130 135 140
Met Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys
145 150 155 160
Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala
165 170 175
Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala Met Lys Lys
180 185 190
Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp Gly Lys Thr Leu Asn
195 200 205
Asp Glu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile
210 215 220
Ser Pro Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln
225 230 235 240
Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser
245 250 255
Ile Val Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro Leu Val
260 265 270
Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu
275 280 285
Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys Ala Pro Gly
290 295 300
Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala Ile Ala Thr
305 310 315 320
Gly Gly Ala Val Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp
325 330 335
Val Gln Pro His Asp Leu Gly Lys Val Gly Glu Val Ile Val Thr Lys
340 345 350
Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu
355 360 365
Lys Arg Ile Gln Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu
370 375 380
Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly
385 390 395 400
Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu
405 410 415
Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val
420 425 430
Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile
435 440 445
Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gln Lys Ile Gly
450 455 460
Ile Glu Ile Ile Lys Arg Thr Leu Lys Ile Pro Ala Met Thr Ile Ala
465 470 475 480
Lys Asn Ala Gly Val Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln
485 490 495
Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn
500 505 510
Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg Thr Ala
515 520 525
Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val
530 535 540
Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala
545 550 555 560
Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe
565 570
<210> 4
<211> 163
<212> PRT
<213> P21[2-164]
<400> 4
Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys Ala
1 5 10 15
Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg Asp
20 25 30
Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg Trp
35 40 45
Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala Trp
50 55 60
Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr Gly
65 70 75 80
Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly Thr
85 90 95
Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp Leu
100 105 110
Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu Gly
115 120 125
Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln Thr
130 135 140
Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser Lys
145 150 155 160
Arg Lys Pro
<210> 5
<211> 9
<212> PRT
<213> DAG[1-9]
<400> 5
Cys Asp Ala Gly Arg Lys Gln Lys Cys
1 5
<210> 6
<211> 140
<212> PRT
<213> DNAJC8[114-253]
<400> 6
Glu Gln Lys Lys Arg Ala Leu Asp Val Ile Gln Ala Gly Lys Glu Tyr
1 5 10 15
Val Glu His Thr Val Lys Glu Arg Lys Lys Gln Leu Lys Lys Glu Gly
20 25 30
Lys Pro Thr Ile Val Glu Glu Asp Asp Pro Glu Leu Phe Lys Gln Ala
35 40 45
Val Tyr Lys Gln Thr Met Lys Leu Phe Ala Glu Leu Glu Ile Lys Arg
50 55 60
Lys Glu Arg Glu Ala Lys Glu Met His Glu Arg Lys Arg Gln Arg Glu
65 70 75 80
Glu Glu Ile Glu Ala Gln Glu Lys Ala Lys Arg Glu Arg Glu Trp Gln
85 90 95
Lys Asn Phe Glu Glu Ser Arg Asp Gly Arg Val Asp Ser Trp Arg Asn
100 105 110
Phe Gln Ala Asn Thr Lys Gly Lys Lys Glu Lys Lys Asn Arg Thr Phe
115 120 125
Leu Arg Pro Pro Lys Val Lys Met Glu Gln Arg Glu
130 135 140
<210> 7
<211> 32
<212> PRT
<213> RAGE[23-54]
<400> 7
Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val Leu Lys Cys
1 5 10 15
Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp Lys Leu Asn
20 25 30
<210> 8
<211> 84
<212> PRT
<213> ASXL1[1-84]
<400> 8
Met Lys Asp Lys Gln Lys Lys Lys Lys Glu Arg Thr Trp Ala Glu Ala
1 5 10 15
Ala Arg Leu Val Leu Glu Asn Tyr Ser Asp Ala Pro Met Thr Pro Lys
20 25 30
Gln Ile Leu Gln Val Ile Glu Ala Glu Gly Leu Lys Glu Met Ser Gly
35 40 45
Thr Ser Pro Leu Ala Cys Leu Asn Ala Met Leu His Ser Asn Ser Arg
50 55 60
Gly Gly Glu Gly Leu Phe Tyr Lys Leu Pro Gly Arg Ile Ser Leu Phe
65 70 75 80
Thr Leu Lys Arg
<210> 9
<211> 160
<212> PRT
<213> JMJD2D[1-160]
<400> 9
Met Glu Thr Met Lys Ser Lys Ala Asn Cys Ala Gln Asn Pro Asn Cys
1 5 10 15
Asn Ile Met Ile Phe His Pro Thr Lys Glu Glu Phe Asn Asp Phe Asp
20 25 30
Lys Tyr Ile Ala Tyr Met Glu Ser Gln Gly Ala His Arg Ala Gly Leu
35 40 45
Ala Lys Ile Ile Pro Pro Lys Glu Trp Lys Ala Arg Glu Thr Tyr Asp
50 55 60
Asn Ile Ser Glu Ile Leu Ile Ala Thr Pro Leu Gln Gln Val Ala Ser
65 70 75 80
Gly Arg Ala Gly Val Phe Thr Gln Tyr His Lys Lys Lys Lys Ala Met
85 90 95
Thr Val Gly Glu Tyr Arg His Leu Ala Asn Ser Lys Lys Tyr Gln Thr
100 105 110
Pro Pro His Gln Asn Phe Glu Asp Leu Glu Arg Lys Tyr Trp Lys Asn
115 120 125
Arg Ile Tyr Asn Ser Pro Ile Tyr Gly Ala Asp Ile Ser Gly Ser Leu
130 135 140
Phe Asp Glu Asn Thr Lys Gln Trp Asn Leu Gly His Leu Gly Thr Ile
145 150 155 160

Claims (3)

1.一种阿尔茨海默症检测用抗原，其特征在于：所述抗原为蛋白片段组合物，所述蛋白片段组合物包括：ADARB1蛋白的蛋白片段、HSP60蛋白的蛋白片段和ASXL1蛋白的蛋白片段；所述ADARB1蛋白的蛋白片段序列如SEQ ID NO.2所示，所述HSP60蛋白的蛋白片段序列如SEQ ID NO.3所示，所述ASXL1蛋白的蛋白片段序列如SEQ ID NO.8所示。
2.利用权利要求1所述的抗原制备的检测阿尔茨海默症的试剂盒。
3.根据权利要求2所述的试剂盒，其特征在于：所述试剂盒所用样本为全血或血清。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109738653A (zh) * 2019-01-11 2019-05-10 湖南诺琪生物科技有限公司 用于阿尔茨海默症的检测、诊断或风险预测的抗原蛋白组合以及包含其的试剂盒

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0507598D0 (en) * 2005-04-14 2005-05-18 Trojantec Technologies Ltd Composition
US20110312059A1 (en) * 2008-08-20 2011-12-22 Oligomerix Inc. Tau protease compositions and methods
JP2013528799A (ja) * 2010-05-13 2013-07-11 ユニバーシティ オブ メディスン アンド デンティストリー オブ ニュー ジャージー 神経変性疾患の検知および診断のための診断用自己抗体プロファイル
US20120232016A1 (en) * 2011-03-08 2012-09-13 Coleman Paul D Method and system to detect and diagnose alzheimer's disease
WO2013010003A1 (en) * 2011-07-12 2013-01-17 University Of Medicine And Dentistry Of New Jersey Diagnostic biomarker profiles for the detection and diagnosis of alzheimer's disease
WO2014205213A1 (en) * 2013-06-19 2014-12-24 University Of Utah Research Foundation Substituted (e)-n'-(1-phenylethylidene) benzohydrazide analogs as histone demethylase inhibitors
EP2899543A1 (en) * 2014-01-28 2015-07-29 Predemtec GmbH Biomarker and methods for early diagnosis of Alzheimer's disease
EP3283655A4 (en) * 2015-04-15 2018-12-05 Promedior, Inc. Methods for treating myeloproliferative disorders
EP3390700A4 (en) * 2015-12-14 2019-12-18 Rowan University ANTIBODY BIOMARKERS FOR MORBUS ALZHEIMER IN THE EARLY STADIUM, TARGETED AND DIAGNOSTIC USES THEREOF
US11571485B2 (en) * 2016-11-16 2023-02-07 Sanford Burnham Prebys Medical Discovery Institute Peptides and antibodies for detecting changes in alzheimer's disease brain and methods of use thereof
EP3376230A1 (en) * 2017-03-13 2018-09-19 Biocross, S.L. Identification of signatures for neurodegeneration diseases diagnoses
CN109342738B (zh) * 2018-11-19 2022-04-15 深圳大学 一组血清差异蛋白组合在制备用于检测阿尔茨海默症的试剂中的应用
CN111471096B (zh) * 2020-01-15 2022-02-18 上海众启生物科技有限公司 用于阿尔茨海默症自身抗体检测的含有adarb1蛋白片段组合物

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109738653A (zh) * 2019-01-11 2019-05-10 湖南诺琪生物科技有限公司 用于阿尔茨海默症的检测、诊断或风险预测的抗原蛋白组合以及包含其的试剂盒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XP_029402533.1：microtubule-associated protein tau isoform X15 [Mus pahari];Johannes G et al.;《NCBI》;20190618;全文 *

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