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WO2023000688A1 - 一种阿尔茨海默病的外周血tcr标志物及其检测试剂盒和应用 - Google Patents

一种阿尔茨海默病的外周血tcr标志物及其检测试剂盒和应用 Download PDF

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WO2023000688A1
WO2023000688A1 PCT/CN2022/080368 CN2022080368W WO2023000688A1 WO 2023000688 A1 WO2023000688 A1 WO 2023000688A1 CN 2022080368 W CN2022080368 W CN 2022080368W WO 2023000688 A1 WO2023000688 A1 WO 2023000688A1
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disease
alzheimer
tcr
marker
peripheral blood
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张志新
乐卫东
杨鑫
卓越
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Chengdu Exab Biotechnology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a peripheral blood TCR marker for Alzheimer's disease, a detection kit and application thereof.
  • AD Alzheimer's disease
  • a neurodegenerative disease with insidious onset and progressive development Clinically, it is characterized by comprehensive dementia such as memory impairment, aphasia, apraxia, agnosia, impairment of visuospatial skills, executive dysfunction, and personality and behavior changes.
  • the etiology is still unknown.
  • Those with onset before the age of 65 are called Alzheimer's disease; those with onset after the age of 65 are called senile dementia.
  • Alzheimer's disease With the aging of the population, Alzheimer's disease has become an increasingly significant social problem. According to the "World Alzheimer's Disease 2018 Report" released by the International Alzheimer's Association, there were about 50 million Alzheimer's patients in the world in 2018, and it is estimated that 150 million people will suffer from Alzheimer's by 2050. According to incomplete statistics, there are currently more than 10 million dementia patients in China, of which 60% are Alzheimer's disease, the incidence rate of people over 65 years old is 5%, and the incidence rate of people over 80 years old exceeds 30%. It is estimated that by 2050, there will be more than 20 million dementia patients in China.
  • senile dementia is mainly divided into dementia caused by brain degenerative diseases (including Alzheimer's disease, frontotemporal dementia, Lewy body type, Parkinson's dementia syndrome, etc.), dementia caused by cerebrovascular disease and mixed dementia. kind.
  • the clinical diagnosis of Alzheimer's disease is mainly based on its special clinical evolution process, combined with relevant examinations to exclude other organic diseases that cause dementia, and the final diagnosis requires pathological examination results.
  • NINCDS-ADRDA Alzheimer's Disease and Related Disorders Association
  • Lumbar puncture pressure is normal in laboratory examination
  • the NINCDS-ADRDA diagnostic criteria have a good consistency with the pathological results.
  • the standard emphasizes that "the degree of cognitive impairment must affect the patient's daily life ability and social activity function before the diagnosis can be established", and emphasizes that other related system or brain diseases that may cause dementia must be excluded, and the latter is often the same as Alzheimer's disease.
  • the degree of cognitive impairment must affect the patient's daily life ability and social activity function before the diagnosis can be established
  • other related system or brain diseases that may cause dementia must be excluded, and the latter is often the same as Alzheimer's disease.
  • silent disease there is a lack of clear and easily detectable biomarkers. This brings difficulties to the early identification and diagnosis of Alzheimer's disease patients.
  • MRI nuclear magnetic resonance
  • PET Electron emission tomography
  • MRI shows that the hippocampus, entorhinal cortex, and amygdala shrink in size (compared with the same age group);
  • Abnormal cerebrospinal fluid biomarkers decreased A ⁇ 42, increased total tau or phosphorylated tau protein, or the presence of all three at the same time;
  • PET Special manifestations of PET: such as decreased glucose metabolism in bilateral temporal lobes, pathological changes of AD revealed by the imaging agent 18F-FDDNP*, etc.;
  • Alzheimer's disease caused by proven autosomal dominant inheritance in immediate family members.
  • FDDNP An analogue of the non-steroidal anti-inflammatory drug naproxen, which has a strong affinity for senile plaques in vitro.
  • PET Positron Emission Tomography
  • Glucose metabolism PET can show the metabolic decline of the limbic system in patients with Alzheimer's disease in the early stage, and the diagnostic sensitivity for mild Alzheimer's disease reaches 84%, and the specificity reaches 93%.
  • Amyloid PET Amyloid PET
  • Amyloid PET found that when patients with familial Alzheimer's disease did not have brain atrophy and brain metabolism decreased in the asymptomatic period, amyloid deposition in the brain had already occurred, indicating that amyloid clusters (plaques) Block) may be the earliest biomarker, however, studies have also pointed out that other diseases other than Alzheimer's disease may also lead to amyloid deposition in the brain.
  • CSF markers included A ⁇ 42, total tau (t-tau), and phosphorylated tau (p-tau).
  • a ⁇ 42 reflects the deposition of cortical amyloid protein
  • t-tau protein reflects the density of neurodegeneration
  • p-tau is related to the pathological changes of neurofibrillary tangles.
  • Peripheral blood markers include A ⁇ 42 dimer content in peripheral blood cell membranes, plasma gelsolin (GSN) that can inhibit A ⁇ fibrosis and depolymerize A ⁇ fibers, and GSN main degrading enzyme MMP3, etc.
  • GSN plasma gelsolin
  • MMP3 GSN main degrading enzyme
  • structural magnetic resonance can measure regional or whole brain volume, reflecting structural atrophy caused by cell damage, axonal degeneration, and synaptic dysregulation.
  • the earliest and progressive atrophy of the medial temporal lobe structure may be a characteristic change in the early stage of AD.
  • Functional magnetic resonance fMRI
  • Diffusion tensor imaging (DTI), T2 relaxation time imaging and other nuclear magnetic resonance parameters have also shown potential as early diagnostic criteria for Alzheimer's disease in some recent studies.
  • DTI diffusion tensor imaging
  • T2 relaxation time imaging and other nuclear magnetic resonance parameters have also shown potential as early diagnostic criteria for Alzheimer's disease in some recent studies.
  • MRI detection is still more suitable for measuring and monitoring the disease process of Alzheimer's disease so far, and whether it is suitable for early diagnosis of the disease needs further research and further verification.
  • the present invention provides a peripheral blood TCR marker for Alzheimer's disease and its detection kit and application, which can non-invasively and accurately and quickly determine whether there is a high Alzheimer's disease in the sample to be tested. Patients at risk for Haimer's disease.
  • a peripheral blood TCR marker for Alzheimer's disease includes at least one of the proteins whose sequences are shown in SEQ ID NO.1-50, and the specific sequence is shown in Table 1.
  • protein sequence of the marker is a protein that can achieve the same function after the sequence shown in SEQ ID NO.1-50 is substituted, deleted and/or replaced by one or more amino acids.
  • the marker is peripheral blood TCR CDR3 sequence.
  • the preparation includes the T cell receptor containing the marker, or a plasmid, virus vector or nucleic acid fragment capable of expressing the T cell receptor producing the marker.
  • a reagent kit for detecting Alzheimer's disease including an antibody capable of specifically binding to the above-mentioned marker.
  • a preparation including an antibody capable of specifically binding to the above-mentioned marker; the preparation can be used for diagnosing, predicting, detecting or screening Alzheimer's disease.
  • the protein chip for detecting Alzheimer's disease.
  • the protein chip includes a substrate and a specific antibody spotted on the substrate.
  • the specific antibody is an antibody that can specifically combine with the above-mentioned markers.
  • B lymphocytes and T lymphocytes in the human body are two important types of cells in the acquired immune system.
  • B cells recognize antigens through the B cell receptor (BCR) on the cell surface.
  • BCR B cell receptor
  • TCR T cell receptor
  • the diversity of BCR and TCR is the basis for establishing the acquired immune system.
  • the theoretical value of BCR diversity is 10 18
  • the theoretical value of TCR diversity is 10 14 .
  • antigenic determinant 3 (CDR3) is the most important part to determine the antigenic specificity, so the sequence of CDR3 is considered to represent the characteristics of BCR and TCR sequences.
  • BCR and TCR will change with different antigen stimulation. Therefore, the occurrence and development of diseases can be tracked by using the results of high-throughput sequencing of BCR or TCR.
  • MCHII histocompatibility antigen II
  • Antigen fragments presented by normal cells will not cause an immune response due to immune tolerance.
  • the abnormal protein expressed by the mutated gene, and its fragments are presented on the cell surface, which will cause the human immune system to produce a targeted immune response. Therefore, analyzing the changes of BCR or TCR can detect the occurrence and development of tumors or other diseases.
  • a large data analysis model was first established using 1725 non-Alzheimer's disease control samples and TCR high-throughput sequencing data of 11 Alzheimer's disease patients.
  • the comparison of TCR sequences specific to Alzheimer's disease can clearly determine whether there is a person with a higher risk of Alzheimer's disease in the sample to be tested.
  • TCR changes through high-throughput sequencing can detect very early Alzheimer's disease, using the unique TCR CDR3 sequence of Alzheimer's disease to analyze the response of T cells in the human immune system to Alzheimer's disease, It is a new detection method.
  • the present invention uses high-throughput sequencing technology to simultaneously compare a large number of specific TCR sequences, which has higher specificity and accuracy than detecting one or several markers alone.
  • the cost of high-throughput sequencing equipment used in the present invention is lower than that of large-scale imaging equipment, and can be outsourced to third parties.
  • the labor cost of sampling and processing is lower than that of simultaneous detection of multiple markers, and lower than that of a large number of cytology detection, therefore, the present invention greatly reduces the detection cost.
  • the present invention only needs to take a small amount of peripheral blood, and the sampling is simple and safe, and it is a non-invasive testing method.
  • TCR CDR3 sequence described in the present invention can be used for immunotherapy of Alzheimer's disease.
  • Figure 1 shows the discovery of the characteristic TCR sequence of Alzheimer's disease using the immune-based big data analysis system in the present invention.
  • the abscissa represents the order in which the CDR3 sequence of a specific amino acid combination is added to the control sequence set or the Alzheimer's disease characteristic sequence set, and the ordinate represents the logarithmic value of the sequence's repeated occurrence times C X in a sample;
  • the immune map of patients with Alzheimer's disease has multiple types and high repetitions of Alzheimer's disease characteristic sequences, while healthy people have very few Alzheimer's disease characteristic sequences, and the comparison of Alzheimer's disease characteristics in unknown subjects Obviously, the risk of Alzheimer's disease is higher.
  • Fig. 2 is a comparative analysis of Alzheimer's disease and other diseases using Alzheimer's disease characteristic index in the present invention.
  • the Alzheimer's disease characteristic index of healthy people, non-neurological disease patients, and non-Alzheimer's disease patients with other neurological diseases is significantly different from that of Alzheimer's disease patients, which proves the characteristic sequence of Alzheimer's disease Set specificity. Based on this, it can be determined whether the unknown subject suffers from Alzheimer's disease.
  • Example 1 Obtaining the sequence set of Alzheimer's disease TCR marker CDR3 through immunographic analysis
  • Collect 1726 control groups including healthy people and non-nervous disease patients, 1725 people for model building, 1 healthy person for verification), 12 Alzheimer's disease patients (11 people for model building, 1 person for for verification) and the peripheral blood of a subject with unknown health status (10mL per person), the amino acid sequence of the TCR epitope 3 (CDR3) of the subject and the control group was obtained by high-throughput sequencing, ensuring that each sample The total number of CDR3 sequences of the functional TCR is not less than 25,000;
  • the immune map of a healthy person, a patient with Alzheimer's disease and a subject with unknown health status are compared with reference to the control sequence set and the Alzheimer's disease characteristic sequence set.
  • Figures see Figures 1B-D. It can be seen from the figure that the immune profiles of patients with Alzheimer's disease contain more types of Alzheimer's disease characteristic sequences with a high number of repetitions (Figure 1B); in the immune profiles of healthy people, only a very small amount of Al The characteristic sequence of Alzheimer's disease (Fig. 1C); while the subject with unknown health status has a higher characteristic sequence of Alzheimer's disease than the healthy person, indicating that this person has a higher risk of suffering from Alzheimer's disease (Fig. 1D) .
  • the antigenic determinant 3 (CDR3) amino acid sequence of the TCR of the patients and the control group to ensure that the total number of functional TCR CDR3 sequences of each sample is not less than 25,000; the total number of CDR3 sequences of each functional TCR is higher than 30,000
  • the sequence of the sample is randomly sampled without replacement, so that the total number of CDR3 sequences of the sample is 30,000. So far, the total number of functional TCR CDR3 sequences contained in all samples is 25,000-30,000.
  • Example 2 According to the 11 Alzheimer's patients from Example 1, and 97 healthy people, 44 non-nervous disease patients, 26 epilepsy patients, 20 Parkinson's patients, and 5 newly acquired patients from Example 2 Immune profiles of subjects with unknown health status analyzed for Alzheimer's disease characteristic indices.
  • the characteristic index of Alzheimer's disease is defined as: in a certain sample, the sum of the repeated times C X of all CDR3 sequences belonging to the characteristic sequence set of Alzheimer's disease in the sample.
  • the analysis results are shown in Table 2 and accompanying drawing 2 below.
  • the Alzheimer's disease TCR marker CDR3 sequence described in the present invention does have significant Alzheimer's disease specificity, and can not only be used for Alzheimer's disease to predict subjects suffering from Alzheimer's disease It can also be used in the biological immunotherapy of Alzheimer's disease in the future.

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Abstract

一种阿尔茨海默病的外周血TCR标志物及其检测试剂盒和应用。阿尔茨海默病的外周血TCR标志物包括序列为SEQ ID NO.1～50所示的蛋白中的至少一种。基于高通量测序方法，只需要采取少量外周血，提取RNA，通过对样本的处理建立免疫图谱文库，再经过高通量测序和TCR数据分析，首先确定阿尔茨海默病外周血中特征性TCR序列，然后将待测样本测试结果与该特征性TCR序列比对，从而确定是否患有阿尔茨海默病。该方法能够同时比较巨大数量的阿尔茨海默病特异性TCR序列，相比单独检测一种或几种标记物，具有更高的特异性和准确性，提高了诊断效率。

Description

一种阿尔茨海默病的外周血TCR标志物及其检测试剂盒和应用 技术领域
本发明属于基因工程技术领域，具体涉及一种阿尔茨海默病的外周血TCR标志物及其检测试剂盒和应用。
背景技术
阿尔茨海默病(Alzheimer Disease，AD)是一种起病隐匿的进行性发展的神经系统退行性疾病。临床上以记忆障碍、失语、失用、失认、视空间技能损害、执行功能障碍以及人格和行为改变等全面性痴呆表现为特征，病因迄今未明。65岁以前发病者，称早老性痴呆；65岁以后发病者称老年性痴呆。
随着人口老龄化加剧，老年痴呆症已经成为日益显著的社会问题。根据国际阿尔茨海默病协会发布的《世界阿尔茨海默病2018年报告》，2018年全球约有5千万老年痴呆患者，预计到2050年将有1.5亿人罹患老年痴呆。据不完全统计，目前中国痴呆症患者超过1000万人，其中有60％为阿尔茨海默症，65岁以上人群发病率为5％，80岁以上发病率超过30％。预计到2050年，中国痴呆患者将超过2000万。
老年痴呆根据其病因主要分为脑变性疾病引起的痴呆(包括阿尔茨海默病、额颞叶痴呆、路易体型、帕金森痴呆综合症等)、脑血管病引起的痴呆和混合型痴呆三大类。阿尔茨海默病的临床诊断主要依据其特殊临床演变过程，并结合有关检查排除引起痴呆的其他器质性疾病，而最终确诊需要病理检验结果。
目前，阿尔茨海默病的临床诊断广泛应用NINCDS-ADRDA诊断标准，由美国国立神经病语言障碍卒中研究所(NINCDS)和阿尔茨海默病及相关疾病协会(ADRDA)建立的NINCDS-ADRDA专题工作组推荐(1984年发表于Neurology杂志)，其主要内容如下：
1、诊断标准：
1)临床检查和认知量表测查确定有痴呆。
2)两个或两个以上认知功能缺损，且进行性恶化。
3)无意识障碍。
4)40-90岁起病，多见于65岁以后。
5)排除其他引起进行性记忆和认知功能损害的系统性疾病和脑部疾病。
2、支持标准：
1)特殊性认知功能如言语(失语症)、运动技能(失用症)、知觉(失认症)的进行性损害。
2)日常生活功能损害或行为方式的改变。
3)家庭中有类似疾病史，特别是有神经病理学或实验室证据者。
4)实验室检查腰穿压力正常；
5)脑电图正常或无特殊性的改变如慢波增加；
6)CT或MRI证实有脑萎缩，且随诊检查有进行性加重。
3、排除标准：
1)突然起病或卒中样发作。
2)早期有局灶性神经系统体征，如偏瘫、感觉丧失、视野缺损、共济失调。
3)起病或疾病早期有癫痫发作或步态异常。
NINCDS-ADRDA诊断标准经过多年临床实践，与病理结果有很好的一致性。但该标准强调“认知功能损害程度一定要影响患者日常生活能力和社会活动功能，诊断才能成立”，而且强调要排除其它可能导致痴呆的相关系统或脑部疾病，而后者往往同阿尔茨海默病一样，缺乏明确且易检测的生物学标志物。这给阿尔茨海默病患者的早识别、早诊断带来困难。
针对这一不足，随着阿尔茨海默病相关领域研究的迅速进展，NINCDS-ADRDA诊断标准于2007年进行了修订，一些生物学标志物，包括核磁共振(MRI)示内侧颞叶萎缩，正电子发射断层扫描(PET)可见颞顶叶脑血流下降以及脑脊液中Aβ和tau蛋白的异常改变等，被纳入诊断标准。这提高了阿尔茨海默病诊断的特异性和敏感性，对早期诊断帮助较大：
1、核心标准
1)早期、显著的情景记忆障碍；
2)支持表现内颞叶萎缩：MRI显示海马、内嗅皮质、杏仁核体积缩小(与同年龄人群比较)；
3)脑脊液生物标记异常：Aβ42降低、总tau或磷酸化tau蛋白增高，或3者同时存在；
4)PET的特殊表现：如双侧颞叶糖代谢减低，显像剂18F-FDDNP*显示AD病理的改变等；
5)直系亲属中有已证实的常染色体显性遗传导致的阿尔茨海默病。
注：*FDDNP：非甾体类抗炎药物甲氧萘普酸的类似物，在体外证实其与老年斑有很强的亲和力。
2、排除标准
1)病史：突然起病；早期出现下列症状：步态不稳、癫痫、行为异常。
2)临床特点：局灶性神经系统症状体征：偏瘫、感觉缺失、视野损害；早期的锥体外系体征。
3)其它疾病状态严重到足以解释记忆和相关症状：非阿尔茨海默病痴呆；严重的抑郁；脑血管病；中毒或代谢异常(要求特殊检查证实)；MRI的FLAIR或T2加权相内颞叶信号异常与感染或血管损害一致。
目前实验室中对阿尔茨海默病的检验技术有：
1、正电子发射断层扫描(PET)
目前诊断阿尔茨海默病最有力的方法。葡萄糖代谢PET(FDG PET)在早期能显示阿尔茨海默病患者边缘系统的代谢下降，对轻度阿尔茨海默病诊断灵敏度达到84％，特异性达到93％。而淀粉样蛋白PET(Amyloid PET)研究发现家族性阿尔茨海默病患者在无症状期未出现脑萎缩和脑代谢减低时，已出现了脑内淀粉样蛋白沉积，说明淀粉样蛋白簇(斑块)有可能是最早出现的生物标志物，然而也有研究指出，非阿尔茨海默病的其它疾病也有可能导致脑补淀粉样蛋白沉积。近年来的最新研究表明，早期临床阿尔茨海默症患者的Tau蛋白PET脑部扫描能准确地预测1-2年后核磁共振测量的脑萎缩的位置，而淀粉样蛋白PET显像未来脑萎缩的位置。然而，PET检测设备稀缺、检测费用昂贵，而且由于血脑屏障的存在，必须直接向中枢神经系统内注射显影液，因此其应用存在相当大的局限性。
2、脑脊液检测
脑脊液标记物包括Aβ42、总tau蛋白(t-tau)和磷酸化的tau蛋白(p-tau)。其中，Aβ42反映了皮层淀粉样蛋白沉积；t-tau蛋白反映了神经变性的密度；p-tau与神经元纤维缠结病理改变相关。这些核心CSF标记物有较高的诊断准确性，在早期(轻度认知损害期，mild cognitive impairment，MCI)的敏感度和特异性可达85-90％。但脑脊液检验存在取样困难，病人痛苦大的显著缺点。
3、外周血检测
外周血标志物包括外周血细胞膜的Aβ42二聚体含量、可抑制Aβ纤维化和解聚Aβ纤维的血浆凝溶胶蛋白(GSN)和GSN主要降解酶MMP3等。但外周血中这些成分含量较低，且目前对于外周血成分是否能够真正代表中枢神经系统的改变，仍存在一定质疑。
4、核磁共振检测(MRI)
其中，结构磁共振(sMRI)可测量区域性或全脑体积大小，反映因细胞损伤、轴突退变、突触失调引起的结构萎缩。最早出现并进行性加重的内侧颞叶结构萎缩可能是AD早期出现的特征性改变。大脑接受刺激后，神经细胞活动、局部脑血流、区域的耗氧量发生改变，功能磁共振(fMRI)通过血流动力学反应与脑神经细胞活动之间的密切关系，可反映神经元和突触功能异常。弥散张量成像(DTI)、T2弛豫时间成像等核磁共振参数，在最近的一些研究中，也展示出了作为阿尔茨海默病早期诊断标准的潜力。然而根据最新诊断标准，迄今为止核磁 共振检测还是更适合用于测量和监测阿尔茨海默病的疾病过程，是否适用于疾病的早期诊断还需更多研究进一步验证。
5、基因检测
有研究表明，一些基因型的携带者如APOE4基因，有较高几率罹患阿尔茨海默病。然而，这种传统基因的检测，只能作为一种辅助的风险评估，而无法作为阿尔茨海默病的诊断标准。
综上所述，目前针对阿尔茨海默病的临床诊断，仍然严重依赖于评估性的量表标准，缺乏一种方便、准确且相对廉价的确诊检验手段。
发明内容
针对现有技术中的上述不足，本发明提供一种阿尔茨海默病的外周血TCR标志物及其检测试剂盒和应用，能无创且准确快速的判断待测样本中是否有较高阿尔茨海默病风险患者。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种阿尔茨海默病的外周血TCR标志物，该标志物包括序列为SEQ ID NO.1～50所示的蛋白中的至少一种，具体序列见表1。
表1 标志物序列
序列编号 蛋白序列
SEQ ID NO.1 Ala Ser Ser His Gly His Gly Leu Ala Gly Ser Glu Gln Phe
SEQ ID NO.2 Ala Ser Arg Asp Ser Thr Arg Glu Gln Tyr
SEQ ID NO.3 Ser Val Ile Leu Ala Asp Lys Tyr Asn Glu Gln Phe
SEQ ID NO.4 Ala Ser Ser Leu Ala Arg Val Ile Thr Gly Asn Glu Gln Phe
SEQ ID NO.5 Ser Ala Asn Pro Val Gly Gly Val Asn Thr Glu Ala Phe
SEQ ID NO.6 Ala Ser Ser Pro Ala Leu Asp Arg Arg Pro Asp Tyr Gly Tyr Thr
SEQ ID NO.7 Ala Ser Arg Ile Gly Phe Gly Glu Gln Tyr
SEQ ID NO.8 Ala Ser Arg Met Thr Gly Gly Lys Glu Thr Gln Tyr
SEQ ID NO.9 Ala Ser Ser Leu Ser Gly Arg Ala Asn Ile Glu Gln Phe
SEQ ID NO.10 Ala Ser Ser Leu Ala Leu Ala Gly Gly Ala Gln Asn Glu Gln Phe
SEQ ID NO.11 Ala Ser Arg Pro Gly Arg Val Asp Thr Gly Glu Leu Phe
SEQ ID NO.12 Ala Ser Glu Ile Gly Gly Arg Asn Thr Gly Glu Leu Phe
SEQ ID NO.13 Ser Ala Ser Pro Asp Tyr Asp Ser Thr Asp Thr Gln Tyr
SEQ ID NO.14 Ala Thr Ser Arg Asp Pro Ala Gly Leu Ala Gly Glu Gln Phe
SEQ ID NO.15 Ala Ser Ser Val Asp Glu Thr Ser Leu Gly Gln Phe
SEQ ID NO.16 Ala Ser Ser Ala Gly Gln Tyr Gly Thr Asp Thr Gln Tyr
SEQ ID NO.17 Ser Val Glu Ala Glu Thr Ser Gly Arg Gly Gly Thr Gly Glu Leu Phe
SEQ ID NO.18 Ser Ala Ser Ser Pro Ile Gly Gly Ser Ser Tyr Glu Gln Tyr
SEQ ID NO.19 Ala Trp Ser Asp Arg Ala Ala Met Thr Asp Thr Gln Tyr
SEQ ID NO.20 Ser Ala Arg Ala Asp Arg Gln Thr Gly Ser Tyr Glu Gln Tyr
SEQ ID NO.21 Ala Ser Lys Ser Gly Arg Met Asp Asn Glu Gln Phe
SEQ ID NO.22 Ala Ser Ser Gln His Glu Gln Glu Glu Gly Thr Glu Ala Phe
SEQ ID NO.23 Ala Ser Ser Ser Gly Gln Glu Arg Gln Gly Arg Val Tyr Thr Glu Ala
  Phe
SEQ ID NO.24 Ser Ala Arg Asp Gly Gly Ser Thr Asn Glu Gln Phe
SEQ ID NO.25 Ser Ala Tyr Gly Ala Tyr Thr Ile Asn Glu Gln Phe
SEQ ID NO.26 Ala Ser Arg Pro Thr Ser Glu Thr Ser Tyr Glu Gln Tyr
SEQ ID NO.27 Ser Ala Arg Ser Arg Leu Leu Ala Lys Asn Ile Gln Tyr
SEQ ID NO.28 Ser Ala Leu Ser Arg Gly Gln Tyr Asn Glu Gln Phe
SEQ ID NO.29 Ala Thr Arg Gly Ala Ala Glu Leu Thr Tyr Glu Gln Tyr
SEQ ID NO.30 Ala Ser Arg Glu Ala Gly Gly Arg Asp Thr Gln Tyr
SEQ ID NO.31 Ala Ser Arg Gly Ala Gly Val Ala Tyr Asn Glu Gln Phe
SEQ ID NO.32 Ala Ser Ser Leu Val Val Thr Ser Gly Pro Thr Asp Thr Gln Tyr
SEQ ID NO.33 Ala Ser Ser Ser Ser Lys Gly Gly Gln Pro Gln His
SEQ ID NO.34 Ala Ser Ser Phe Ser Cys Ile Cys Val Leu Thr Ala Pro
SEQ ID NO.35 Ala Trp Arg Ile Gly Ser Thr Gly Glu Leu Phe
SEQ ID NO.36 Ala Ser Ser Leu Glu His Gly Arg Asp Thr Gln Tyr
SEQ ID NO.37 Ala Ser Ser Phe Gln Thr Phe Ser Tyr Asn Glu Gln Phe
SEQ ID NO.38 Ala Ser Ser Met Arg Val Lys Tyr Asn Gln Pro Gln His
SEQ ID NO.39 Ala Ser Ser Gln Glu Val Gly Trp Asn Glu Gln Phe
SEQ ID NO.40 Ala Ser Ser Gly Arg Leu Gly Asn Tyr Gly Tyr Thr
SEQ ID NO.41 Ala Ser Ser Leu Asp Gln Gly Thr Gln Gly Gln Tyr
SEQ ID NO.42 Ala Ser Ser Asp Phe Pro Ser Gly Gly Ala Ser Tyr Asn Glu Gln Phe
SEQ ID NO.43 Ala Ser Ser Ile Thr Ser Gly Lys Phe Asn Glu Gln Phe
SEQ ID NO.44 Ala Trp Ser Glu Ala Ser Gly Glu Glu Thr Gln Tyr
SEQ ID NO.45 Ala Ser Ser Val Asp Gln Gln Pro Glu Thr Gln Tyr
SEQ ID NO.46 Ala Ser Ser Tyr Phe Gly Thr Ala Ser Glu Gln Tyr
SEQ ID NO.47 Ala Ile Ser Glu Lys Ala Gly Asp Leu Asn Thr Glu Ala Phe
SEQ ID NO.48 Ala Ser Ser Glu Ser Gly Thr Gly Leu His Tyr Glu Gln Tyr
SEQ ID NO.49 Ala Ser Ser Gln Ala Asp Trp Ala Gly Gly Arg His Asn Gly Gln Phe
SEQ ID NO.50 Ala Ser Ser Arg Thr Gly Gly Gly Gly Arg Glu Leu Phe
进一步地，标志物的蛋白序列为SEQ ID NO.1～50所示的序列经取代、缺失和/或替换一个或多个氨基酸后，能实现相同功能的蛋白。
进一步地，标志物为外周血TCR CDR3序列。
上述标志物在制备治疗阿尔茨海默病的制剂中的应用。
进一步地，制剂中包括含有该标志物的T细胞受体，或能表达产生该标志物的T细胞受体的质粒、病毒载体或核酸片段。
一种用于阿尔茨海默病检测的试剂盒，包括能与上述标志物发生特异性结合的抗体。
一种制剂，包括能与上述标志物发生特异性结合的抗体；所述制剂可用于对阿尔茨海默病进行诊断、预测、检测或筛查。
一种检测阿尔茨海默病的蛋白质芯片，该蛋白质芯片包括基片和点样在基片上特异性抗体，该特异性抗体为能与上述标志物发生特异性结合的抗体。
本发明的原理为：人体内的B淋巴细胞和T淋巴细胞是获得性免疫系统中重要的两类细 胞。B细胞通过细胞表面的B细胞受体(BCR)识别抗原，后期BCR在B细胞分化成浆细胞时，表达成抗体，分泌到细胞外。T细胞通过细胞表面的T细胞受体(TCR)识别抗原。BCR和TCR的多样性是建立获得性免疫系统的基础。BCR多样性的理论值是10 18，TCR多样性的理论值是10 14。BCR与TCR序列中，抗原决定簇3(CDR3)是决定其抗原特异性最重要的部分，因此CDR3的序列被认为可以代表BCR、TCR序列的特性。
在各种疾病中，随着不同的抗原刺激，BCR和TCR的多样性或者表达水平都会发生改变。因此，利用BCR或者TCR高通量测序结果可以追踪疾病的发生、发展。人体内细胞中，衰老蛋白质降解后，其片段会被运输到细胞表面，通过组织相容性抗原II(MCHII)呈递给免疫系统中的T细胞。正常细胞呈递的抗原片段，由于免疫耐受的关系，不会引起免疫反应。一旦当正常细胞出现病变后，突变的基因表达的异常蛋白，其片段被呈递到细胞表面后，就会引起人体免疫系统产生针对性的免疫反应。因此，分析BCR或TCR的变化，能够检测出肿瘤或其它疾病的发生和发展。
本发明的有益效果为：
1、本发明中，首先利用1725个非阿尔茨海默病的对照组样本、和11个阿尔茨海默病病人的TCR高通量测序数据建立了大数据分析模型，通过和这些阿尔茨海默病特异性TCR序列对比，可以清楚的判断待测样本中是否有较高阿尔茨海默病风险者。
2、通过高通量测序分析TCR变化可以发现很早期的阿尔茨海默病，利用阿尔茨海默病特有的TCR CDR3序列分析人的免疫系统中的T细胞对阿尔茨海默病的反应，是一种新型的检测方法。
3、本发明通过采用高通量测序技术同时比较数量巨大的特异性TCR序列，比起单独检测一种或几种标记物，具有更高的特异性和准确性。
4、本发明中使用的高通量测序仪器成本低于大型影像学设备，且可向第三方外包，此外，采样、处理的人力成本低于同时检测多种标记物，也低于大量细胞学检测，因此，本发明大大降低了检测成本。
5、本发明只需要采取少量外周血，采样简便、安全，是一种无创检验方法。
6、本发明中所述TCR CDR3序列，可用于阿尔茨海默病的免疫治疗。
附图说明
图1为本发明中，利用基于免疫大数据分析系统发现阿尔茨海默病特征性TCR序列。横坐标代表某一特定氨基酸组合的CDR3序列被加入对照序列集合或阿尔茨海默病特征序列集合的先后顺序,纵坐标代表该序列在某一样本中重复出现次数C X的对数值；阿尔茨海默病患者的免疫图谱具有多个种类且重复次数较高的阿尔茨海默病特征序列，健康人极少阿尔茨海默 病特征序列，而未知受试者的阿尔茨海默病特征比较明显，说明罹患阿尔茨海默病风险较高。
图2为本发明中，利用阿尔茨海默病特征特征性指数对比分析阿尔茨海默病和其他疾病。健康人、非神经疾病病人、非阿尔茨海默病的其它神经性疾病患者的阿尔茨海默病特征性指数均与阿尔茨海默病患者具有显著差异，证明了阿尔茨海默病特征序列集的特异性。据此可以判断未知受试者是否罹患阿尔茨海默病。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1 通过免疫图谱分析，获得阿尔茨海默病TCR标志物CDR3序列集
1、采样及免疫图谱分析
采集1726名对照组(包括健康人和非神经疾病病人，1725人用于建立模型，1个健康人用于验证)、12名阿尔茨海默病患者(11人用于建立模型，1人用于验证)及1名未知健康状况受试者的外周血(每人10mL)，通过高通量测序得到受试者和对照组的TCR的抗原决定簇3(CDR3)氨基酸序列，保证每个样本的功能性TCR的CDR3序列总数综合不低于25000；
2、对每个功能性TCR的CDR3序列总数综合高于30000的样本的序列进行随机不放回抽样，使该样本的CDR3序列数量总和为30000。至此所有样本包含的功能性TCR的CDR3序列总数为25000-30000。对任一特定CDR3序列X，在单样本测序结果中重复出现次数计为C X；
3、通过分析TCR CDR3数据，确定阿尔茨海默病TCR标志物CDR3序列：
a)将1725名用于建立模型的对照组样本的所有CDR3序列归总去重，设为对照序列集；
b)将11名用于建立模型的阿尔茨海默病样本的所有CDR3序列归总去重，再去除所有与对照序列集中包含序列重复的序列，设为阿尔茨海默病特征序列集。作图如附图1A所示，其中横坐标代表某一特定氨基酸组合的CDR3序列被加入对照序列集合或阿尔茨海默病特征序列集合的先后顺序，纵坐标代表该序列在某一样本中重复出现次数C X的对数值。
c)按照同样作图方法，将1名健康人、1名阿尔茨海默病患者和1名健康状况未知受试者的免疫图谱，参照对照序列集合和阿尔茨海默病特征序列集合进行作图，见附图1B-D。从图中可见，阿尔茨海默病患者的免疫图谱中，含有较多种类且较高重复出现次数的阿尔茨海默病特征序列(图1B)；健康人的免疫图谱中，只有极少量阿尔茨海默病特征序列(图1C)；而未知健康状况受试者，有高于健康人的阿尔茨海默病特征序列，说明此人有较高风险罹患阿尔茨海默病(图1D)。
d)将阿尔茨海默病特征序列集中，将所有出现在两个及以上参与建模阿尔茨海默病样本里的CDR3序列，按“所有参与建模阿尔茨海默病样本里该序列单样本中重复出现次数C X的总和×包含该序列的参与建模阿尔茨海默病样本数”从高到低排序，排名前100者即为阿尔茨海默病TCR标志物CDR3序列，具体序列如SEQ ID NO.1～50所示。
实施例2 验证阿尔茨海默病TCR标志物CDR3序列集的特异性
1、采样及免疫图谱分析
采集97名健康人、44名非神经疾病的患者26名癫痫患者、20名帕金森症患者，5名未知健康状况受试者的外周血(每人10mL)，通过高通量测序得到受试者和对照组的TCR的抗原决定簇3(CDR3)氨基酸序列，保证每个样本的功能性TCR的CDR3序列总数综合不低于25000；对每个功能性TCR的CDR3序列总数综合高于30000的样本的序列进行随机不放回抽样，使该样本的CDR3序列数量总和为30000。至此所有样本包含的功能性TCR的CDR3序列总数为25000-30000。
2、根据来自实施例1的11名阿尔茨海默病患者，以及实施例2新获取的97名健康人、44名非神经疾病患者、26名癫痫患者、20名帕金森症患者，5名未知健康状况受试者的免疫图谱，分析其阿尔茨海默病特征性指数。
其中阿尔茨海默病特征性指数定义为：某个样本中，属于阿尔茨海默病特征序列集的所有CDR3序列在该样本内重复出现次数C X的总和。分析结果见下表2及附图2。阿尔茨海默病组与健康人(p＝2.97E-54)、非神经疾病(p＝1.07E-26)、癫痫(p＝3.09E-17)、帕金森症(p＝4.83E-14)都有显著差异，这证明了阿尔茨海默病特征序列集的特异性。
表2 不同样本组的阿尔茨海默病特征性指数
Figure PCTCN2022080368-appb-000001
Figure PCTCN2022080368-appb-000002
Figure PCTCN2022080368-appb-000003
3、分析各组的阿尔茨海默病特征指数(表3)，5位未知健康状况受试者(检测样本)的阿尔茨海默病特征指数显著高于“非神经疾病”组的平均值+2×SD(2.1+2×2.3＝6.7)、“癫痫”组的平均值+2×SD(2.3+2×3.1＝8.6)或“帕金森症”组的平均值+2×SD(2.1+2×2.9＝7.9)，此5人具有较高风险罹患阿尔茨海默病。与临床体检结果对照后，这2人确为早期阿尔茨海默病患者。此实施例证明了利用阿尔茨海默病特征序列集及阿尔茨海默病特征性指数，预测 受试者罹患阿尔茨海默病风险的可行性。
表3 阿尔茨海默病特征性指数分析
Figure PCTCN2022080368-appb-000004
综上所述，本发明所述阿尔茨海默病TCR标志物CDR3序列，确实具有显著的阿尔茨海默病特异性，不仅可以用于阿尔茨海默病预测受试者罹患阿尔茨海默病风险，未来还可用于阿尔茨海默病的生物免疫治疗。

Claims (7)

  1. 一种阿尔茨海默病的外周血TCR标志物，其特征在于，所述标志物包括序列为SEQ ID NO.1～50所示的蛋白中的至少一种。
  2. 根据权利要求1所述的阿尔茨海默病的外周血TCR标志物，其特征在于，所述标志物的蛋白序列为SEQ ID NO.1～50所示的序列经取代、缺失和/或替换一个或多个氨基酸后，能实现相同功能的蛋白。
  3. 权利要求1所述的标志物在制备治疗阿尔茨海默病的制剂中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述制剂包括含有该TCR标志物的T细胞受体，或能表达产生该标志物的T细胞受体的质粒、病毒载体或核酸片段。
  5. 一种用于阿尔茨海默病检测的试剂盒，其特征在于，包括能与权利要求1所述TCR标志物发生特异性结合的抗体。
  6. 一种制剂，其特征在于，包括能与权利要求1所述TCR标志物发生特异性结合的抗体；所述制剂可用于对阿尔茨海默病进行诊断、预测、检测或筛查。
  7. 一种检测阿尔茨海默病的蛋白质芯片，其特征在于，所述蛋白质芯片包括基片和点样在基片上特异性抗体，所述特异性抗体为能与权利要求1所述TCR标志物发生特异性结合的抗体。
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