WO2023000689A1 - 一种IgA肾病的外周血TCR标志物及其检测试剂盒和应用 - Google Patents

一种IgA肾病的外周血TCR标志物及其检测试剂盒和应用 Download PDF

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WO2023000689A1
WO2023000689A1 PCT/CN2022/080395 CN2022080395W WO2023000689A1 WO 2023000689 A1 WO2023000689 A1 WO 2023000689A1 CN 2022080395 W CN2022080395 W CN 2022080395W WO 2023000689 A1 WO2023000689 A1 WO 2023000689A1
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iga nephropathy
tcr
marker
peripheral blood
sequence
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张志新
李贵森
杨鑫
卓越
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成都益安博生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61P13/00—Drugs for disorders of the urinary system
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/6854—Immunoglobulins
    • 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
    • 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
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • 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 T cell receptor (TCR) marker in peripheral blood of IgA nephropathy, a detection kit and application thereof.
  • TCR T cell receptor
  • IgA nephropathy also known as Berger's disease, is the most common clinical primary glomerular disease, characterized by IgA antibody or IgA-based immune complex deposition in the mesangial region of the glomerulus.
  • the incidence of IgA nephropathy has obvious regional differences, accounting for 15% to 40% of primary glomerular diseases in Europe and Asia, while in my country, the incidence of IgA nephropathy accounts for 26% of primary glomerular diseases—— 34%, and has shown a gradual upward trend in recent years.
  • IgA nephropathy may occur in all age groups, and it is most common in young men aged 20 to 30. IgA nephropathy is mostly a chronic course, and the early symptoms of patients are mild. However, if not treated in time, as many as 50% of the affected patients will slowly progress to different degrees of renal failure, so early diagnosis and timely treatment are required.
  • the etiology and pathogenesis of IgA nephropathy are still being studied, and many evidences show that it is a polygenic, multifactorial and complex disease.
  • the etiology may be caused by family inheritance or abnormal autoimmune regulation, or may be caused by other diseases, especially infectious diseases and autoimmune diseases, such as HIV infection, viral hepatitis, leprosy, allergic purpura, systemic lupus erythematosus, Rheumatoid arthritis, psoriasis, seronegative spondyloarthritis, tumors, and more.
  • IgA nephropathy is mostly caused by immune injury.
  • Recurring gross hematuria usually occurs 1 to 3 days after upper respiratory tract infection, which can turn into microscopic hematuria after several hours to several days, accompanied by abdominal pain, low back pain, muscle pain or low-grade fever, some patients found abnormal urine during physical examination , for asymptomatic proteinuria and (or) microscopic hematuria, a small number of patients have persistent gross hematuria and varying degrees of proteinuria, may be accompanied by edema and hypertension.
  • IgA nephropathy may be confused with urinary system infection, acute glomerulonephritis, and hereditary glomerular diseases, etc., which need to be distinguished based on doctor's experience and cannot be used as a basis for diagnosis.
  • Serum IgA levels were elevated in 50% of patients. Specific circulating immune complexes containing IgA were detected in 37% to 75% of patients. However, a large proportion of patients had serum IgA in the normal range on routine immunological tests.
  • Proteinuria ⁇ 1g/24h is often mild and focal mesangial hyperplasia. Moderate to severe proteinuria is mostly diffuse mesangial hyperplasia, often accompanied by crescents and glomerulosclerosis. Hematuria: The morphology of urinary RBCs is pleomorphic, suggesting that the source of hematuria is glomerular. However, most patients with kidney disease are accompanied by abnormal urine indicators. Urine examination can be used as a basis for judging the condition of patients with confirmed IgA nephropathy, but it cannot be used as a basis for diagnosis.
  • Serum creatinine rises to 1.5mg/dl (132.6umol/L), mostly for disease progression.
  • GFR 20ml/min
  • the pathological changes belong to grade III or above.
  • renal function tests can be used as a basis for judging the condition of patients with confirmed IgA nephropathy, but they cannot be used as a basis for diagnosis.
  • IgA nephropathy The diagnosis of IgA nephropathy must be confirmed by renal biopsy pathology, which needs to be supported by the results of immunofluorescence or immunohistochemistry.
  • the diagnostic features are: diffuse mesangial hyperplasia or focal segmental proliferative glomerulonephritis is common under the light microscope; IgA or IgA-based immune complex deposition in the mesangial area can be seen by immunofluorescence, which is the diagnosis of IgA nephropathy sign.
  • renal biopsy is very painful for patients, and there is a considerable chance of missed diagnosis and misdiagnosis.
  • the present invention provides a method to quickly and accurately judge whether the person to be tested has developed a disease by using the characteristic TCR markers in the peripheral blood of IgA nephropathy patients.
  • IgA nephropathy-specific T cell response detection markers and their detection kits and applications can non-invasively, accurately and quickly determine whether there is a patient with a high risk of IgA nephropathy in the sample to be tested.
  • a peripheral blood TCR marker for IgA nephropathy includes at least one of the proteins whose sequences are shown in SEQ ID NO.1-100, 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.1-100 is substituted, deleted and/or replaced by one or more amino acids.
  • the marker is the protein sequence of peripheral blood TCR epitope 3 (CDR3).
  • the preparation includes the T cell receptor gene sequence containing the marker, or a plasmid, virus vector or nucleic acid fragment capable of expressing the T cell receptor producing the marker.
  • a test kit for detecting IgA nephropathy 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 IgA nephropathy.
  • a protein chip for detecting IgA nephropathy 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 adaptive 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.
  • IgA nephropathy In response to IgA nephropathy, the patient develops a disease-specific T cell immune response. We can use these IgA nephropathy patients' unique immune response characteristics to judge the disease.
  • a large data analysis model is established by using 1627 non-IgA nephropathy control samples and TCR high-throughput sequencing data of 47 IgA nephropathy patients. By comparing with these IgA nephropathy-specific TCR sequences, it can be clearly to determine whether there is a person with a higher risk of IgA nephropathy in the sample to be tested;
  • the present invention uses high-throughput sequencing technology to compare a large number of specific TCR sequences at the same time, 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 IgA nephropathy.
  • Figure 1 shows the discovery of the characteristic TCR sequence of IgA nephropathy by 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 was added to the IgA nephropathy characteristic sequence set, and the ordinate represents the logarithmic value of the number of repeated occurrences of the sequence C X in a sample; the immune big data of IgA nephropathy patients has multiple
  • IgA nephropathy characteristic sequences with high repetition times, healthy people rarely have IgA nephropathy characteristic sequences, but unknown subjects have more obvious IgA nephropathy characteristics, indicating that the risk of suffering from IgA nephropathy is higher.
  • Fig. 2 is a comparative analysis of IgA nephropathy and other diseases using the characteristic index of IgA nephropathy in the present invention.
  • the characteristic indexes of IgA nephropathy in healthy people and patients with other non-tumor diseases were significantly different from those in patients with IgA nephropathy, which proved the specificity of the characteristic sequence set of IgA nephropathy. Based on this, it can be judged whether the unknown subject suffers from IgA nephropathy.
  • Example 1 Obtain the sequence set of IgA nephropathy TCR marker CDR3 through immune big data analysis
  • Collect 1628 control groups including healthy people and other non-tumor and non-renal disease patients, 1627 people are used for model establishment, 1 healthy person is used for verification), 48 IgA nephropathy patients (47 people are used for model building, 1 person is used 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;
  • Peripheral blood (10 mL per person) was collected from 95 healthy people, 44 patients with other non-renal disease and non-tumor diseases, and 3 subjects with unknown health status, and the antigen determination of TCR in the subjects and the control group was obtained by high-throughput sequencing Cluster 3 (CDR3) amino acid sequence, to ensure that the total number of CDR3 sequences of each functional TCR in each sample is not less than 25,000; perform random non-replacement sampling on the sequences of samples whose total number of CDR3 sequences in each functional TCR is higher than 30,000 , so that the total number of CDR3 sequences of this sample is 30000. So far, the total number of functional TCR CDR3 sequences contained in all samples is 25,000-30,000.
  • CDR3 high-throughput sequencing Cluster 3
  • the characteristic index of IgA nephropathy 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 IgA nephropathy in this sample.
  • the analysis results are shown in Table 2 and accompanying drawing 2 below.
  • the IgA nephropathy TCR marker CDR3 sequence described in the present invention does have significant IgA nephropathy specificity, and can not only be used for the early diagnosis of IgA nephropathy, but also for evaluating the curative effect of drugs for the treatment of IgA nephropathy.
  • New biological immunotherapy regimens can be designed based on these immune signatures.

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Abstract

本发明公开了一种IgA肾病的外周血TCR标志物及其检测试剂盒和应用。该标志物包括序列为SEQ ID NO.1～100所示的蛋白中的至少一种。本发明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大数据文库，再经过高通量测序和TCR数据分析，首先确定IgA肾病外周血中特征性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IgA肾病。本发明能够同时比较巨大数量的IgA肾病特异性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IgA肾病的外周血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IgA肾病的外周血中的T细胞受体(TCR)标志物及其检测试剂盒和应用。
背景技术
一)IgA肾病简介
IgA肾病又称Berger病，是临床上最常见的原发性肾小球疾病，其特征是肾小球系膜区出现IgA抗体或以IgA为主的免疫复合物沉积。IgA肾病的发病有明显的地域差别，在欧洲和亚洲占原发性肾小球疾病的15％～40％，而在我国，IgA肾病的发病率占原发性肾小球疾病的26％—34％，且近年呈现逐渐上升的趋势。
各年龄段均可能发生IgA肾病，其中以20～30岁男性青壮年最为多见。IgA肾病多为慢性病程，患者的早期症状较轻，但如不及时治疗，据统计多达50％的受累患者会缓慢进展为不同程度的肾衰竭，因此需要尽早诊断及时治疗。
二)病因和发病机理
IgA肾病的病因和发病机理尚在研究中，诸多证据表明它是一个多基因、多因素复杂性状疾病。病因可能源于家族遗传或自身免疫调节异常，也可能由其它疾病特别是感染性疾病和自身免疫性疾病引起，如HIV感染、病毒性肝炎、麻风病、过敏性紫癜、系统性红斑狼疮、类风湿关节炎、银屑病、血清阴性脊柱关节炎、肿瘤等等。
IgA肾病多由免疫性损伤所致，通过刺激机体产生过多的糖基化异常的IgA并诱发自身抗体形成免疫复合物、沉积于肾小球的系膜区，使系膜细胞增生、系膜基质增多、局灶性节段性增生或硬化，少数患者可有较多新月体的形成等，从而导致IgA肾病。
三)现有诊断方法
1、常见临床表现
多在上呼吸道感染1～3天后出现易反复发作的肉眼血尿，持续数小时至数天后可转为镜下血尿，可伴有腹痛，腰痛，肌肉痛或低热，部分患者在体检时发现尿异常，为无症状性蛋白尿和(或)镜下血尿，少数患者有持续性肉眼血尿和不同程度蛋白尿，可伴有水肿和高血压。然而，IgA肾病的临床表现，有可能与泌尿系统感染、急性肾小球肾炎、遗传性肾小球疾病等发生混淆，需要依赖医生经验加以区分，无法作为确诊依据。
2、实验室检查
1)血清免疫学检查
50％的病人血清IgA水平升高。37％～75％病人测到含有IgA的特异性循环免疫复合物。然 而，也有很大比例的病人常规免疫学检查中血清IgA处于正常范围。
2)尿液检查
蛋白尿定量和分型对IgA肾病病情判断、估计预后很重要。蛋白尿<1g/24h者常为轻微及病灶性系膜增生为主。中～重度蛋白尿多为弥漫性系膜增生，常伴新月体及肾小球硬化。血尿：尿RBC形态呈多形性，提示血尿来源是肾小球源性。然而，肾病患者多伴有尿液指标异常，尿液检查可以作为判断IgA肾病确诊患者病情的依据，却无法作为诊断依据。
3)肾功能检查
血肌酐上升到1.5mg/dl(132.6umol/L)多为病情进展。GFR<20ml/min时，病理改变属Ⅲ级以上。与尿液检查类似，肾功能检查可以作为判断IgA肾病确诊患者病情的依据，却无法作为诊断依据。
3、活检病理诊断
IgA肾病的确诊必须要有肾活检病理，需要免疫荧光或免疫组化的结果支持。其诊断特点是：光镜下常见弥漫性系膜增生或局灶节段增生性肾小球肾炎；免疫荧光可见系膜区IgA或以IgA为主的免疫复合物沉积，这是IgA肾病的诊断标志。然而，肾脏穿刺活检对病人痛苦大，且存在相当大漏诊、误诊几率。
综上所述，目前临床上亟需一种方便、准确且便利的针对IgA肾病的检验诊断方法。
发明内容
针对目前对于IgA肾病临床诊断的迫切需要，以及现有技术中的上述不足，本发明提供一种利用IgA肾病人的外周血中特征性的TCR标志物来快速准确的判断待测人员是否已经发生IgA肾病特有的T细胞反应的检测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IgA肾病风险患者。
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IgA肾病的外周血TCR标志物，该标志物包括序列为SEQ ID NO.1～100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
Figure PCTCN2022080395-appb-000001
Figure PCTCN2022080395-appb-000002
Figure PCTCN2022080395-appb-000003
Figure PCTCN2022080395-appb-000004
进一步地，标志物的蛋白序列为SEQ ID NO.1～100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抗原决定簇3(CDR3)的蛋白序列。
上述标志物在制备治疗IgA肾病的制剂中的应用。
进一步地，制剂中包括含有该标志物的T细胞受体基因序列，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IgA肾病检测的试剂盒，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IgA肾病进行诊断、预测、检测或筛查。
一种检测IgA肾病的蛋白质芯片，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。
针对IgA肾病，病人体内产生了疾病特有的T细胞免疫反应。我们就可以利用这些IgA 肾病病人特有免疫反应特征来判断疾病。
本发明的有益效果为：
1、本发明中，首先利用1627个非IgA肾病的对照组样本、和47个IgA肾病病人的TCR高通量测序数据建立大数据分析模型，通过和这些IgA肾病特异性TCR序列对比，可以清楚的判断待测样本中是否有较高IgA肾病风险者；
2、通过高通量测序分析TCR变化可以发现很早期的IgA肾病，利用IgA肾病特有的TCR CDR3序列分析人的免疫系统中的T细胞对IgA肾病的反应，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IgA肾病的免疫治疗。
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IgA肾病特征性TCR序列。横坐标代表某一特定氨基酸组合的CDR3序列被加入IgA肾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；IgA肾病患者的免疫大数据具有多个种类且重复次数较高的IgA肾病特征序列，健康人极少IgA肾病特征序列，而未知受试者的IgA肾病特征比较明显，说明罹患IgA肾病风险较高。
图2为本发明中，利用IgA肾病特征特征性指数对比分析IgA肾病和其他疾病。健康人、其它非肿瘤疾病病人的IgA肾病特征性指数均与IgA肾病患者具有显著差异，证明了IgA肾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IgA肾病。
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 通过免疫大数据分析，获得IgA肾病TCR标志物CDR3序列集
1、采样及免疫大数据分析
采集1628名对照组(包括健康人和其它非肿瘤非肾病疾病病人，1627人用于建立模型， 1个健康人用于验证)、48名IgA肾病患者(47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IgA肾病TCR标志物CDR3序列：
a)将1627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47名用于建立模型的IgA肾病样本的所有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IgA肾病特征序列集。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IgA肾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IgA肾病患者和1名健康状况未知受试者的免疫大数据，参照IgA肾病特征序列集合进行作图，见附图1B-D。从图中可见，IgA肾病患者的免疫大数据中，含有较多种类且较高重复出现次数的IgA肾病特征序列(图1B)；健康人的免疫大数据中，只有极少量IgA肾病特征序列(图1C)；而未知健康状况受试者，有高于健康人的IgA肾病特征序列，说明此人有较高风险罹患IgA肾病(图1D)。
d)将IgA肾病特征序列集中，将所有出现在两个及以上参与建模IgA肾病样本里的CDR3序列，按“所有参与建模IgA肾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IgA肾病样本数”从高到低排序，排名前100者即为IgA肾病TCR标志物CDR3序列，具体序列如SEQ ID NO.1～100所示。
实施例2 验证IgA肾病TCR标志物CDR3序列集的特异性
1、采样及免疫大数据分析
采集95名健康人、44名其它非肾病非肿瘤疾病患者和3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。
3、根据来自实施例1的47名IgA肾病患者，以及实施例2新获取的95名健康人、44名其它非肾病非肿瘤疾病患者、3名未知健康状况受试者的免疫大数据，分析其IgA肾病特征性指数。
其中IgA肾病特征性指数定义为：某个样本中，属于IgA肾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。IgA肾病组与健康人(p＝6.96E-138)、非肾病非肿瘤疾病(p＝4.89E-82)都有显著差异，这证明了IgA肾病特征序列集的特异性。
表2 不同样本组的IgA肾病特征性指数
Figure PCTCN2022080395-appb-000005
Figure PCTCN2022080395-appb-000006
Figure PCTCN2022080395-appb-000007
Figure PCTCN2022080395-appb-000008
4、分析各组的IgA肾病特征指数(表3)，3位未知健康状况受试者(检测样本)的IgA肾病特征指数显著高于“健康人”组的平均值+2×SD(45.1+2×15.8＝76.6)、“非肾病非肿瘤疾病”组的平均值+2×SD(49.8+2×32.5＝114.8)，此3人具有较高风险罹患IgA肾病。与临床体检结果对照后，这3人确为早期IgA肾病患者。此实施例证明了利用IgA肾病特征序列集及IgA肾病特征性指数，预测受试者罹患IgA肾病风险的可行性。
表3 IgA肾病特征性指数分析
Figure PCTCN2022080395-appb-000009
综上所述，本发明所述IgA肾病TCR标志物CDR3序列，确实具有显著的IgA肾病特异性，不仅可以用于IgA肾病的早期诊断，还可用于评估治疗IgA肾病的药物的疗效，未来还可以根据这些免疫特征设计新的生物免疫治疗方案。

Claims (7)

  1. 一种IgA肾病的外周血TCR标志物，其特征在于，所述标志物包括序列为SEQ ID NO.1～100所示TCR蛋白序列中的至少一种。
  2. 根据权利要求1所述的IgA肾病的外周血TCR标志物，其特征在于，所述标志物的蛋白序列为SEQ ID NO.1～100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TCR标志物在制备治疗IgA肾病的制剂中的应用。
  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IgA肾病检测的试剂盒，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IgA肾病进行诊断、预测、检测或筛查。
  7. 一种检测IgA肾病的蛋白质芯片，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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