WO2022194033A1 - Peripheral blood tcr marker for diffuse large b-cell lymphoma, and detection kit and use therefor - Google Patents

Peripheral blood tcr marker for diffuse large b-cell lymphoma, and detection kit and use therefor Download PDF

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WO2022194033A1
WO2022194033A1 PCT/CN2022/080282 CN2022080282W WO2022194033A1 WO 2022194033 A1 WO2022194033 A1 WO 2022194033A1 CN 2022080282 W CN2022080282 W CN 2022080282W WO 2022194033 A1 WO2022194033 A1 WO 2022194033A1
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cell lymphoma
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卓越
杨鑫
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成都益安博生物技术有限公司
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    • 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
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
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    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a peripheral blood TCR marker of diffuse large B-cell lymphoma and a detection kit and application thereof.
  • Non-Hodgkin Lymphoma is a general term for a group of malignant tumors originating from lymph nodes and lymphoid tissues. NHL lesions mainly occur in lymph nodes, spleen, thymus and other lymphoid organs, and can also occur outside lymph nodes. Malignant tumors of the lymphoid tissues and organs of the lymphohematopoietic system. They are divided into three basic types according to their cell origin: B cells, T cells, and NK/T cell NHL.
  • non-Hodgkin lymphoma accounts for 4% of new tumors each year, and accounts for 90% of all malignant lymphomas. About 54,000 people are diagnosed with non-Hodgkin lymphoma every year nationwide, and the annual number of deaths About 19,000 people. The incidence of the disease increases significantly with age, and it is more common in men than women.
  • DLBCL Diffuse Large B-Cell Lymphoma
  • NHL Diffuse Large B-Cell Lymphoma
  • DLBCL is a diffuse growth of tumorous large B lymphocytes.
  • the nuclei of tumor cells are similar in size to those of normal tissue cells or larger than those of normal tissue cells, usually 2 times larger than those of normal lymphocytes.
  • This cancer can occur at any age, but is common in older adults, and diffuse large B-cell lymphoma can occur almost anywhere in the body. Its incidence accounts for 31% to 34% of non-Hodgkin's lymphoma (NHL), and is generally greater than 40% in Asian countries.
  • NDL non-Hodgkin's lymphoma
  • non-Hodgkin lymphoma The etiology and pathogenesis of non-Hodgkin lymphoma are not yet fully understood, and it is generally believed to be related to risk factors such as infection, immune status, environment, and diet. Under the influence of these risk factors, it can cause the activation of cancer genes in B cells and T cells and the inactivation of tumor suppressor genes, so that lymphocyte proliferation is out of control, and eventually non-Hodgkin lymphoma is formed.
  • the survival rate of lymphoma is relatively high. In the case of standardized comprehensive treatment, the cure rate of lymphoma can exceed 60%. Among them, the cure rate of Hodgkin's lymphoma is 70%-90%, and 50% of non-Hodgkin's lymphoma cancer patients can achieve long-term survival. However, according to the 2018 statistics of The Lancet, the 5-year survival rate of lymphoma patients in my country is about 38.3%, compared with 57.3% and 68.1% in Japan and the United States, there is still a significant gap. There are several reasons. The first is related to the accuracy of diagnosis, and different subtypes have different treatment options. Histological subtype is a major factor in determining the clinical presentation, prognosis and treatment of patients.
  • Traumatic examinations are mostly performed by taking histological specimens for diagnosis, while non-invasive examinations are mostly performed to find the location of lesions, assess the extent of lesions and evaluate the efficacy of treatment.
  • the method of operation is determined mainly by the size of the lesion, the site of growth, and the possible risk of trauma examination.
  • tissue and cell specimens can be taken under the assistance of endoscopy such as bronchoscopy, mediastinoscopy, gastroscope, and colonoscopy.
  • endoscopy such as bronchoscopy, mediastinoscopy, gastroscope, and colonoscopy.
  • Various surgical methods are used to obtain lymph node or lesion cell specimens.
  • Cerebrospinal fluid cytology was performed by lumbar puncture.
  • Laboratory tests include complete blood count, erythrocyte sedimentation rate, lactate dehydrogenase, ⁇ 2-microglobulin, and liver and kidney function.
  • Imaging examinations include whole-body CT, magnetic resonance, and positron emission tomography (PET/CT).
  • PET/CT positron emission tomography
  • Imaging detection also has its own advantages and disadvantages. Its detection requires the tumor to reach a certain size before it can be detected. There is a hysteresis, and it cannot be used as a means of real-time monitoring due to radiation and other reasons. Therefore, there is an urgent need for a more convenient and sensitive screening and testing method that can track the occurrence and development of diffuse large B-cell lymphoma.
  • the present invention provides a peripheral blood TCR marker for diffuse large B-cell lymphoma and a detection kit and application thereof, which can accurately and quickly determine whether a sample to be tested has a higher diffuse large B-cell lymphoma Cellular lymphoma risk patients.
  • a peripheral blood TCR marker for diffuse large B-cell lymphoma includes at least one of the proteins whose sequences are shown in SEQ ID NO. 1 to 100, and the specific sequence is shown in Table 1.
  • protein sequence of the marker is that the sequence shown in SEQ ID NO. 1-100 can express a protein with the same function after one or more bases are substituted, deleted and/or replaced.
  • the marker is the peripheral blood TCR CDR3 sequence.
  • the preparation includes a T cell receptor containing the marker, or a plasmid, viral vector or nucleic acid fragment capable of expressing the T cell receptor producing the marker.
  • a kit for detecting diffuse large B-cell lymphoma comprising antibodies that can specifically bind to the above markers.
  • a preparation comprising an antibody that can specifically bind to the above markers; the preparation can be used for diagnosis, prediction, detection or screening of diffuse large B-cell lymphoma.
  • a protein chip for detecting diffuse large B-cell lymphoma the protein chip comprises a substrate and a specific antibody spotted on the substrate, the specific antibody being an antibody that can specifically bind to the above marker.
  • 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. Later, when B cells differentiate into plasma cells, BCR is expressed as an antibody and secreted outside the cell.
  • T cells recognize antigens through T cell receptors (TCRs) on the cell surface.
  • BCR and TCR The diversity of BCR and TCR is the basis for the establishment of the adaptive immune system.
  • the theoretical value of BCR diversity is 10 18 and the theoretical value of TCR diversity is 10 14 .
  • antigenic determinant 3 CDR3
  • CDR3 antigenic determinant 3
  • BCR and TCR will change with different antigenic stimulation. Therefore, the occurrence and development of diseases can be tracked by using BCR or TCR high-throughput sequencing results.
  • MCHII histocompatibility antigen II
  • Antigen fragments presented by normal cells do 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.
  • an artificial intelligence analysis model was established by first using 1300 non-diffuse large B-cell lymphoma control samples and 40 diffuse large B-cell lymphoma patients' TCR high-throughput sequencing data to establish an artificial intelligence analysis model.
  • Large B-cell lymphoma-specific TCR sequence comparison can clearly determine whether there is a higher risk of diffuse large B-cell lymphoma in the sample to be tested.
  • TCR CDR3 sequence specific to diffuse large B-cell lymphoma to analyze the effect of T cells in the human immune system on diffuse large B-cell lymphoma.
  • the reaction is a new detection method.
  • the present invention simultaneously compares a huge number of specific TCR sequences, and has higher specificity and accuracy than detecting one or several markers alone.
  • the cost of the high-throughput sequencing instrument used in the present invention is lower than that of large-scale imaging equipment, and it can be outsourced to a third party.
  • the labor cost of sampling and processing is lower than the simultaneous detection of multiple markers, and it is also 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.
  • TCR CDR3 sequence described in the present invention can be used for immunotherapy of diffuse large B-cell lymphoma.
  • Fig. 1 shows that in the present invention, the characteristic TCR sequence of diffuse large B-cell lymphoma is found by using an immune-based big data analysis system; wherein, the abscissa represents that the CDR3 sequence of a certain amino acid combination is added to the control sequence set or diffuse large B-cell lymphoma
  • the order of the tumor characteristic sequence set, the ordinate represents the logarithm of the number of repeated occurrences of the sequence in a sample, C X ;
  • the immune map of patients with diffuse large B-cell lymphoma has multiple types of diffuse large B cells with a high number of repetitions Characteristic sequences of diffuse large B-cell lymphoma in healthy people are rare, while the characteristics of diffuse large B-cell lymphoma in unknown subjects are more obvious, indicating a higher risk of developing diffuse large B-cell lymphoma;
  • Fig. 2 is a comparative analysis of diffuse large B-cell lymphoma and other diseases using the characteristic index of diffuse large B-cell lymphoma in the present invention.
  • the characteristic indices of DLBCL were significantly different from those of patients with DLBCL, proving the specificity of the DLBCL signature sequence set, which can be used to determine whether an unknown subject has DLBCL .
  • Collected 1301 controls including healthy and non-tumor disease patients, 1300 were used for model establishment, 1 healthy person was used for validation), 41 diffuse large B-cell lymphoma patients (40 were used for model establishment, 1 For verification) and peripheral blood of 1 subject with unknown health status (10mL per person), the antigenic determinant 3 (CDR3) amino acid sequences of TCR of the subjects and the control group were obtained by high-throughput sequencing to ensure that each The total number of CDR3 sequences of functional TCRs in the sample should not be less than 25000;
  • CDR3 amino acid sequence ensure that the total number of CDR3 sequences of functional TCRs in each sample is not less than 25,000; the sequences of samples whose total number of CDR3 sequences of each functional TCR is more than 30,000 are randomly sampled without replacement, so that The total number of CDR3 sequences for this sample is 30,000.
  • the total number of CDR3 sequences of functional TCRs contained in all samples so far is 25,000-30,000.
  • the characteristic index of diffuse large B-cell lymphoma is defined as: in a certain sample, the sum of the repeated occurrences C X of all CDR3 sequences belonging to the characteristic sequence set of diffuse large B-cell lymphoma in the sample.
  • the analysis results are shown in Table 2 below and accompanying drawing 2.
  • the CDR3 sequence of the diffuse large B-cell lymphoma TCR marker of the present invention does have significant specificity for diffuse large B-cell lymphoma, and it can not only be used for the prediction of diffuse large B-cell lymphoma in subjects suffering from diffuse large B-cell lymphoma.
  • the risk of large B-cell lymphoma can also be used for biological immunotherapy of diffuse large B-cell lymphoma in the future.

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Abstract

A peripheral blood TCR marker for diffuse large B-cell lymphoma, and a detection kit and use therefor. The marker comprises at least one among proteins having sequences shown in SEQ ID NO. 1-100. According to the high-throughput sequencing method: only requiring a small amount of peripheral blood, extracting RNA is extracted, establishing an immune map library by means of processing samples, then carrying out high-throughput sequencing and TCR data analysis, first determining a characteristic TCR sequence in the peripheral blood of the diffuse large B-cell lymphoma, and then comparing a test result of a sample to be tested with the characteristic TCR sequence, so as to determine whether diffuse large B cell lymphoma exists. The present solution compares a large number of diffuse large B cell lymphoma specific TCR sequences at the same time, and compared with separately detecting one or more markers, has higher specificity and accuracy, and improves diagnosis efficiency.

Description

一种弥漫大B细胞淋巴瘤的外周血TCR标志物及其检测试剂盒和应用A peripheral blood TCR marker of diffuse large B-cell lymphoma and its detection kit and application 技术领域technical field
本发明属于基因工程技术领域，具体涉及一种弥漫大B细胞淋巴瘤的外周血TCR标志物及其检测试剂盒和应用。The invention belongs to the technical field of genetic engineering, and in particular relates to a peripheral blood TCR marker of diffuse large B-cell lymphoma and a detection kit and application thereof.
背景技术Background technique
非霍奇金淋巴瘤(Non-Hodgkin Lymphoma，NHL)是一组起源于***和淋巴组织的恶性肿瘤的总称，NHL病变是主要发生在***、脾脏、胸腺等淋巴器官，也可发生在***外的淋巴组织和器官的淋巴造血***的恶性肿瘤。依据细胞来源将其分为三种基本类型：B细胞、T细胞和NK/T细胞NHL。Non-Hodgkin Lymphoma (NHL) is a general term for a group of malignant tumors originating from lymph nodes and lymphoid tissues. NHL lesions mainly occur in lymph nodes, spleen, thymus and other lymphoid organs, and can also occur outside lymph nodes. Malignant tumors of the lymphoid tissues and organs of the lymphohematopoietic system. They are divided into three basic types according to their cell origin: B cells, T cells, and NK/T cell NHL.
流行病学显示，我国非霍奇金淋巴瘤患者相对较少，但近年来发病率逐渐上升。最近的一项调查显示，非霍奇金淋巴瘤占每年新发肿瘤的4％，占所有恶性淋巴瘤的90％，全国每年约有54000人被诊断为非霍奇金淋巴瘤，每年死亡人数约19000人。该病发病率随年龄的增长而显著上升，男性患者比女性多见。Epidemiology shows that there are relatively few patients with non-Hodgkin lymphoma in my country, but the incidence has gradually increased in recent years. According to a recent survey, non-Hodgkin lymphoma accounts for 4% of new tumors each year, and accounts for 90% of all malignant lymphomas. About 54,000 people are diagnosed with non-Hodgkin lymphoma every year nationwide, and the annual number of deaths About 19,000 people. The incidence of the disease increases significantly with age, and it is more common in men than women.
弥漫大B细胞淋巴瘤(Diffuse Large B-Cell Lymphoma，DLBCL)是NHL中最常见的类型，在临床上最常见的病理学类型、最具代表性的侵袭性淋巴瘤。DLBCL是肿瘤性大B淋巴细胞呈弥漫性生长，肿瘤细胞的核与正常组织细胞的核大小相近或大于组织细胞的核，通常大于正常淋巴细胞的2倍。该癌种可发生于任何年龄段，但常见于老年人，几乎全身任何部位均可发生弥漫性大B细胞淋巴瘤。其发病率占非霍奇金淋巴瘤(NHL)的31％～34％，在亚洲国家一般大于40％。我国2011年一项由24个中心联合进行、共收集10002例病例样本的分析报告指出，在中国DLBCL占所有NHL的45.8％，占所有淋巴瘤的40.1％。Diffuse Large B-Cell Lymphoma (DLBCL) is the most common type of NHL, the most common pathological type in clinic, and the most representative aggressive lymphoma. DLBCL is a diffuse growth of tumorous large B lymphocytes. The nuclei of tumor cells are similar in size to those of normal tissue cells or larger than those of normal tissue cells, usually 2 times larger than those of normal lymphocytes. This cancer can occur at any age, but is common in older adults, and diffuse large B-cell lymphoma can occur almost anywhere in the body. Its incidence accounts for 31% to 34% of non-Hodgkin's lymphoma (NHL), and is generally greater than 40% in Asian countries. In 2011, an analysis report of 10,002 case samples collected by 24 centers in China indicated that DLBCL accounted for 45.8% of all NHLs and 40.1% of all lymphomas in China.
非霍奇金淋巴瘤的病因和发病机制目前尚未完全清楚，一般认为其与感染、免疫状态、环境和饮食等危险因素相关。在这些危险因素影响下，可引起B细胞和T细胞内癌症基因的激活及肿瘤抑制基因的失活，使淋巴细胞增殖失控，最终形成非霍奇金淋巴瘤。The etiology and pathogenesis of non-Hodgkin lymphoma are not yet fully understood, and it is generally believed to be related to risk factors such as infection, immune status, environment, and diet. Under the influence of these risk factors, it can cause the activation of cancer genes in B cells and T cells and the inactivation of tumor suppressor genes, so that lymphocyte proliferation is out of control, and eventually non-Hodgkin lymphoma is formed.
淋巴瘤生存率相对较高，在规范化综合治疗的情况下，淋巴瘤治愈率可超过60％，其中霍奇金氏淋巴瘤治愈率为70％-90％，50％的非霍奇金氏淋巴瘤患者可获得长期生存。但根据《柳叶刀》2018统计数据，我国淋巴瘤患者的5年生存率约为38.3％，相比日本和美国的57.3％，68.1％，仍存在明显差距。原因有好几个。首先是与诊断的准确性有关，不同亚型的确定治疗方案不同。组织学亚型是决定患者临床表现、预后和治疗的主要因素。2019淋巴瘤***数据显示，参与调研的患者中，约43％患者曾有过误诊经历，51％的患者辗转多家医院后方得以确诊。患者从初次诊断到最终确定所患亚型，平均耗费时长为2.5个月，而一些亚型患者确诊所需等待时间更为漫长。同时，调研表明，大部分患者为了确诊，需奔赴北上广等医疗资源集中的大城市；其次是患者的就诊意识较低。很多患者前来就诊时就已经是晚期，治疗效果肯定不如早期。所以早诊断、早治疗，是提高淋巴瘤治疗效果，改善患者预后，节约社会资源的迫切需求。The survival rate of lymphoma is relatively high. In the case of standardized comprehensive treatment, the cure rate of lymphoma can exceed 60%. Among them, the cure rate of Hodgkin's lymphoma is 70%-90%, and 50% of non-Hodgkin's lymphoma cancer patients can achieve long-term survival. However, according to the 2018 statistics of The Lancet, the 5-year survival rate of lymphoma patients in my country is about 38.3%, compared with 57.3% and 68.1% in Japan and the United States, there is still a significant gap. There are several reasons. The first is related to the accuracy of diagnosis, and different subtypes have different treatment options. Histological subtype is a major factor in determining the clinical presentation, prognosis and treatment of patients. According to the data of the 2019 Lymphoma White Paper, about 43% of the patients who participated in the survey had experienced misdiagnosis, and 51% of the patients were diagnosed after going to multiple hospitals. It took an average of 2.5 months for patients to go from initial diagnosis to final determination of their subtype, with some subtypes waiting longer for diagnosis. At the same time, the survey shows that most patients need to go to big cities with concentrated medical resources such as Beijing, Shanghai and Guangzhou in order to make a diagnosis; secondly, the patients' awareness of seeking medical treatment is low. Many patients are already in the late stage when they come to see the doctor, and the treatment effect is definitely not as good as the early stage. Therefore, early diagnosis and early treatment are urgent needs to improve the treatment effect of lymphoma, improve the prognosis of patients, and save social resources.
弥漫大B细胞淋巴瘤的诊断检查可分为创伤性和非创伤性检查。创伤性检查多是取组织细胞学标本为诊断而进行，非创伤性检查多是为寻找病变位置、评估病变范围和评估治疗疗效而进行。Diagnostic tests for diffuse large B-cell lymphoma can be divided into invasive and non-invasive tests. Traumatic examinations are mostly performed by taking histological specimens for diagnosis, while non-invasive examinations are mostly performed to find the location of lesions, assess the extent of lesions and evaluate the efficacy of treatment.
1、创伤性检查1. Traumatic examination
主要根据病变的大小、生长的部位和创伤检查可能带来的风险而决定操作的方式。The method of operation is determined mainly by the size of the lesion, the site of growth, and the possible risk of trauma examination.
浅表肿大***直接穿刺；或在超声、CT定位引导下经皮进行深部***及占位病变穿刺针吸细胞获取标本。此种方法价值存在一定的争议。Direct puncture of superficial enlarged lymph nodes; or percutaneous deep lymph node and space-occupying lesion puncture needle aspiration under the guidance of ultrasound and CT positioning to obtain specimens. The value of this method is controversial.
根据病变部位可在支气管镜、纵隔镜、胃镜、结肠镜等内镜辅助下取组织细胞标本。各种手术方式获取***或病变部位细胞标本。According to the lesion site, tissue and cell specimens can be taken under the assistance of endoscopy such as bronchoscopy, mediastinoscopy, gastroscope, and colonoscopy. Various surgical methods are used to obtain lymph node or lesion cell specimens.
骨髓穿刺和活检。Bone marrow aspiration and biopsy.
腰椎穿刺进行脑脊液细胞学检查。Cerebrospinal fluid cytology was performed by lumbar puncture.
2、非创伤性检查2. Non-invasive examination
实验室检查包括全血细胞计数、血沉、乳酸脱氢酶、β2微球蛋白和肝肾功能等。Laboratory tests include complete blood count, erythrocyte sedimentation rate, lactate dehydrogenase, β2-microglobulin, and liver and kidney function.
影像学检查包括全身CT、磁共振、正电子发射型断层扫描技术(PET/CT)等。Imaging examinations include whole-body CT, magnetic resonance, and positron emission tomography (PET/CT).
综上所述，非创伤性检查中的血液检测只能大致判断有疾病存在，无法分辨针对具体疾病；创伤性检查中的骨髓穿刺和活检，需要穿刺活检，取样困难，不能作为实时监测手段，对患者身体也会造成损伤。往往需将血液和骨髓穿刺检查结果结合在一起，不能通过检测一项而判断疾病，这也增加了患者负担。影像学检测也都有各自优缺点，其检测都需要肿瘤达到一定体积大小才能检测到，存在滞后性，因辐射等原因不能作为实时监控的手段。因此，亟需一种能更便捷灵敏，且可以追踪弥漫大B细胞淋巴瘤发生、发展状况的筛查、检验方法。To sum up, blood testing in non-invasive examinations can only roughly determine the presence of diseases, and cannot identify specific diseases; bone marrow aspirate and biopsy in invasive examinations require puncture biopsy, which is difficult to sample, and cannot be used as a real-time monitoring method. It can also cause damage to the patient's body. It is often necessary to combine the results of blood and bone marrow aspirate examinations, and the disease cannot be judged by one test, which also increases the burden on patients. Imaging detection also has its own advantages and disadvantages. Its detection requires the tumor to reach a certain size before it can be detected. There is a hysteresis, and it cannot be used as a means of real-time monitoring due to radiation and other reasons. Therefore, there is an urgent need for a more convenient and sensitive screening and testing method that can track the occurrence and development of diffuse large B-cell lymphoma.
发明内容SUMMARY OF THE INVENTION
针对现有技术中的上述不足，本发明提供一种弥漫大B细胞淋巴瘤的外周血TCR标志物及其检测试剂盒和应用，能准确快速的判断待测样本中是否有较高弥漫大B细胞淋巴瘤风险患者。In view of the above deficiencies in the prior art, the present invention provides a peripheral blood TCR marker for diffuse large B-cell lymphoma and a detection kit and application thereof, which can accurately and quickly determine whether a sample to be tested has a higher diffuse large B-cell lymphoma Cellular lymphoma risk patients.
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：For realizing the above-mentioned purpose, the technical scheme that the present invention solves its technical problem adopts is:
一种弥漫大B细胞淋巴瘤的外周血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。A peripheral blood TCR marker for diffuse large B-cell lymphoma, the marker includes at least one of the proteins whose sequences are shown in SEQ ID NO. 1 to 100, and the specific sequence is shown in Table 1.
表1标志物序列Table 1 Marker sequences
序列编号serial number 蛋白序列protein sequence
SEQ ID NO.1SEQ ID NO.1 Ala Ser Ser Tyr Ser Glu Ser Ala His Glu Gln PheAla Ser Ser Tyr Ser Glu Ser Ala His Glu Gln Phe
SEQ ID NO.2SEQ ID NO.2 Ala Ser Ser Glu Pro Glu Gly Lys Glu Glu Ala PheAla Ser Ser Glu Pro Glu Gly Lys Glu Glu Ala Phe
SEQ ID NO.3SEQ ID NO.3 Ala Ser Ser Arg Leu Ile Gly Gly Asn Glu Gln PheAla Ser Ser Arg Leu Ile Gly Gly Asn Glu Gln Phe
SEQ ID NO.4SEQ ID NO.4 Ala Ser Ser Leu Gln Arg Glu Glu Tyr Glu Gln TyrAla Ser Ser Leu Gln Arg Glu Glu Tyr Glu Gln Tyr
SEQ ID NO.5SEQ ID NO.5 Ala Thr Ser Gly Pro Gln Gly Thr Asp Thr Gln TyrAla Thr Ser Gly Pro Gln Gly Thr Asp Thr Gln Tyr
SEQ ID NO.6SEQ ID NO.6 Ala Ser Ser Leu Tyr Gly Pro Gly Ala Asn Thr Glu Ala PheAla Ser Ser Leu Tyr Gly Pro Gly Ala Asn Thr Glu Ala Phe
SEQ ID NO.7SEQ ID NO.7 Ala Ser Ser Ile Gly Gly Ser Met Val Gln Pro Gln HisAla Ser Ser Ile Gly Gly Ser Met Val Gln Pro Gln His
SEQ ID NO.8SEQ ID NO.8 Ala Thr Gln Asp Pro Ser Arg Pro Gln HisAla Thr Gln Asp Pro Ser Arg Pro Gln His
SEQ ID NO.9SEQ ID NO.9 Ser Ala Leu Gly Gln Thr Val Lys Thr Tyr Glu Gln TyrSer Ala Leu Gly Gln Thr Val Lys Thr Tyr Glu Gln Tyr
SEQ ID NO.10SEQ ID NO.10 Pro Pro Ala Val Pro Pro Glu Arg Pro Ser ThrPro Pro Ala Val Pro Pro Glu Arg Pro Ser Thr
SEQ ID NO.11SEQ ID NO.11 Ala Ser Ser Ser Ala Thr Ser Gly Val Tyr Glu Gln TyrAla Ser Ser Ser Ala Thr Ser Gly Val Tyr Glu Gln Tyr
SEQ ID NO.12SEQ ID NO.12 Ala Ser Ser Gln Glu Pro Ser Gly Ser Ser Gln Glu Thr Gln TyrAla Ser Ser Gln Glu Pro Ser Gly Ser Ser Gln Glu Thr Gln Tyr
SEQ ID NO.13SEQ ID NO.13 Ala Ser Ser Asn Ser Arg Val Asp Gln Pro Gln HisAla Ser Ser Asn Ser Arg Val Asp Gln Pro Gln His
SEQ ID NO.14SEQ ID NO.14 Ala Ser Thr Leu Leu His Arg Asp Asn Glu Gln PheAla Ser Thr Leu Leu His Arg Asp Asn Glu Gln Phe
SEQ ID NO.15SEQ ID NO.15 Ala Ser Ser Tyr Ser Leu Pro Gly Leu Ala Gly Lys Glu Gly Gln Glu Thr Gln TyrAla Ser Ser Tyr Ser Leu Pro Gly Leu Ala Gly Lys Glu Gly Gln Glu Thr Gln Tyr
SEQ ID NO.16SEQ ID NO.16 Ala Ser Arg His Arg Glu Ser Phe Thr Asp Thr Gln TyrAla Ser Arg His Arg Glu Ser Phe Thr Asp Thr Gln Tyr
SEQ ID NO.17SEQ ID NO.17 Ala Ser Ser Asp Asp Gly Ala Trp Glu Asp Glu Gln PheAla Ser Ser Asp Asp Gly Ala Trp Glu Asp Glu Gln Phe
SEQ ID NO.18SEQ ID NO.18 Ala Asn Thr Leu Pro Glu Gly Gly Ser Glu Gln PheAla Asn Thr Leu Pro Glu Gly Gly Ser Glu Gln Phe
SEQ ID NO.19SEQ ID NO.19 Ala Ser Ser His Arg Thr Ser Gly Arg Ala Leu Tyr Glu Gln TyrAla Ser Ser His Arg Thr Ser Gly Arg Ala Leu Tyr Glu Gln Tyr
SEQ ID NO.20SEQ ID NO.20 Ala Ser Ser Gln Gln Val Pro Pro Thr Asn Thr Gly Glu Leu PheAla Ser Ser Gln Gln Val Pro Pro Thr Asn Thr Gly Glu Leu Phe
SEQ ID NO.21SEQ ID NO.21 Ser Val Glu Pro His Ser Gly Arg Ser Glu Thr Gln TyrSer Val Glu Pro His Ser Gly Arg Ser Glu Thr Gln Tyr
SEQ ID NO.22SEQ ID NO.22 Ala Ser Ser Ser Gly Thr Gly Pro Gly Leu Pro Gln HisAla Ser Ser Ser Gly Thr Gly Pro Gly Leu Pro Gln His
SEQ ID NO.23SEQ ID NO.23 Ser Val Glu Glu Val Arg Glu Gln TyrSer Val Glu Glu Val Arg Glu Gln Tyr
SEQ ID NO.24SEQ ID NO.24 Ala Ser Ile Ser Gly His Ser Tyr Glu Gln TyrAla Ser Ile Ser Gly His Ser Tyr Glu Gln Tyr
SEQ ID NO.25SEQ ID NO.25 Ala Ser Ser Thr Arg Asp Arg Val Glu Met Asn Thr Glu Ala PheAla Ser Ser Thr Arg Asp Arg Val Glu Met Asn Thr Glu Ala Phe
SEQ ID NO.26SEQ ID NO.26 Ala Ser Ser Leu Gly Thr Thr Ala Thr Asp Thr Gly Glu Leu PheAla Ser Ser Leu Gly Thr Thr Ala Thr Asp Thr Gly Glu Leu Phe
SEQ ID NO.27SEQ ID NO.27 Ala Ser Lys Gly Trp Thr Thr Ser Pro Leu HisAla Ser Lys Gly Trp Thr Thr Ser Pro Leu His
SEQ ID NO.28SEQ ID NO.28 Ala Ile Arg Arg Asp Ile Val Lys Thr Glu Ala PheAla Ile Arg Arg Asp Ile Val Lys Thr Glu Ala Phe
SEQ ID NO.29SEQ ID NO.29 Ala Ser Ser Glu Pro Ala Ser Leu Asp Thr Gln TyrAla Ser Ser Glu Pro Ala Ser Leu Asp Thr Gln Tyr
SEQ ID NO.30SEQ ID NO.30 Ala Ser Gly Leu Ser Gly Gly Gly Gln TyrAla Ser Gly Leu Ser Gly Gly Gly Gln Tyr
SEQ ID NO.31SEQ ID NO.31 Ala Ser Thr Arg Gln Ala Gln Met Ser Asn Gln Pro Gln HisAla Ser Thr Arg Gln Ala Gln Met Ser Asn Gln Pro Gln His
SEQ ID NO.32SEQ ID NO.32 Gln Gln Gln Arg Thr Gly Gly Ala His Arg Tyr Ala ValGln Gln Gln Arg Thr Gly Gly Ala His Arg Tyr Ala Val
SEQ ID NO.33SEQ ID NO.33 Ala Ser Thr Ser Ser Arg Gly Gly Ser Gly Ala Asn Val Leu ThrAla Ser Thr Ser Ser Ser Arg Gly Gly Ser Gly Ala Asn Val Leu Thr
SEQ ID NO.34SEQ ID NO.34 Ala Ser Ser Leu Met Arg Val Lys Ser His Glu Gln TyrAla Ser Ser Leu Met Arg Val Lys Ser His Glu Gln Tyr
SEQ ID NO.35SEQ ID NO.35 Ala Ser Ser Pro Ala Gln Arg Gly Val Glu Gln PheAla Ser Ser Pro Ala Gln Arg Gly Val Glu Gln Phe
SEQ ID NO.36SEQ ID NO.36 Ala Thr Ser Gly Asp Ser Leu Ala Glu Gln PheAla Thr Ser Gly Asp Ser Leu Ala Glu Gln Phe
SEQ ID NO.37SEQ ID NO.37 Ser Asp Leu Leu Leu Gly Thr Gln TyrSer Asp Leu Leu Leu Gly Thr Gln Tyr
SEQ ID NO.38SEQ ID NO.38 Ala Ile Ser Glu Pro Thr Tyr Ala Ala Ser Ala SerAla Ile Ser Glu Pro Thr Tyr Ala Ala Ser Ala Ser
SEQ ID NO.39SEQ ID NO.39 Ala Ser Ser Ala Pro Arg Leu Ala Asp Asn Glu Gln PheAla Ser Ser Ala Pro Arg Leu Ala Asp Asn Glu Gln Phe
SEQ ID NO.40SEQ ID NO.40 Ser Ala Thr Gln Thr Asp Ile Tyr Asn Glu Gln PheSer Ala Thr Gln Thr Asp Ile Tyr Asn Glu Gln Phe
SEQ ID NO.41SEQ ID NO.41 Ala Ser Thr Thr Gly Gly Gly Pro Ile Ala Gln TyrAla Ser Thr Thr Gly Gly Gly Pro Ile Ala Gln Tyr
SEQ ID NO.42SEQ ID NO.42 Ala Ser Arg Asp Gly Ser Gln Gly Asn Ser Pro Leu HisAla Ser Arg Asp Gly Ser Gln Gly Asn Ser Pro Leu His
SEQ ID NO.43SEQ ID NO.43 Ala Ser Ser Gly Gly Thr Gly Ala Leu Asn Glu Gln PheAla Ser Ser Gly Gly Thr Gly Ala Leu Asn Glu Gln Phe
SEQ ID NO.44SEQ ID NO.44 Ala Ser Thr Gln Ala Gly Ala Gln Ala Asp Thr Gln TyrAla Ser Thr Gln Ala Gly Ala Gln Ala Asp Thr Gln Tyr
SEQ ID NO.45SEQ ID NO.45 Ala Ser Ser Ile Ser Gly Gly Gly Ala Val Thr Glu Gln PheAla Ser Ser Ile Ser Gly Gly Gly Ala Val Thr Glu Gln Phe
SEQ ID NO.46SEQ ID NO.46 Ala Ser Arg Lys Gly Ser Gln Glu Gly Thr Gln TyrAla Ser Arg Lys Gly Ser Gln Glu Gly Thr Gln Tyr
SEQ ID NO.47SEQ ID NO.47 Ala Ser Ser Leu Gly Gly Arg Gly Val Ala Gly Glu Leu PheAla Ser Ser Leu Gly Gly Arg Gly Val Ala Gly Glu Leu Phe
SEQ ID NO.48SEQ ID NO.48 Ala Ser Ser Asp Thr Gly Thr Ser Tyr His Glu Gln TyrAla Ser Ser Asp Thr Gly Thr Ser Tyr His Glu Gln Tyr
SEQ ID NO.49SEQ ID NO.49 Ala Ser Ser Leu Asp Gly Asp Tyr Gln Glu Thr Gln TyrAla Ser Ser Leu Asp Gly Asp Tyr Gln Glu Thr Gln Tyr
SEQ ID NO.50SEQ ID NO.50 Ala Thr Gly Leu Arg Gln Gly Asn Thr Gly Glu Leu PheAla Thr Gly Leu Arg Gln Gly Asn Thr Gly Glu Leu Phe
SEQ ID NO.51SEQ ID NO.51 Ala Ser His Ser Gly Arg Ile Asn Thr Glu Ala PheAla Ser His Ser Gly Arg Ile Asn Thr Glu Ala Phe
SEQ ID NO.52SEQ ID NO.52 Ala Ser Ser Lys Ala Lys Gly Thr Gly Ser Tyr Asn Glu Gln PheAla Ser Ser Lys Ala Lys Gly Thr Gly Ser Tyr Asn Glu Gln Phe
SEQ ID NO.53SEQ ID NO.53 Ala Ser Ser Pro Leu Thr Gly Thr Ala Gly Ser Tyr ThrAla Ser Ser Pro Leu Thr Gly Thr Ala Gly Ser Tyr Thr
SEQ ID NO.54SEQ ID NO.54 Ala Thr Ser Glu Glu Ala Gln Glu Thr Gln TyrAla Thr Ser Glu Glu Ala Gln Glu Thr Gln Tyr
SEQ ID NO.55SEQ ID NO.55 His Gln Gln Tyr Arg Arg Arg Asp Pro ValHis Gln Gln Tyr Arg Arg Arg Asp Pro Val
SEQ ID NO.56SEQ ID NO.56 Ala Thr Ser Arg Gly Glu Thr Asp Tyr Gln Glu Thr Gln TyrAla Thr Ser Arg Gly Glu Thr Asp Tyr Gln Glu Thr Gln Tyr
SEQ ID NO.57SEQ ID NO.57 Ala Trp Asn Leu Lys Asp Thr Arg Gln Glu Thr Gln TyrAla Trp Asn Leu Lys Asp Thr Arg Gln Glu Thr Gln Tyr
SEQ ID NO.58SEQ ID NO.58 Ala Ser Ser Leu Ala Gly Gln Gly Thr Gly Glu Gln TyrAla Ser Ser Leu Ala Gly Gln Gly Thr Gly Glu Gln Tyr
SEQ ID NO.59SEQ ID NO.59 Ala Thr Ser Leu Ile Gln Gly Arg Thr Glu Ala PheAla Thr Ser Leu Ile Gln Gly Arg Thr Glu Ala Phe
SEQ ID NO.60SEQ ID NO.60 Pro Ala Asp His Arg Phe Pro Thr Ser Ser ThrPro Ala Asp His Arg Phe Pro Thr Ser Ser Ser Thr
SEQ ID NO.61SEQ ID NO.61 Ala Ser Ser Ser Ile Leu Gly Thr Asn Glu Gln PheAla Ser Ser Ser Ile Leu Gly Thr Asn Glu Gln Phe
SEQ ID NO.62SEQ ID NO.62 Ala Ser Arg Ala Ala Asn Gly Gln Lys Glu Thr Gln TyrAla Ser Arg Ala Ala Asn Gly Gln Lys Glu Thr Gln Tyr
SEQ ID NO.63SEQ ID NO.63 Ala Ser Ser Leu Leu Gln Met Asn Asn Glu Gln PheAla Ser Ser Leu Leu Gln Met Asn Asn Glu Gln Phe
SEQ ID NO.64SEQ ID NO.64 Ser Ala Glu Thr Gly Phe Ser Asn Gln Pro Gln HisSer Ala Glu Thr Gly Phe Ser Asn Gln Pro Gln His
SEQ ID NO.65SEQ ID NO.65 Ala Ser Ser Leu Gly Glu Thr Gly Thr His Asn Ser Pro Leu HisAla Ser Ser Leu Gly Glu Thr Gly Thr His Asn Ser Pro Leu His
SEQ ID NO.66SEQ ID NO.66 Ala Trp Arg Asp Thr Tyr Arg Gly Ala Glu Ala PheAla Trp Arg Asp Thr Tyr Arg Gly Ala Glu Ala Phe
SEQ ID NO.67SEQ ID NO.67 Ala Ser Ser Ser Ile Arg Gly Gln Gly Asn Ser Asn Glu Gln PheAla Ser Ser Ser Ile Arg Gly Gln Gly Asn Ser Asn Glu Gln Phe
SEQ ID NO.68SEQ ID NO.68 Ala Ser Ser Pro Asp Gly Thr Ser Gly Met Glu Thr Gln TyrAla Ser Ser Pro Asp Gly Thr Ser Gly Met Glu Thr Gln Tyr
SEQ ID NO.69SEQ ID NO.69 Ala Ser Ser Thr Gln Gly Ser Val Ser Tyr Glu Gln TyrAla Ser Ser Thr Gln Gly Ser Val Ser Tyr Glu Gln Tyr
SEQ ID NO.70SEQ ID NO.70 Ala Ser Ser Gln Met Thr Ser Gly Glu Gln PheAla Ser Ser Gln Met Thr Ser Gly Glu Gln Phe
SEQ ID NO.71SEQ ID NO.71 Ala Ser Ser Leu Ser Trp Gly Ser Ser Gly Ala Asn Val Leu ThrAla Ser Ser Leu Ser Trp Gly Ser Ser Gly Ala Asn Val Leu Thr
SEQ ID NO.72SEQ ID NO.72 Ala Ser Ser Gln Thr Val Gly Pro Asp Glu Gln TyrAla Ser Ser Gln Thr Val Gly Pro Asp Glu Gln Tyr
SEQ ID NO.73SEQ ID NO.73 Ala Ser Leu Met Gly His Arg Pro Gly Asn Glu Gln PheAla Ser Leu Met Gly His Arg Pro Gly Asn Glu Gln Phe
SEQ ID NO.74SEQ ID NO.74 Ala Cys Gly Arg Ala Asp Asn Glu Gln PheAla Cys Gly Arg Ala Asp Asn Glu Gln Phe
SEQ ID NO.75SEQ ID NO.75 Ala Ser Ser Phe Leu Ser Asn Pro Gly Asn Thr Ile TyrAla Ser Ser Phe Leu Ser Asn Pro Gly Asn Thr Ile Tyr
SEQ ID NO.76SEQ ID NO.76 Ala Ser Ser Ile Leu Gly Gly Ala Ala Asp Thr Gln TyrAla Ser Ser Ile Leu Gly Gly Ala Ala Asp Thr Gln Tyr
SEQ ID NO.77SEQ ID NO.77 Ala Asp Pro Phe Asp Arg Gly Leu Glu Ala PheAla Asp Pro Phe Asp Arg Gly Leu Glu Ala Phe
SEQ ID NO.78SEQ ID NO.78 Ala Ser Ser Leu Gly Thr Ile Ile Gly Ser Gly Asn Thr Ile TyrAla Ser Ser Leu Gly Thr Ile Ile Gly Ser Gly Asn Thr Ile Tyr
SEQ ID NO.79SEQ ID NO.79 Ala Ser Asp Trp Gly Pro Ala Leu His Glu Gln TyrAla Ser Asp Trp Gly Pro Ala Leu His Glu Gln Tyr
SEQ ID NO.80SEQ ID NO.80 Ala Ser Ser Ser Ser Thr Gly Leu Ala Gly Gly Pro Tyr Glu Gln TyrAla Ser Ser Ser Ser Ser Thr Gly Leu Ala Gly Gly Pro Tyr Glu Gln Tyr
SEQ ID NO.81SEQ ID NO.81 Ala Ser Ser Glu Leu Gly Gly Val Trp Ser Glu Ala PheAla Ser Ser Glu Leu Gly Gly Val Trp Ser Glu Ala Phe
SEQ ID NO.82SEQ ID NO.82 Ala Ser Ser Gly Pro Thr Thr Gly Val Arg Asn Asn Glu Gln PheAla Ser Ser Gly Pro Thr Thr Gly Val Arg Asn Asn Glu Gln Phe
SEQ ID NO.83SEQ ID NO.83 Ala Thr Ser Ser Tyr Trp Pro Gln HisAla Thr Ser Ser Tyr Trp Pro Gln His
SEQ ID NO.84SEQ ID NO.84 Ala Ser Ser Ile Asp Val Val Ser Gly Ser Phe Pro Tyr Glu Gln TyrAla Ser Ser Ile Asp Val Val Ser Gly Ser Phe Pro Tyr Glu Gln Tyr
SEQ ID NO.85SEQ ID NO.85 Ala Ser Ser Ser Thr Gly Gly Thr Gly Ile Tyr Asn Glu Gln PheAla Ser Ser Ser Thr Gly Gly Thr Gly Ile Tyr Asn Glu Gln Phe
SEQ ID NO.86SEQ ID NO.86 Ala Ser Ser Ser Ser Phe Asp Thr Gly Glu Leu PheAla Ser Ser Ser Ser Ser Phe Asp Thr Gly Glu Leu Phe
SEQ ID NO.87SEQ ID NO.87 Ala Ser Ser Pro Val Ala Glu Gly Glu Glu Thr Gln TyrAla Ser Ser Pro Val Ala Glu Gly Glu Glu Thr Gln Tyr
SEQ ID NO.88SEQ ID NO.88 Ala Ser Ser Leu Ala Asp Phe Leu Ser Tyr Asn Glu Gln PheAla Ser Ser Leu Ala Asp Phe Leu Ser Tyr Asn Glu Gln Phe
SEQ ID NO.89SEQ ID NO.89 Ala Ile Ser Glu Pro Met Val Gly Gly Thr Glu Ala PheAla Ile Ser Glu Pro Met Val Gly Gly Thr Glu Ala Phe
SEQ ID NO.90SEQ ID NO.90 Ala Ser Ser Pro Gly Gln Gly Glu Pro Thr Ile TyrAla Ser Ser Pro Gly Gln Gly Glu Pro Thr Ile Tyr
SEQ ID NO.91SEQ ID NO.91 Ala Ser Thr Ser Glu Thr Ser Phe Tyr Glu Gln PheAla Ser Thr Ser Glu Thr Ser Phe Tyr Glu Gln Phe
SEQ ID NO.92SEQ ID NO.92 Pro Ala Val Thr Arg Gly Val Ser Thr Ser Ser ThrPro Ala Val Thr Arg Gly Val Ser Thr Ser Ser Ser Thr
SEQ ID NO.93SEQ ID NO.93 Ala Ser Ser Gln Glu Tyr Ser Gly Ser Ala Tyr Glu Gln TyrAla Ser Ser Gln Glu Tyr Ser Gly Ser Ala Tyr Glu Gln Tyr
SEQ ID NO.94SEQ ID NO.94 Ala Thr Lys Arg Arg Asp Arg Gly Asn Glu Gln PheAla Thr Lys Arg Arg Asp Arg Gly Asn Glu Gln Phe
SEQ ID NO.95SEQ ID NO.95 Ala Ser Thr Pro His Leu Ser Ser Tyr Glu Gln TyrAla Ser Thr Pro His Leu Ser Ser Tyr Glu Gln Tyr
SEQ ID NO.96SEQ ID NO.96 Ala Ser Ser Thr Asp Arg Phe Ala Gln HisAla Ser Ser Thr Asp Arg Phe Ala Gln His
SEQ ID NO.97SEQ ID NO.97 Ala Ile Ser Glu Pro Thr Thr Ser Gly Gly Gly Leu Leu Glu Thr Gln TyrAla Ile Ser Glu Pro Thr Thr Ser Gly Gly Gly Leu Leu Glu Thr Gln Tyr
SEQ ID NO.98SEQ ID NO.98 Ala Ser Ser Lys Gly Thr Ser Trp Phe Leu Ala Tyr Glu Gln TyrAla Ser Ser Lys Gly Thr Ser Trp Phe Leu Ala Tyr Glu Gln Tyr
SEQ ID NO.99SEQ ID NO.99 Ala Ser Ser Leu Val Val Thr Gln Gly Leu Thr Asp Thr Gln TyrAla Ser Ser Leu Val Val Val Thr Gln Gly Leu Thr Asp Thr Gln Tyr
SEQ ID NO.100SEQ ID NO.100 Ala Thr Thr Ser Gly Leu Ala Gly Gly Tyr Leu Ser Gly Glu Leu PheAla Thr Thr Ser Gly Leu Ala Gly Gly Tyr Leu Ser Gly Glu Leu Phe
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/或替换一个或多个碱基后，能表达相同功能的蛋白。Further, the protein sequence of the marker is that the sequence shown in SEQ ID NO. 1-100 can express a protein with the same function after one or more bases are substituted, deleted and/or replaced.
进一步地，标志物为外周血TCR CDR3序列。Further, the marker is the peripheral blood TCR CDR3 sequence.
上述标志物在制备治疗弥漫大B细胞淋巴瘤的制剂中的应用。The application of the above markers in the preparation of preparations for treating diffuse large B-cell lymphoma.
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。Further, the preparation includes a T cell receptor containing the marker, or a plasmid, viral vector or nucleic acid fragment capable of expressing the T cell receptor producing the marker.
一种用于弥漫大B细胞淋巴瘤检测的试剂盒，包括能与上述标志物发生特异性结合的抗体。A kit for detecting diffuse large B-cell lymphoma, comprising antibodies that can specifically bind to the above markers.
一种制剂，包括能与上述标志物发生特异性结合的抗体；所述制剂可用于对弥漫大B细胞淋巴瘤进行诊断、预测、检测或筛查。A preparation comprising an antibody that can specifically bind to the above markers; the preparation can be used for diagnosis, prediction, detection or screening of diffuse large B-cell lymphoma.
一种检测弥漫大B细胞淋巴瘤的蛋白质芯片，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。A protein chip for detecting diffuse large B-cell lymphoma, the protein chip comprises a substrate and a specific antibody spotted on the substrate, the specific antibody being an antibody that can specifically bind to the above marker.
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***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 ***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。 The principle of the present invention is as follows: 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. Later, when B cells differentiate into plasma cells, BCR is expressed as an antibody and secreted outside the cell. T cells recognize antigens through T cell receptors (TCRs) on the cell surface. The diversity of BCR and TCR is the basis for the establishment of the adaptive immune system. The theoretical value of BCR diversity is 10 18 and the theoretical value of TCR diversity is 10 14 . Among BCR and TCR sequences, antigenic determinant 3 (CDR3) is the most important part in determining its antigenic specificity, so the sequence of CDR3 is considered to represent the properties of BCR and TCR sequences.
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***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癌变后，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***产生针对性的免疫反应。因此，分析BCR或TCR的变化，能够检测出肿瘤的发生和发展。In various diseases, the diversity or expression levels of BCR and TCR will change with different antigenic stimulation. Therefore, the occurrence and development of diseases can be tracked by using BCR or TCR high-throughput sequencing results. In human cells, after degradation of senescent proteins, their fragments are transported to the cell surface and presented to T cells in the immune system through histocompatibility antigen II (MCHII). Antigen fragments presented by normal cells do not cause an immune response due to immune tolerance. Once normal cells become cancerous, 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.
本发明的有益效果为：The beneficial effects of the present invention are:
1、本发明中，首先利用1300个非弥漫大B细胞淋巴瘤的对照组样本、和40个弥漫大B细胞淋巴瘤病人的TCR高通量测序数据建立了人工智能分析模型，通过和这些弥漫大B细胞淋巴瘤特异性TCR序列对比，可以清楚的判断待测样本中是否有较高弥漫大B细胞淋巴瘤风险者。1. In the present invention, an artificial intelligence analysis model was established by first using 1300 non-diffuse large B-cell lymphoma control samples and 40 diffuse large B-cell lymphoma patients' TCR high-throughput sequencing data to establish an artificial intelligence analysis model. Large B-cell lymphoma-specific TCR sequence comparison can clearly determine whether there is a higher risk of diffuse large B-cell lymphoma in the sample to be tested.
2、通过高通量测序分析TCR变化可以发现很早期的弥漫大B细胞淋巴瘤，利用弥漫大B细胞淋巴瘤特有的TCR CDR3序列分析人的免疫***中的T细胞对弥漫大B细胞淋巴瘤的反应，是一种新型的检测方法。2. Analysis of TCR changes by high-throughput sequencing can find very early diffuse large B-cell lymphoma, and use the TCR CDR3 sequence specific to diffuse large B-cell lymphoma to analyze the effect of T cells in the human immune system on diffuse large B-cell lymphoma. The reaction is a new detection method.
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。3. By using high-throughput sequencing technology, the present invention simultaneously compares a huge number of specific TCR sequences, and has higher specificity and accuracy than detecting one or several markers alone.
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。4. The cost of the high-throughput sequencing instrument used in the present invention is lower than that of large-scale imaging equipment, and it can be outsourced to a third party. In addition, the labor cost of sampling and processing is lower than the simultaneous detection of multiple markers, and it is also lower than that of a large number of cytology detection, therefore, the present invention greatly reduces the detection cost.
5、本发明只需要采取少量外周血，采样简便、安全。5. The present invention only needs to take a small amount of peripheral blood, and the sampling is simple and safe.
6、本发明中所述TCR CDR3序列，可用于弥漫大B细胞淋巴瘤的免疫治疗。6. The TCR CDR3 sequence described in the present invention can be used for immunotherapy of diffuse large B-cell lymphoma.
附图说明Description of drawings
图1为本发明中，利用基于免疫大数据分析***发现弥漫大B细胞淋巴瘤特征性TCR序列；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弥漫大B细胞淋巴瘤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；弥漫大B细胞淋巴瘤患者的免疫图谱具有多个种类且重复次数较高的弥漫大B细胞淋巴瘤特征序列，健康人极少弥漫大B细胞淋巴瘤特征序列，而未知受试者的弥漫大B细胞淋巴瘤特征比较明显，说明罹患弥漫大B细胞淋巴瘤风险较高； Fig. 1 shows that in the present invention, the characteristic TCR sequence of diffuse large B-cell lymphoma is found by using an immune-based big data analysis system; wherein, the abscissa represents that the CDR3 sequence of a certain amino acid combination is added to the control sequence set or diffuse large B-cell lymphoma The order of the tumor characteristic sequence set, the ordinate represents the logarithm of the number of repeated occurrences of the sequence in a sample, C X ; the immune map of patients with diffuse large B-cell lymphoma has multiple types of diffuse large B cells with a high number of repetitions Characteristic sequences of diffuse large B-cell lymphoma in healthy people are rare, while the characteristics of diffuse large B-cell lymphoma in unknown subjects are more obvious, indicating a higher risk of developing diffuse large B-cell lymphoma;
图2为本发明中，利用弥漫大B细胞淋巴瘤特征特征性指数对比分析弥漫大B细胞淋巴瘤和其他疾病；健康人、非肿瘤病人、非弥漫大B细胞淋巴瘤肿瘤患者的弥漫大B细胞淋巴瘤特征性指数均与弥漫大B细胞淋巴瘤患者具有显著差异，证明了弥漫大B细胞淋巴瘤特征序列集的特异性，据此可以判断未知受试 者是否罹患弥漫大B细胞淋巴瘤。Fig. 2 is a comparative analysis of diffuse large B-cell lymphoma and other diseases using the characteristic index of diffuse large B-cell lymphoma in the present invention; The characteristic indices of DLBCL were significantly different from those of patients with DLBCL, proving the specificity of the DLBCL signature sequence set, which can be used to determine whether an unknown subject has DLBCL .
具体实施方式Detailed ways
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。The specific embodiments of the present invention are described below to facilitate those skilled in the art to understand the present invention, but it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes Such changes are obvious within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the inventive concept are within the scope of protection.
实施例1 通过免疫图谱分析，获得弥漫大B细胞淋巴瘤TCR标志物CDR3序列集Example 1 Obtaining the Diffuse Large B-Cell Lymphoma TCR Marker CDR3 Sequence Set by Immunographic Analysis
1、采样及免疫图谱分析(方法参考申请号为201910300069.9的专利)1. Sampling and immunochromatographic analysis (method reference patent application number 201910300069.9)
采集1301名对照组(包括健康人和非肿瘤疾病病人，1300人用于建立模型，1个健康人用于验证)、41名弥漫大B细胞淋巴瘤患者(40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；Collected 1301 controls (including healthy and non-tumor disease patients, 1300 were used for model establishment, 1 healthy person was used for validation), 41 diffuse large B-cell lymphoma patients (40 were used for model establishment, 1 For verification) and peripheral blood of 1 subject with unknown health status (10mL per person), the antigenic determinant 3 (CDR3) amino acid sequences of TCR of the subjects and the control group were obtained by high-throughput sequencing to ensure that each The total number of CDR3 sequences of functional TCRs in the sample should not be less than 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； 2. Perform random non-replacement sampling on the sequences of the samples whose total number of CDR3 sequences of each functional TCR is higher than 30,000, so that the total number of CDR3 sequences of this sample is 30,000. The total number of CDR3 sequences of functional TCRs contained in all samples so far is 25,000-30,000. For any specific CDR3 sequence X, the number of repetitions in the single-sample sequencing result is counted as C X ;
3、通过分析TCR CDR3数据，确定弥漫大B细胞淋巴瘤TCR标志物CDR3序列：3. Determine the CDR3 sequence of diffuse large B-cell lymphoma TCR marker by analyzing TCR CDR3 data:
a)将1300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；a) All the CDR3 sequences of the 1300 control samples used to build the model were aggregated and deduplicated, and set as a control sequence set;
b)将40名用于建立模型的弥漫大B细胞淋巴瘤样本的所有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，设为弥漫大B细胞淋巴瘤特征序列集。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弥漫大B细胞淋巴瘤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。 b) Summarize and deduplicate all CDR3 sequences of the 40 diffuse large B-cell lymphoma samples used to establish the model, and then remove all sequences containing sequence duplication with the control sequence set, and set it as the characteristic sequence set of diffuse large B-cell lymphoma . The drawing is shown in Figure 1A, in which the abscissa represents the sequence in which the CDR3 sequence of a specific amino acid combination is added to the control sequence set or the characteristic sequence set of diffuse large B-cell lymphoma, and the ordinate represents the sequence in a certain sample. The logarithm of the number of repetitions C X.
c)按照同样作图方法，将1名健康人、1名弥漫大B细胞淋巴瘤患者和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弥漫大B细胞淋巴瘤特征序列集合进行作图，见附图1B-D。从图中可见，弥漫大B细胞淋巴瘤患者的免疫图谱中，含有较多种类且较高重复出现次数的弥漫大B细胞淋巴瘤特征序列(图1B)；健康人的免疫图谱中，只有极少量弥漫大B细胞淋巴瘤特征序列(图1C)；而未知健康状况受试者，有高于健康人的弥漫大B细胞淋巴瘤特征序列，说明此人有较高风险罹患弥漫大B细胞淋巴瘤(图1D)。c) According to the same mapping method, the immune profiles of a healthy person, a patient with diffuse large B-cell lymphoma, and a subject with unknown health status were compared with the reference sequence set and the characteristic sequence set of diffuse large B-cell lymphoma. For mapping, see Figures 1B-D. It can be seen from the figure that in the immune maps of patients with diffuse large B-cell lymphoma, there are more types and high repetitions of the characteristic sequences of diffuse large B-cell lymphoma (Figure 1B); in the immune maps of healthy people, only extremely A small number of diffuse large B-cell lymphoma signature sequences (Fig. 1C); while subjects of unknown health status had higher DLBCL signature sequences than healthy subjects, indicating that this person is at higher risk for diffuse large B-cell lymphoma tumor (Figure 1D).
d)将弥漫大B细胞淋巴瘤特征序列集中，将所有出现在两个及以上参与建模弥漫大B细胞淋巴瘤样本里的CDR3序列，按“所有参与建模弥漫大B细胞淋巴瘤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弥漫大B细胞淋巴瘤样本数”从高到低排序，排名前100者即为弥漫大B细胞淋巴瘤TCR标志物CDR3序列，具体序列如SEQ ID NO.1～100所示。 d) Concentrate the characteristic sequences of diffuse large B-cell lymphoma, and group all CDR3 sequences that appear in two or more samples of diffuse large B-cell lymphoma participating in modeling, and click “All samples of diffuse large B-cell lymphoma participating in modeling”. The sum of the number of repeated occurrences C X in a single sample of the sequence × the number of samples of diffuse large B-cell lymphoma participating in the modeling of the sequence” is sorted from high to low, and the top 100 are the TCR markers of diffuse large B-cell lymphoma The CDR3 sequence, the specific sequence is shown in SEQ ID NO.1-100.
实施例2 验证弥漫大B细胞淋巴瘤TCR标志物CDR3序列集的特异性Example 2 Verification of the specificity of the diffuse large B-cell lymphoma TCR marker CDR3 sequence set
1、采样及免疫图谱分析(方法参考申请号为201910300069.9的专利)1. Sampling and immunochromatographic analysis (method reference patent application number 201910300069.9)
采集577名非弥漫大B细胞淋巴瘤的肿瘤患者、7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。The peripheral blood (10 mL per person) of 577 non-diffuse large B-cell lymphoma tumor patients and 7 subjects with unknown health status was collected, and the TCR epitope 3 of the subjects and the control group was obtained by high-throughput sequencing. (CDR3) amino acid sequence, ensure that the total number of CDR3 sequences of functional TCRs in each sample is not less than 25,000; the sequences of samples whose total number of CDR3 sequences of each functional TCR is more than 30,000 are randomly sampled without replacement, so that The total number of CDR3 sequences for this sample is 30,000. The total number of CDR3 sequences of functional TCRs contained in all samples so far is 25,000-30,000.
2、从实施例1的对照组中随机挑选100名健康人及43名非肿瘤疾病病人。2. 100 healthy people and 43 non-tumor disease patients were randomly selected from the control group in Example 1.
3、根据来自实施例1的100名健康人、43名非肿瘤疾病病人、38名弥漫大B细胞淋巴瘤患者，以及实施例2新获取的577名非弥漫大B细胞淋巴瘤肿瘤患者、7名未知健康状况受试者的免疫图谱，分析其弥漫大B细胞淋巴瘤特征性指数。3. According to 100 healthy people, 43 non-tumor disease patients, 38 diffuse large B-cell lymphoma patients from Example 1, and 577 non-diffuse large B-cell lymphoma tumor patients newly obtained in Example 2, 7 Immune profiling of subjects with unknown health status for the characteristic index of diffuse large B-cell lymphoma.
其中弥漫大B细胞淋巴瘤特征性指数定义为：某个样本中，属于弥漫大B细胞淋巴瘤特征序列集的所有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。弥漫大B细胞淋巴瘤组与健康人(p＝3.1E-93)、非肿瘤疾病(p＝8.6E-50)、其它肿瘤(p趋近于0)都有显著差异，这证明了弥漫大B细胞淋巴瘤特征序列集的特异性。 Among them, the characteristic index of diffuse large B-cell lymphoma is defined as: in a certain sample, the sum of the repeated occurrences C X of all CDR3 sequences belonging to the characteristic sequence set of diffuse large B-cell lymphoma in the sample. The analysis results are shown in Table 2 below and accompanying drawing 2. The diffuse large B-cell lymphoma group was significantly different from healthy people (p=3.1E-93), non-tumor disease (p=8.6E-50), and other tumors (p approached 0), which proved that diffuse large B-cell lymphoma Specificity of the B-cell lymphoma signature sequence set.
表2不同样本组的弥漫大B细胞淋巴瘤特征性指数Table 2 Diffuse large B-cell lymphoma characteristic indices in different sample groups
Figure PCTCN2022080282-appb-000001
Figure PCTCN2022080282-appb-000001
Figure PCTCN2022080282-appb-000002
Figure PCTCN2022080282-appb-000002
Figure PCTCN2022080282-appb-000003
Figure PCTCN2022080282-appb-000003
Figure PCTCN2022080282-appb-000004
Figure PCTCN2022080282-appb-000004
Figure PCTCN2022080282-appb-000005
Figure PCTCN2022080282-appb-000005
Figure PCTCN2022080282-appb-000006
Figure PCTCN2022080282-appb-000006
Figure PCTCN2022080282-appb-000007
Figure PCTCN2022080282-appb-000007
Figure PCTCN2022080282-appb-000008
Figure PCTCN2022080282-appb-000008
Figure PCTCN2022080282-appb-000009
Figure PCTCN2022080282-appb-000009
Figure PCTCN2022080282-appb-000010
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Figure PCTCN2022080282-appb-000011
Figure PCTCN2022080282-appb-000011
4、分析各组的弥漫大B细胞淋巴瘤特征指数(表3)，7位未知健康状况受试者(检测样本)的弥漫大B细胞淋巴瘤特征指数显著高于“其它肿瘤”组的平均值+2×SD(152.7+2×587.4＝1327.5)，此7人具有较高风险罹患弥漫大B细胞淋巴瘤。与临床体检结果对照后，这7人确为早期弥漫大B细胞淋巴瘤患者。此实施例证明了利用弥漫大B细胞淋巴瘤特征序列集及弥漫大B细胞淋巴瘤特征性指数，预测受试者罹患弥漫大B细胞淋巴瘤风险的可行性。4. The characteristic index of diffuse large B-cell lymphoma in each group was analyzed (Table 3), and the characteristic index of diffuse large B-cell lymphoma in 7 subjects of unknown health status (test samples) was significantly higher than the average of the "other tumors" group value + 2 x SD (152.7 + 2 x 587.4 = 1327.5), these 7 people have a higher risk of developing diffuse large B-cell lymphoma. Compared with the clinical physical examination results, these 7 people were indeed patients with early diffuse large B-cell lymphoma. This example demonstrates the feasibility of using a diffuse large B-cell lymphoma signature sequence set and a diffuse large B-cell lymphoma signature index to predict a subject's risk of developing a diffuse large B-cell lymphoma.
表3弥漫大B细胞淋巴瘤特征性指数分析Table 3 Characteristic index analysis of diffuse large B-cell lymphoma
Figure PCTCN2022080282-appb-000012
Figure PCTCN2022080282-appb-000012
综上所述，本发明所述弥漫大B细胞淋巴瘤TCR标志物CDR3序列，确实具有显著的弥漫大B细胞 淋巴瘤特异性，不仅可以用于弥漫大B细胞淋巴瘤预测受试者罹患弥漫大B细胞淋巴瘤风险，未来还可用于弥漫大B细胞淋巴瘤的生物免疫治疗。To sum up, the CDR3 sequence of the diffuse large B-cell lymphoma TCR marker of the present invention does have significant specificity for diffuse large B-cell lymphoma, and it can not only be used for the prediction of diffuse large B-cell lymphoma in subjects suffering from diffuse large B-cell lymphoma. The risk of large B-cell lymphoma can also be used for biological immunotherapy of diffuse large B-cell lymphoma in the future.

Claims (7)

  1. 一种弥漫大B细胞淋巴瘤的外周血TCR标志物，其特征在于，所述标志物包括序列为SEQ ID NO.1～100所示的蛋白中的至少一种。A peripheral blood TCR marker for diffuse large B-cell lymphoma, characterized in that the marker comprises at least one of the proteins whose sequences are shown in SEQ ID NO. 1-100.
  2. 根据权利要求1所述的弥漫大B细胞淋巴瘤的外周血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/或替换一个或多个碱基后，能表达相同功能的蛋白。The peripheral blood TCR marker for diffuse large B-cell lymphoma according to claim 1, wherein the protein sequence of the marker is the sequence shown in SEQ ID NO. 1-100 with substitution, deletion and/or After replacing one or more bases, the protein with the same function can be expressed.
  3. 权利要求1所述的标志物在制备治疗弥漫大B细胞淋巴瘤的制剂中的应用。The application of the marker of claim 1 in the preparation of a preparation for the treatment of diffuse large B-cell lymphoma.
  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。The use according to claim 3, wherein the preparation comprises a T cell receptor containing the marker, or a plasmid, viral vector or nucleic acid fragment capable of expressing the T cell receptor producing the marker.
  5. 一种用于弥漫大B细胞淋巴瘤检测的试剂盒，其特征在于，包括能与权利要求1所述标志物发生特异性结合的抗体。A kit for detecting diffuse large B-cell lymphoma, characterized in that it comprises an antibody that can specifically bind to the marker of claim 1.
  6. 一种制剂，其特征在于，包括能与权利要求1所述标志物发生特异性结合的抗体；所述制剂可用于对弥漫大B细胞淋巴瘤进行诊断、预测、检测或筛查。A preparation is characterized by comprising an antibody that can specifically bind to the marker of claim 1; the preparation can be used for diagnosis, prediction, detection or screening of diffuse large B-cell lymphoma.
  7. 一种检测弥漫大B细胞淋巴瘤的蛋白质芯片，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所述标志物发生特异性结合的抗体。A protein chip for detecting diffuse large B-cell lymphoma, characterized in that the protein chip comprises a substrate and a specific antibody spotted on the substrate, and the specific antibody is capable of producing the marker described in claim 1. specific binding antibodies.
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