WO2022012279A1 - Peripheral blood tcr marker sequence for colorectal cancer, detection kit therefor and application thereof - Google Patents
Peripheral blood tcr marker sequence for colorectal cancer, detection kit therefor and application thereof Download PDFInfo
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- the invention belongs to the technical field of genetic engineering, and in particular relates to a peripheral blood TCR marker of colorectal cancer and a detection kit and application thereof.
- Colorectal cancer also known as colorectal cancer (Carcinoma of Colon and Rectum), is a common malignant tumor in the gastrointestinal tract and the third most common malignant tumor worldwide, with an annual incidence of about 1 million and an annual death of about 500,000. people. Colorectal cancer is also one of the most common malignant tumors in my country, and its morbidity and mortality are second only to gastric cancer, esophageal cancer and primary liver cancer among digestive system malignant tumors. The incidence of malignant tumors in my country ranks third (29/100,000), and the mortality rate ranks fifth (14/100,000), and both the morbidity and mortality are on the rise in recent years.
- Colorectal cancer mostly occurs in middle-aged and older men, and it is most common between the ages of 40 and 70, but it is not uncommon to find people under the age of 30 at the end of the 20th century.
- the male to female incidence ratio is approximately 2:1.
- Colorectal cancer can occur in any part of the colon or rectum, but the rectum and sigmoid colon are the most common, and the rest are found in the cecum, ascending colon, descending colon and transverse colon.
- the majority of carcinomas are adenocarcinomas, and a few are squamous carcinomas and mucinous carcinomas.
- colorectal cancer With the enlargement of the tumor, the clinical manifestations of colorectal cancer include changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain. However, because the early symptoms are not obvious, most patients are already in the middle and late stages when they are found. The key to colorectal cancer treatment is early detection, timely diagnosis and radical surgery. Generally, cancers confined to the intestinal wall have a better prognosis, and those with infiltration outside the intestine have a poor prognosis.
- the main detection and diagnosis methods for colorectal cancer are:
- Colonoscopy an important method for diagnosing colon cancer, which can be morphologically diagnosed by the naked eye and biopsy.
- Barium enema The traditional method for diagnosing colorectal cancer has advantages in the localization of lesions, but it is easy to miss small tumors.
- CT virtual colonoscopy non-invasive, relatively simple and safe, suitable for patients who cannot undergo colonoscopy, especially patients with intestinal stenosis caused by malignant tumors, but cannot obtain lesion tissue for pathological examination.
- Intracavitary B-ultrasound, CT, MRI It is suitable for determining the local invasion, lymph node metastasis and distant metastasis of the tumor, which can improve the staging of colorectal cancer.
- Biopsy including fiberoptic colonoscopy biopsy specimens and postoperative specimens of surgical patients, suitable for early lesions or when biopsy of stenotic sites is difficult.
- CEA CarcinoEmbryonic Antigen, Carcinoembryonic Antigen
- CA199 Carbohydrate Antigen 199, Carbohydrate Antigen 199
- the purpose of the present invention is to provide a peripheral blood TCR marker of colorectal cancer and its detection kit and application in view of the deficiencies in the prior art.
- a peripheral blood TCR marker for colorectal cancer comprising at least one of the proteins shown in the sequence SpecSeq1-100, as shown in Table 1:
- protein sequence of the marker is the sequence shown in SpecSeq1-100 after one or more bases are substituted, deleted and/or replaced, and a protein with the same function can be expressed.
- the preparation includes a plasmid, viral vector or nucleic acid fragment of the T cell receptor containing the marker.
- a kit for colorectal cancer detection includes antibodies that can specifically bind to the above markers.
- a preparation includes antibodies that can specifically bind to the above markers; the preparation can be used for diagnosing, predicting, detecting or screening colorectal cancer.
- a protein chip for detecting colorectal cancer comprises a substrate and a specific antibody spotted on the substrate.
- the specific antibody is 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.
- CDR3 antigenic determinant 3
- TCR CDR3 data analysis Determine the CDR3 sequence of colorectal cancer TCR marker by analysis:
- the colorectal cancer characteristic index is defined as: in a certain sample, the sum of the repeated occurrences C X of all CDR3 sequences belonging to the colorectal cancer characteristic sequence set in the sample;
- an artificial intelligence analysis model is first established by using the non-colorectal cancer control group samples and the TCR high-throughput sequencing data of colorectal cancer patients. By comparing with these colorectal cancer-specific TCR sequences, it is possible to clearly determine the test Whether there is a higher risk of colorectal cancer in the sample;
- 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 sequences described in the present invention can also be used for immunotherapy of colorectal cancer.
- Figure 1 shows the CDR3 sequence of the control group and the characteristic sequence of colorectal cancer in the present invention.
- the abscissa represents the sequence in which the CDR3 sequence of a specific amino acid combination was added to the control sequence set or the colorectal cancer characteristic sequence set, and the ordinate represents the logarithm of the number of repetitions of the sequence in a sample, C X ; the immune system of colorectal cancer patients
- the atlas has multiple types of colorectal cancer feature sequences with a high number of repetitions. Healthy people rarely have colorectal cancer feature sequences, while unknown subjects have more obvious colorectal cancer features, indicating a higher risk of colorectal cancer.
- Fig. 2 shows the colorectal cancer characteristic index of healthy people, non-tumor patients, non-colorectal cancer tumor patients and colorectal cancer patients according to the colorectal cancer feature sequence set in the present invention, healthy people, non-tumor patients, non-colorectal cancer tumor patients
- the colorectal cancer characteristic indices of CRC patients were significantly different from those of colorectal cancer patients, proving the specificity of the colorectal cancer characteristic sequence set. Based on this, it can be determined whether the unknown subject suffers from colorectal cancer.
- Collect 1301 control groups including healthy people and non-tumor disease patients, 1300 people are used for model building, 1 healthy person is used for validation), 101 colorectal cancer patients (100 people are used for model building, 1 person is used for validation)
- peripheral blood (10mL per person) of a subject with unknown health status, the TCR antigenic determinant 3 (CDR3) amino acid sequence of the subject and the control group was obtained by high-throughput sequencing to ensure the functionality of each sample
- the total number of CDR3 sequences of TCR is not less than 30000;
- the immune profiles of 1 healthy person, 1 colorectal cancer patient and 1 subject with unknown health status are mapped with reference to the control sequence set and the colorectal cancer characteristic sequence set, as shown in Figure 1B- D. It can be seen from the figure that the immune maps of patients with colorectal cancer contain more types of colorectal cancer feature sequences with high repetitions; the immune maps of healthy people contain only a very small number of colorectal cancer feature sequences; while subjects with unknown health status , have a higher colorectal cancer characteristic sequence than healthy people, indicating that this person has a higher risk of colorectal cancer.
- CDR3 sequences of TCR of each sample are randomly sampled without replacement, so that the total number of CDR3 sequences of each sample is 30,000.
- Example 3 According to 100 healthy people, 45 non-tumor disease patients, 100 colorectal cancer patients from Example 1, and 358 non-colorectal cancer tumor patients and 3 subjects with unknown health conditions newly obtained in Example 2. The immune profile was analyzed to analyze the characteristic index of colorectal cancer.
- the colorectal cancer characteristic index is defined as: in a certain sample, the sum of the repetition times C X of all CDR3 sequences belonging to the colorectal cancer characteristic sequence set in the sample.
- the analysis results are shown in Table 2 and Figure 2 below.
- the colorectal cancer TCR marker CDR3 sequence of the present invention does have significant colorectal cancer specificity, and can not only be used for colorectal cancer to predict the risk of colorectal cancer in subjects, but also can be used for colorectal cancer biological immunity in the future. treat.
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Abstract
Disclosed are a peripheral blood TCR marker for colorectal cancer, a detection kit therefor and an application thereof. The marker comprises at least one of the proteins set forth in the sequence SpecSeq1-100. In the present invention, on the basis of a high-throughput sequencing method, only a small amount of peripheral blood needs to be taken. RNA is extracted, and an immune profile library is established by means of processing samples. Next, a characteristic TCR sequence in the peripheral blood of colorectal cancer is first determined by means of high-throughput sequencing and TCR data analysis, and then test results of a sample to be tested are compared to the characteristic TCR sequence so as to determine whether a patient has colorectal cancer. In the present invention, a huge number of colorectal cancer-specific TCR sequences may be compared at the same time, which has higher specificity and accuracy than detecting one or several markers separately, thereby increasing the diagnosis efficiency.
Description
本发明属于基因工程技术领域，尤其涉及一种大肠癌的外周血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 colorectal cancer and a detection kit and application thereof.
大肠癌，又称结肠直肠癌(Carcinoma of Colon and Rectum)是胃肠道中常见的恶性肿瘤，在世界范围内是第三位常见的恶性肿瘤，年发病率约100万人，年死亡约50万人。大肠癌也是我国最常见的恶性肿瘤之一，其发病率和病死率在消化系统恶性肿瘤中仅次于胃癌、食管癌和原发性肝癌。在我国恶性肿瘤发病率中居第三位(29/10万)，死亡率居第五位(14/10万)，而且近年来发病率和死亡率均呈上升趋势。在2012年诊断的全球136万例大肠癌患者中，中国大肠癌新发病例数达25.3万例，占18.6％。而2015中国癌症统计数据则显示，我国大肠癌发病率、死亡率在全部恶性肿瘤中均居第5位，其中新发病例37.6万例，死亡病例19.1万例。中国已成为全球大肠癌每年新发病例数最多的国家。Colorectal cancer, also known as colorectal cancer (Carcinoma of Colon and Rectum), is a common malignant tumor in the gastrointestinal tract and the third most common malignant tumor worldwide, with an annual incidence of about 1 million and an annual death of about 500,000. people. Colorectal cancer is also one of the most common malignant tumors in my country, and its morbidity and mortality are second only to gastric cancer, esophageal cancer and primary liver cancer among digestive system malignant tumors. The incidence of malignant tumors in my country ranks third (29/100,000), and the mortality rate ranks fifth (14/100,000), and both the morbidity and mortality are on the rise in recent years. Among the 1.36 million cases of colorectal cancer diagnosed worldwide in 2012, the number of new cases of colorectal cancer in China reached 253,000, accounting for 18.6%. The 2015 Chinese cancer statistics showed that the incidence and mortality of colorectal cancer in my country ranked fifth among all malignant tumors, including 376,000 new cases and 191,000 deaths. China has become the country with the largest number of new cases of colorectal cancer every year in the world.
大肠癌多发生在中年以上的男性，以40～70岁最为多见，但20世纪末发现30岁以下者亦不少见。男女两性发病比例约为2:1。大肠癌可以发生在结肠或直肠的任何部位，但以直肠、乙状结肠最为多见，其余依次见于盲肠、升结肠、降结肠及横结肠。癌瘤大多数为腺癌，少数为鳞状上皮癌及粘液癌。Colorectal cancer mostly occurs in middle-aged and older men, and it is most common between the ages of 40 and 70, but it is not uncommon to find people under the age of 30 at the end of the 20th century. The male to female incidence ratio is approximately 2:1. Colorectal cancer can occur in any part of the colon or rectum, but the rectum and sigmoid colon are the most common, and the rest are found in the cecum, ascending colon, descending colon and transverse colon. The majority of carcinomas are adenocarcinomas, and a few are squamous carcinomas and mucinous carcinomas.
其发病原因仍不清楚，但有报道认为与饮食习惯相关。高脂、高蛋白、低纤维素饮食会增加患大肠癌的风险。在20％-30％的大肠癌患者中，遗传因素起着重要作用，大肠癌患者的家族成员发生大肠癌的风险增高。慢性溃疡性结肠炎、家族性结肠息肉、遗传性腺瘤病患者，发生大肠癌的概率增高。Its pathogenesis is still unclear, but it has been reported that it is related to eating habits. A high-fat, high-protein, low-fiber diet increases the risk of colorectal cancer. In 20%-30% of colorectal cancer patients, genetic factors play an important role, and family members of colorectal cancer patients have an increased risk of colorectal cancer. Patients with chronic ulcerative colitis, familial colon polyps, and hereditary adenomatosis are more likely to develop colorectal cancer.
随着癌肿的增大，大肠癌的临床表现为排便习惯改变、便血、腹泻、腹泻与便秘交替、局部腹痛等症状，晚期则表现贫血、体重减轻等全身症状。但因为早期症状不明显，多数患者发现时已属中晚期。大肠癌治疗的关键在于早期发现、及时诊断和手术根治。一般癌肿只限于肠壁者预后较好，浸润到肠外者预后较差，年轻患者、癌瘤浸润广泛、有转移者或有并发症者预后不良。With the enlargement of the tumor, the clinical manifestations of colorectal cancer include changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain. However, because the early symptoms are not obvious, most patients are already in the middle and late stages when they are found. The key to colorectal cancer treatment is early detection, timely diagnosis and radical surgery. Generally, cancers confined to the intestinal wall have a better prognosis, and those with infiltration outside the intestine have a poor prognosis.
当前对大肠癌主要的检测诊断方法有：At present, the main detection and diagnosis methods for colorectal cancer are:
1、影像学检查：1. Imaging examination:
1)纤维结肠镜：诊断结肠癌的重要方法，可以通过肉眼进行形态学诊断，并进行活检。1) Colonoscopy: an important method for diagnosing colon cancer, which can be morphologically diagnosed by the naked eye and biopsy.
2)钡剂灌肠：诊断大肠癌诊断的传统方法，在病灶定位上具有优势，但易漏诊较小的肿瘤。2) Barium enema: The traditional method for diagnosing colorectal cancer has advantages in the localization of lesions, but it is easy to miss small tumors.
3)CT仿真结肠镜：无创伤性，相对简单安全，适用于无法接受结肠镜检查的患者，尤其是恶性肿瘤引起肠腔狭窄的患者，但无法取得病灶组织进行病理检查。3) CT virtual colonoscopy: non-invasive, relatively simple and safe, suitable for patients who cannot undergo colonoscopy, especially patients with intestinal stenosis caused by malignant tumors, but cannot obtain lesion tissue for pathological examination.
4)腔内B超、CT、MRI：适用于明确肿瘤的局部浸润、淋巴结转移和远处转移，可完善大肠癌的分期。4) Intracavitary B-ultrasound, CT, MRI: It is suitable for determining the local invasion, lymph node metastasis and distant metastasis of the tumor, which can improve the staging of colorectal cancer.
2、病理学或细胞学检查：2. Pathological or cytological examination:
1)直肠指检：早期诊断的重要方法，但无法发现和诊断结肠部位肿瘤。1) Digital rectal examination: an important method for early diagnosis, but it cannot detect and diagnose colon tumors.
2)活检：包括纤维结肠镜活检标本和手术患者的术后标本，适用于早期病变，或狭窄部位活检有困难时。2) Biopsy: including fiberoptic colonoscopy biopsy specimens and postoperative specimens of surgical patients, suitable for early lesions or when biopsy of stenotic sites is difficult.
3、生物化学或分子生物学检验：3. Biochemical or molecular biology test:
1)CEA(CarcinoEmbryonic Antigen，癌胚抗原)和CA199(Carbohydrate Antigen 199，糖类抗原199)：较为广泛应用在大肠癌的辅助诊断中，但由于在大肠癌中的敏感性和特异性不够理想，因此目前不推荐用于进行大肠癌的筛查。1) CEA (CarcinoEmbryonic Antigen, Carcinoembryonic Antigen) and CA199 (Carbohydrate Antigen 199, Carbohydrate Antigen 199): It is widely used in the auxiliary diagnosis of colorectal cancer, but due to the unsatisfactory sensitivity and specificity in colorectal cancer, Therefore, it is not currently recommended for colorectal cancer screening.
2)其它分子标志物：包括RAS、BRAF、PIK3CA、MSI等，大量研究报道表面，这些标志物与大肠癌的预后相关，可以用作治疗效果的评估。2) Other molecular markers: including RAS, BRAF, PIK3CA, MSI, etc. A large number of studies have reported that these markers are related to the prognosis of colorectal cancer and can be used to evaluate the therapeutic effect.
上述检验方法都具有一定局限性。一方面，因大肠癌早期无明显症状，或与痔疮、胃肠炎等下消化道疾病症状类似，因此很多患者会因抗拒直肠指检、肠镜需要麻醉等原因，错失早期发现癌变的时机。另一方面，根据美国的研究发现，已知肿瘤标志物对大肠癌缺乏敏感性和特异性，不能作为筛查和早期诊断标准。因此，亟需一种能更便捷灵敏，且可以追踪大肠癌发生、发展状况的筛查、检验方法。The above test methods all have certain limitations. On the one hand, because colorectal cancer has no obvious symptoms in the early stage, or has similar symptoms to lower gastrointestinal diseases such as hemorrhoids and gastroenteritis, many patients will miss the opportunity of early detection of cancer due to resisting digital rectal examination and colonoscopy requiring anesthesia. On the other hand, according to research in the United States, known tumor markers lack sensitivity and specificity for colorectal cancer and cannot be used as screening and early diagnosis criteria. Therefore, there is an urgent need for a more convenient and sensitive screening and testing method that can track the occurrence and development of colorectal cancer.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于：针对现有技术中存在的不足，提供一种大肠癌的外周血TCR标志物及其检测试剂盒和应用。The purpose of the present invention is to provide a peripheral blood TCR marker of colorectal cancer and its detection kit and application in view of the deficiencies in the prior art.
本发明采用的技术方案如下：The technical scheme adopted in the present invention is as follows:
一种大肠癌的外周血TCR标志物，包括序列SpecSeq1-100所示蛋白中的至少一种，如表1所示：A peripheral blood TCR marker for colorectal cancer, comprising at least one of the proteins shown in the sequence SpecSeq1-100, as shown in Table 1:
表1标志物序列Table 1 Marker sequences
| 序列编号serial number | 大肠癌的外周血TCR标志物CDR3序列Sequence of peripheral blood TCR marker CDR3 in colorectal cancer |
| SpecSeq1SpecSeq1 | Ala Gly Thr Gly Ile Ala Ser Ala Gly Glu Leu PheAla Gly Thr Gly Ile Ala Ser Ala Gly Glu Leu Phe |
| SpecSeq2SpecSeq2 | Ser Val Thr Glu Ser Leu Asp Thr Gln TyrSer Val Thr Glu Ser Leu Asp Thr Gln Tyr |
| SpecSeq3SpecSeq3 | Ala Ser Ser Gln Arg Phe Arg Pro Gln HisAla Ser Ser Gln Arg Phe Arg Pro Gln His |
| SpecSeq4SpecSeq4 | Ala Ser Ser Glu Gly Gln Gly Ser Ile Ile Glu Thr Gln TyrAla Ser Ser Glu Gly Gln Gly Ser Ile Ile Glu Thr Gln Tyr |
| SpecSeq5SpecSeq5 | Ser Ala Arg Gly Arg Leu Ala Ala Lys Pro Tyr Glu Gln TyrSer Ala Arg Gly Arg Leu Ala Ala Lys Pro Tyr Glu Gln Tyr |
| SpecSeq6SpecSeq6 | Ala Ser Ser Gly Phe Thr Thr Asn Gln Pro Gln HisAla Ser Ser Gly Phe Thr Thr Asn Gln Pro Gln His |
| SpecSeq7SpecSeq7 | Ala Ser Ser Phe Gly Asp Gln Gly Asp Glu Gln TyrAla Ser Ser Phe Gly Asp Gln Gly Asp Glu Gln Tyr |
| SpecSeq8SpecSeq8 | Ala Ile Ser Arg Ala Thr Val Arg Ala Asp Thr Gln TyrAla Ile Ser Arg Ala Thr Val Arg Ala Asp Thr Gln Tyr |
| SpecSeq9SpecSeq9 | Ala Thr Met Gly Thr Glu Leu Leu Ser Asn Ile Gln TyrAla Thr Met Gly Thr Glu Leu Leu Ser Asn Ile Gln Tyr |
| SpecSeq10SpecSeq10 | Ala Ser Ser Pro Leu Ala Ala Gly Glu Gln TyrAla Ser Ser Pro Leu Ala Ala Gly Glu Gln Tyr |
| SpecSeq11SpecSeq11 | Ser Val Glu Ile Ser Thr Ser Gly His Pro Leu Leu Ser Asn Glu Gln PheSer Val Glu Ile Ser Thr Ser Gly His Pro Leu Leu Ser Asn Glu Gln Phe |
| SpecSeq12SpecSeq12 | Ala Ser Ser Gln Lys Gly Thr Gly Thr Leu HisAla Ser Ser Gln Lys Gly Thr Gly Thr Leu His |
| SpecSeq13SpecSeq13 | Ala Ser Ser Val Glu Leu Arg Ala Gly Glu Gly Asn Glu Gln PheAla Ser Ser Val Glu Leu Arg Ala Gly Glu Gly Asn Glu Gln Phe |
| SpecSeq14SpecSeq14 | Ala Ser Ser Phe Thr Arg Ser Val Ser Tyr Glu Gln TyrAla Ser Ser Phe Thr Arg Ser Val Ser Tyr Glu Gln Tyr |
| SpecSeq15SpecSeq15 | Ala Thr Ser Ser Val Ala Gly Glu Ile Gln TyrAla Thr Ser Ser Val Ala Gly Glu Ile Gln Tyr |
| SpecSeq16SpecSeq16 | Ala Thr Ser Arg Asp Asp Gly Phe Glu Gln PheAla Thr Ser Arg Asp Asp Gly Phe Glu Gln Phe |
| SpecSeq17SpecSeq17 | Ala Ser Ser Pro Tyr Pro Gly Tyr Ser Thr Asp Thr Gln TyrAla Ser Ser Pro Tyr Pro Gly Tyr Ser Thr Asp Thr Gln Tyr |
| SpecSeq18SpecSeq18 | Ala Ile Ser Glu Ser Ile Ala Ser Gly Glu Glu Thr Gln TyrAla Ile Ser Glu Ser Ile Ala Ser Gly Glu Glu Thr Gln Tyr |
| SpecSeq19SpecSeq19 | Ser Ala Ser Ser Ala Leu Ser Phe Tyr Asn Glu Gln PheSer Ala Ser Ser Ala Leu Ser Phe Tyr Asn Glu Gln Phe |
| SpecSeq20SpecSeq20 | Ala Ser Ser Val Ile Gln Gly Phe Tyr Gly Tyr ThrAla Ser Ser Val Ile Gln Gly Phe Tyr Gly Tyr Thr |
| SpecSeq21SpecSeq21 | Ala Ser Ser Ser Pro Gly Gln Gly Pro Ser Asn Tyr Gly Tyr ThrAla Ser Ser Ser Pro Gly Gln Gly Pro Ser Asn Tyr Gly Tyr Thr |
| SpecSeq22SpecSeq22 | Ala Ser Ser Leu Ser Gln Pro Ser Gln Tyr Glu Gln TyrAla Ser Ser Leu Ser Gln Pro Ser Gln Tyr Glu Gln Tyr |
| SpecSeq23SpecSeq23 | Ala Ile Ser Asp Arg Tyr Arg Gly Asn Thr Gly Glu Leu PheAla Ile Ser Asp Arg Tyr Arg Gly Asn Thr Gly Glu Leu Phe |
| SpecSeq24SpecSeq24 | Ser Ala Arg Ser Trp Thr Gly Arg Ile Ile Asp Glu Gln PheSer Ala Arg Ser Trp Thr Gly Arg Ile Ile Asp Glu Gln Phe |
| SpecSeq25SpecSeq25 | Ala Ser Ser Glu Ala Arg Arg Ala Leu Asn Glu Gln PheAla Ser Ser Glu Ala Arg Arg Ala Leu Asn Glu Gln Phe |
| SpecSeq26SpecSeq26 | Ala Ser Ser Pro Ser Gly Val Trp Tyr Gly Tyr ThrAla Ser Ser Pro Ser Gly Val Trp Tyr Gly Tyr Thr |
| SpecSeq27SpecSeq27 | Ala Ile Ser Arg Asp Glu Thr Glu Ala PheAla Ile Ser Arg Asp Glu Thr Glu Ala Phe |
| SpecSeq28SpecSeq28 | Ala Ser Ser Leu Ala Leu Ser Trp Pro Gln HisAla Ser Ser Leu Ala Leu Ser Trp Pro Gln His |
| SpecSeq29SpecSeq29 | Ala Ser Arg Pro Thr Ser Gly Trp Val Asp Glu Gln PheAla Ser Arg Pro Thr Ser Gly Trp Val Asp Glu Gln Phe |
| SpecSeq30SpecSeq30 | Ser Val Ala Ile Asp Glu Arg Gly Gly Ile Glu Gln PheSer Val Ala Ile Asp Glu Arg Gly Gly Ile Glu Gln Phe |
| SpecSeq31SpecSeq31 | Ala Ser Ser Phe Thr Ser Asn His Glu Gln TyrAla Ser Ser Phe Thr Ser Asn His Glu Gln Tyr |
| SpecSeq32SpecSeq32 | Ala Ser Arg Gly Glu Glu Met Gly Gly Thr Asp Thr Gln TyrAla Ser Arg Gly Glu Glu Met Gly Gly Gly Thr Asp Thr Gln Tyr |
| SpecSeq33SpecSeq33 | Thr Glu Gln Gly Ala Gly Thr Glu Ala PheThr Glu Gln Gly Ala Gly Thr Glu Ala Phe |
| SpecSeq34SpecSeq34 | Ser Ala Leu Pro Trp Thr Gly Asp Thr Glu Ala PheSer Ala Leu Pro Trp Thr Gly Asp Thr Glu Ala Phe |
| SpecSeq35SpecSeq35 | Ala Ser Ser Phe Thr Ser Arg Met Glu PheAla Ser Ser Phe Thr Ser Arg Met Glu Phe |
| SpecSeq36SpecSeq36 | Ala Ser Ser Pro Gly Thr Ser Gly Pro Gly Val Thr Asp Thr Gln TyrAla Ser Ser Pro Gly Thr Ser Gly Pro Gly Val Thr Asp Thr Gln Tyr |
| SpecSeq37SpecSeq37 | Ala Ile Ser Val Ser Gln Asp Ile Gly Thr Gln TyrAla Ile Ser Val Ser Gln Asp Ile Gly Thr Gln Tyr |
| SpecSeq38SpecSeq38 | Ala Ser Ser Leu Ala Pro Gly Asp Arg Gly Tyr Asn Glu Gln PheAla Ser Ser Leu Ala Pro Gly Asp Arg Gly Tyr Asn Glu Gln Phe |
| SpecSeq39SpecSeq39 | Ala Ser Ser Gln Phe Gly Glu Glu Thr Val Glu Thr Gln TyrAla Ser Ser Gln Phe Gly Glu Glu Thr Val Glu Thr Gln Tyr |
| SpecSeq40SpecSeq40 | Ala Thr Ser Ser Gly Gly Thr Ser Gly Lys Trp Glu Gln PheAla Thr Ser Ser Gly Gly Thr Ser Gly Lys Trp Glu Gln Phe |
| SpecSeq41SpecSeq41 | Ala Trp Glu Asp Pro Asn Tyr Gly Tyr ThrAla Trp Glu Asp Pro Asn Tyr Gly Tyr Thr |
| SpecSeq42SpecSeq42 | Ala Ser Ser His Ile Gln Thr Ser Asp Arg Asp Gly Tyr ThrAla Ser Ser His Ile Gln Thr Ser Asp Arg Asp Gly Tyr Thr |
| SpecSeq43SpecSeq43 | Ser Ala Ile Pro Ser Glu Gly Ser Asp Glu Gln PheSer Ala Ile Pro Ser Glu Gly Ser Asp Glu Gln Phe |
| SpecSeq44SpecSeq44 | Ala Ser Ser Pro His Gly Arg Leu Asn Thr Glu Ala PheAla Ser Ser Pro His Gly Arg Leu Asn Thr Glu Ala Phe |
| SpecSeq45SpecSeq45 | Ala Trp Ser Pro Trp Asp Ser Gly Asn Tyr Gly Tyr ThrAla Trp Ser Pro Trp Asp Ser Gly Asn Tyr Gly Tyr Thr |
| SpecSeq46SpecSeq46 | Ser Ala Arg Asp Ile Tyr Ser Thr Ser Gly Ser Tyr Glu Gln TyrSer Ala Arg Asp Ile Tyr Ser Thr Ser Gly Ser Tyr Glu Gln Tyr |
| SpecSeq47SpecSeq47 | Ala Ser Ser Gln ArgAla Thr Gly Gly Arg Tyr Thr Glu Ala PheAla Ser Ser Gln ArgAla Thr Gly Gly Arg Tyr Thr Glu Ala Phe |
| SpecSeq48SpecSeq48 | Ala Ser Ser Tyr Lys Gly Gly Ile Lys Gly Tyr Glu Gln PheAla Ser Ser Tyr Lys Gly Gly Ile Lys Gly Tyr Glu Gln Phe |
| SpecSeq49SpecSeq49 | Ser Ala Ala Ala Tyr Ser Gly Glu Gly Pro Asn Glu Gln PheSer Ala Ala Ala Tyr Ser Gly Glu Gly Pro Asn Glu Gln Phe |
| SpecSeq50SpecSeq50 | Ala Ser Ser Leu Pro PheAla Ser Ser Leu Pro Phe |
| SpecSeq51SpecSeq51 | Ser Ala Tyr Arg Pro Gly Leu Ala Gly Gly Lys Asn Tyr Asn Glu Gln PheSer Ala Tyr Arg Pro Gly Leu Ala Gly Gly Lys Asn Tyr Asn Glu Gln Phe |
| SpecSeq52SpecSeq52 | Ala Ser Trp Ala Gly Gly Gln Ala PheAla Ser Trp Ala Gly Gly Gln Ala Phe |
| SpecSeq53SpecSeq53 | Gln Gln Tyr Gly Gly Ala Gly Arg Tyr Ala ValGln Gln Tyr Gly Gly Ala Gly Arg Tyr Ala Val |
| SpecSeq54SpecSeq54 | Ser Val Gly Ser Ala Gly Thr Pro Pro ProSer Val Gly Ser Ala Gly Thr Pro Pro Pro |
| SpecSeq55SpecSeq55 | Ala Ser Ser Pro Ile Gly Asp Ala Asn Asp Glu Gln PheAla Ser Ser Pro Ile Gly Asp Ala Asn Asp Glu Gln Phe |
| SpecSeq56SpecSeq56 | Ala Ile Ser Glu Ser Thr Phe Ala Ser Glu Glu Thr Gln TyrAla Ile Ser Glu Ser Thr Phe Ala Ser Glu Glu Thr Gln Tyr |
| SpecSeq57SpecSeq57 | Ala Ser Thr Asn Lys Arg Asn Glu Gln PheAla Ser Thr Asn Lys Arg Asn Glu Gln Phe |
| SpecSeq58SpecSeq58 | Ala Ser Ser Thr Phe Glu Glu Arg Ser Gly Asn Thr Ile TyrAla Ser Ser Thr Phe Glu Glu Arg Ser Gly Asn Thr Ile Tyr |
| SpecSeq59SpecSeq59 | Ala Ser Ser Lys Gly Pro Pro Gly Gln Gly Glu Asp Thr Gln TyrAla Ser Ser Lys Gly Pro Pro Gly Gln Gly Glu Asp Thr Gln Tyr |
| SpecSeq60SpecSeq60 | Ala Ser Ser Gln Gln Ile Thr Gly Asp Leu Leu Tyr Gly Tyr ThrAla Ser Ser Gln Gln Ile Thr Gly Asp Leu Leu Tyr Gly Tyr Thr |
| SpecSeq61SpecSeq61 | Ala Ser Glu Glu Thr Gly Tyr Asp Gly Asn Thr Glu Ala PheAla Ser Glu Glu Thr Gly Tyr Asp Gly Asn Thr Glu Ala Phe |
| SpecSeq62SpecSeq62 | Ala Ser Ser Ala Gln His Leu Asn Thr Glu Ala PheAla Ser Ser Ala Gln His Leu Asn Thr Glu Ala Phe |
| SpecSeq63SpecSeq63 | Ala Ser Ser Leu Gly Gly Gln Gly Arg Tyr Thr Glu Ala PheAla Ser Ser Leu Gly Gly Gln Gly Arg Tyr Thr Glu Ala Phe |
| SpecSeq64SpecSeq64 | Ala Ser Ser Leu Glu Gly Asn Ser Pro Ser Tyr Glu Gln TyrAla Ser Ser Leu Glu Gly Asn Ser Pro Ser Tyr Glu Gln Tyr |
| SpecSeq65SpecSeq65 | Ala Ser Ser Arg Pro Gln Gly Ala Leu Ile Tyr Glu Gln PheAla Ser Ser Arg Pro Gln Gly Ala Leu Ile Tyr Glu Gln Phe |
| SpecSeq66SpecSeq66 | Ala Ser Ser Leu Tyr Lys Lys Gly Thr Glu Ala PheAla Ser Ser Leu Tyr Lys Lys Gly Thr Glu Ala Phe |
| SpecSeq67SpecSeq67 | Ala Ser Lys Phe Ala Met Gly Asn Gln Pro Gln HisAla Ser Lys Phe Ala Met Gly Asn Gln Pro Gln His |
| SpecSeq68SpecSeq68 | Ala Ser Ser Ile Trp Gly Gln Ser Thr Asp Thr Gln TyrAla Ser Ser Ile Trp Gly Gln Ser Thr Asp Thr Gln Tyr |
| SpecSeq69SpecSeq69 | Ala Ser Ser Leu Glu Arg Arg Gly Lys Asp Gly Tyr ThrAla Ser Ser Leu Glu Arg Arg Gly Lys Asp Gly Tyr Thr |
| SpecSeq70SpecSeq70 | Ala Ile Ser Asp Asp Ser Leu Glu Thr Gln TyrAla Ile Ser Asp Asp Ser Leu Glu Thr Gln Tyr |
| SpecSeq71SpecSeq71 | Ala Ser Ser Gln Asp Ser Arg Val Ser Gly Arg Asp Glu Gln TyrAla Ser Ser Gln Asp Ser Arg Val Ser Gly Arg Asp Glu Gln Tyr |
| SpecSeq72SpecSeq72 | Ser Ala Ser Pro Met Thr Gly Pro Thr Tyr Glu Gln TyrSer Ala Ser Pro Met Thr Gly Pro Thr Tyr Glu Gln Tyr |
| SpecSeq73SpecSeq73 | Ala Gly Met Gly Leu Thr Gly Ala Asn Val Leu ThrAla Gly Met Gly Leu Thr Gly Ala Asn Val Leu Thr |
| SpecSeq74SpecSeq74 | Ala Thr Ser Ser Arg Asp Gly Ser Gln Glu Thr Gln TyrAla Thr Ser Ser Arg Asp Gly Ser Gln Glu Thr Gln Tyr |
| SpecSeq75SpecSeq75 | Ala Ile Ser Pro Pro Gly Gln Gly Gly Ser Glu Gln PheAla Ile Ser Pro Pro Gly Gln Gly Gly Ser Glu Gln Phe |
| SpecSeq76SpecSeq76 | Ala Thr Ser Trp Arg Pro Gly Leu Ala Val Asn Asn Glu Gln PheAla Thr Ser Trp Arg Pro Gly Leu Ala Val Asn Asn Glu Gln Phe |
| SpecSeq77SpecSeq77 | Ala Ser Ser Tyr Ser Gly Thr Gly Ala Val Thr Glu Ala PheAla Ser Ser Tyr Ser Gly Thr Gly Ala Val Thr Glu Ala Phe |
| SpecSeq78SpecSeq78 | Ala Ser Ser Gln Asp Gly Ala Glu Gln Glu Thr Gln TyrAla Ser Ser Gln Asp Gly Ala Glu Gln Glu Thr Gln Tyr |
| SpecSeq79SpecSeq79 | Ala Ser Ser Glu Asn Lys Ala Tyr ThrAla Ser Ser Glu Asn Lys Ala Tyr Thr |
| SpecSeq80SpecSeq80 | Ala Thr Ser Asp Leu Trp Gly Pro Leu Lys Gly Glu Gln PheAla Thr Ser Asp Leu Trp Gly Pro Leu Lys Gly Glu Gln Phe |
| SpecSeq81SpecSeq81 | Ala Ser Ser Pro Pro Asp Met Pro Tyr Glu Gln PheAla Ser Ser Pro Pro Asp Met Pro Tyr Glu Gln Phe |
| SpecSeq82SpecSeq82 | Ala Ser Gly Gln Ala Tyr Gly Asp Thr Glu Ala PheAla Ser Gly Gln Ala Tyr Gly Asp Thr Glu Ala Phe |
| SpecSeq83SpecSeq83 | Ser Ala Arg Gly Pro Leu His Arg Gly Arg Thr Asp Thr Gln TyrSer Ala Arg Gly Pro Leu His Arg Gly Arg Thr Asp Thr Gln Tyr |
| SpecSeq84SpecSeq84 | Ala His Arg Ser Arg Gly Asp Glu Gln TyrAla His Arg Ser Arg Gly Asp Glu Gln Tyr |
| SpecSeq85SpecSeq85 | Ala Ser Ser Ser Gly Gln Asp Glu PheAla Ser Ser Ser Ser Gly Gln Asp Glu Phe |
| SpecSeq86SpecSeq86 | Ser Val Glu Trp Gly Phe Gly Gln Glu Thr Gln TyrSer Val Glu Trp Gly Phe Gly Gln Glu Thr Gln Tyr |
| SpecSeq87SpecSeq87 | Ser Ala Ser Asp Arg Asp Asn Tyr Gly Tyr ThrSer Ala Ser Asp Arg Asp Asn Tyr Gly Tyr Thr |
| SpecSeq88SpecSeq88 | Ser Ala Arg Gln Leu Asp Gly Thr Ser Ala Tyr Glu Gln PheSer Ala Arg Gln Leu Asp Gly Thr Ser Ala Tyr Glu Gln Phe |
| SpecSeq89SpecSeq89 | Ala Ser Thr Ala Asp Gly Gln Gln Tyr Asn Glu Gln PheAla Ser Thr Ala Asp Gly Gln Gln Tyr Asn Glu Gln Phe |
| SpecSeq90SpecSeq90 | Ala Ser Ser Glu Ser Thr Ser Gly Arg Glu Glu Glu Thr Gln TyrAla Ser Ser Glu Ser Thr Ser Gly Arg Glu Glu Glu Thr Gln Tyr |
| SpecSeq91SpecSeq91 | Ala Ile Val Met Gly Asn Thr Ile TyrAla Ile Val Met Gly Asn Thr Ile Tyr |
| SpecSeq92SpecSeq92 | Ala Ser Ser Gln Glu Gly Thr Gly Gly Lys Ala Gln HisAla Ser Ser Gln Glu Gly Thr Gly Gly Lys Ala Gln His |
| SpecSeq93SpecSeq93 | Ala Trp Ser Pro Lys Gly Arg Lys Ala PheAla Trp Ser Pro Lys Gly Arg Lys Ala Phe |
| SpecSeq94SpecSeq94 | Ala Ser Ser Gln Val Thr Ile Thr Ser Ala Asn Thr Gly Glu Leu PheAla Ser Ser Gln Val Thr Ile Thr Ser Ala Asn Thr Gly Glu Leu Phe |
| SpecSeq95SpecSeq95 | Ala Thr Ser Val Gln Thr Gly Ala Leu Ile Tyr Glu Gln TyrAla Thr Ser Val Gln Thr Gly Ala Leu Ile Tyr Glu Gln Tyr |
| SpecSeq96SpecSeq96 | Ala Ser Ser Pro Asp ArgAsp Arg Gly AsnGlnProGlnHisAla Ser Ser Pro Asp ArgAsp Arg Gly AsnGlnProGlnHis |
| SpecSeq97SpecSeq97 | Ala Trp Lys Gly Arg Glu Tyr Glu Gln TyrAla Trp Lys Gly Arg Glu Tyr Glu Gln Tyr |
| SpecSeq98SpecSeq98 | Ser Ala Arg Gly Ser Gly Asp Asn Thr Gln TyrSer Ala Arg Gly Ser Gly Asp Asn Thr Gln Tyr |
| SpecSeq99SpecSeq99 | Ser Ala Pro Ala Ser Tyr Thr Glu Ala PheSer Ala Pro Ala Ser Tyr Thr Glu Ala Phe |
| SpecSeq100SpecSeq100 | Ala Ser Ser Trp Ala Gly Leu Ser Tyr Glu Gln TyrAla Ser Ser Trp Ala Gly Leu Ser Tyr Glu Gln Tyr |
进一步地，标志物的蛋白序列为SpecSeq1-100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 the sequence shown in SpecSeq1-100 after one or more bases are substituted, deleted and/or replaced, and a protein with the same function can be expressed.
上述的标志物在制备检测或治疗大肠癌的制剂中的应用。Use of the above markers in the preparation of preparations for detecting or treating colorectal cancer.
进一步地，制剂包括含有该标志物的T细胞受体的质粒、病毒载体或核酸片段。Further, the preparation includes a plasmid, viral vector or nucleic acid fragment of the T cell receptor containing the marker.
一种用于大肠癌检测的试剂盒，包括能与上述标志物发生特异性结合的抗体。A kit for colorectal cancer detection includes 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；制剂可用于对大肠癌进行诊断、预测、检测或筛查。A preparation includes antibodies that can specifically bind to the above markers; the preparation can be used for diagnosing, predicting, detecting or screening colorectal cancer.
一种检测大肠癌的蛋白质芯片，蛋白质芯片包括基片和点样在基片上特异性抗体，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 colorectal cancer. The protein chip comprises a substrate and a specific antibody spotted on the substrate. The specific antibody is 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。
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.
本发明应用时，采用基于高通量测序分析外周血TCR序列方法以检测是否患有大肠癌，具体步骤如下：When the present invention is applied, a method based on high-throughput sequencing analysis of peripheral blood TCR sequence is used to detect whether there is colorectal cancer, and the specific steps are as follows:
(1)采集受试者和对照组外周血，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30000；(1) Collect the peripheral blood of the subjects and the control group, and obtain the antigenic determinant 3 (CDR3) amino acid sequences of the TCRs of the subjects and the control group through high-throughput sequencing to ensure the total number of CDR3 sequences of functional TCRs in each sample. Comprehensive not less than 30000;
(2)对每个样本的TCR的CDR3序列进行随机不放回抽样，使每个样本的CDR3序列数量总和均为30000；对任一特定CDR3序列X，在该样本中重复出现次数计为C
X；
(2) Randomly sample the CDR3 sequences of the TCR of each sample without replacement, so that the total number of CDR3 sequences in each sample is 30,000; for any specific CDR3 sequence X, the number of repeated occurrences in this sample is counted as C X ;
(3)TCR CDR3数据分析：通过分析确定大肠癌TCR标志物CDR3序列：(3) TCR CDR3 data analysis: Determine the CDR3 sequence of colorectal cancer TCR marker by analysis:
a)将对照组样本所有CDR3序列归总、去重，设为对照序列集；a) Summarize and deduplicate all CDR3 sequences of the control group samples, and set them as a control sequence set;
b)将大肠癌样本所有CDR3序列归总、去重，再去除所有与对照序列集中包含CDR3序列重复的序列，设为大肠癌特征序列集；b) Summarize and deduplicate all CDR3 sequences of the colorectal cancer sample, and then remove all sequences that duplicate the CDR3 sequences in the control sequence set, and set it as a colorectal cancer characteristic sequence set;
c)将大肠癌特征序列集中，出现于两个及以上样本中的CDR3序列，按“在所有大肠癌样本中该序列重复出现次数C
X的总和×包含该序列的大肠癌样本数”从高到低排序，以筛选出大肠癌TCR标志物CDR3序列。
c) Collecting colorectal cancer characteristic sequences, CDR3 sequences that appear in two or more samples, according to " the sum of the number of repeated occurrences of this sequence in all colorectal cancer samples C X × the number of colorectal cancer samples containing this sequence" from high To low ranking to screen out the colorectal cancer TCR marker CDR3 sequence.
(4)利用大肠癌特征性指数判断未知受试者罹患大肠癌风险：(4) Use the colorectal cancer characteristic index to determine the risk of colorectal cancer in unknown subjects:
a)根据健康对照组、非肿瘤病人、非大肠癌肿瘤患者、大肠癌患者以及未知健康状况受试者的免疫图谱，分析其大肠癌特征性指数；a) According to the immune profiles of healthy controls, non-tumor patients, non-colorectal cancer tumor patients, colorectal cancer patients, and subjects with unknown health conditions, analyze their colorectal cancer characteristic indices;
其中大肠癌特征性指数定义为：某个样本中，属于大肠癌特征序列集的所有CDR3序列在该样本内重复出现次数C
X的总和；
The colorectal cancer characteristic index is defined as: in a certain sample, the sum of the repeated occurrences C X of all CDR3 sequences belonging to the colorectal cancer characteristic sequence set in the sample;
b)当未知健康状况受试者的大肠癌特征性指数高于或接近“其它肿瘤”组的平均值+2×SD时，此人具有较高风险罹患大肠癌；如其大肠癌特征指数接近健康人或非肿瘤疾病组平均值，则表明其患大肠癌风险低。b) When the colorectal cancer characteristic index of a subject of unknown health status is higher than or close to the mean value of the "other tumors" group + 2 × SD, this person has a higher risk of colorectal cancer; if the colorectal cancer characteristic index is close to healthy The average value of the human or non-tumor disease group indicates a low risk of colorectal cancer.
综上所述，由于采用了上述技术方案，本发明的有益效果是：To sum up, due to the adoption of the above-mentioned technical solutions, the beneficial effects of the present invention are:
1、本发明中，首先利用非大肠癌的对照组样本和大肠癌病人的TCR高通量测序数据建立了人工智能分析模型，通过和这些大肠癌特异性TCR序列对比，可以清楚的判断待测样本中是否有较高大肠癌风险者；1. In the present invention, an artificial intelligence analysis model is first established by using the non-colorectal cancer control group samples and the TCR high-throughput sequencing data of colorectal cancer patients. By comparing with these colorectal cancer-specific TCR sequences, it is possible to clearly determine the test Whether there is a higher risk of colorectal cancer in the sample;
2、通过高通量测序分析TCR变化可以发现很早期的大肠癌，利用大肠癌特有的TCR CDR3序列分析人的免疫系统中的T细胞对大肠癌的反应，是一种新型的检测方法；2. Analysis of TCR changes by high-throughput sequencing can detect very early colorectal cancer. Using the unique TCR CDR3 sequence of colorectal cancer to analyze the response of T cells in the human immune system to colorectal cancer is a new type of detection method;
3、本发明中，由于采用高通量测序技术，同时比较巨大数量的特异性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. In the present invention, due to the use of high-throughput sequencing technology and the simultaneous comparison of a huge number of specific TCR sequences, it 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，也可用于大肠癌的免疫治疗。6. The TCR CDR3 sequences described in the present invention can also be used for immunotherapy of colorectal cancer.
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings required in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.
图1为本发明中，对照组CDR3序列及大肠癌特征序列。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大肠癌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；大肠癌患者的免疫图谱具有多个种类且重复次数较高的大肠癌特征序列，健康人极少大肠癌特征序列，而未知受试者的大肠癌特征比较明显，说明罹患大肠癌风险较高。
Figure 1 shows the CDR3 sequence of the control group and the characteristic sequence of colorectal cancer in the present invention. The abscissa represents the sequence in which the CDR3 sequence of a specific amino acid combination was added to the control sequence set or the colorectal cancer characteristic sequence set, and the ordinate represents the logarithm of the number of repetitions of the sequence in a sample, C X ; the immune system of colorectal cancer patients The atlas has multiple types of colorectal cancer feature sequences with a high number of repetitions. Healthy people rarely have colorectal cancer feature sequences, while unknown subjects have more obvious colorectal cancer features, indicating a higher risk of colorectal cancer.
图2为本发明中，针对大肠癌特征序列集，计算健康人、非肿瘤病人、非大肠癌肿瘤患者和大肠癌患者的大肠癌特征性指数，健康人、非肿瘤病人、非大肠癌肿瘤患者的大肠癌特征性指数均与大肠癌患者具有显著差异，证明了大肠癌特征序列集的特异性。据此可以判断未知受试者是否罹患大肠癌。Fig. 2 shows the colorectal cancer characteristic index of healthy people, non-tumor patients, non-colorectal cancer tumor patients and colorectal cancer patients according to the colorectal cancer feature sequence set in the present invention, healthy people, non-tumor patients, non-colorectal cancer tumor patients The colorectal cancer characteristic indices of CRC patients were significantly different from those of colorectal cancer patients, proving the specificity of the colorectal cancer characteristic sequence set. Based on this, it can be determined whether the unknown subject suffers from colorectal cancer.
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Thus, the following detailed description of the embodiments of the invention provided in the accompanying drawings are not intended to limit the scope of the invention as claimed, but are merely representative of selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present invention.
以下结合实施例对本发明的特征和性能作进一步的详细描述。The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.
实施例1Example 1
通过免疫图谱分析，获得大肠癌TCR标志物CDR3序列集：Obtained the colorectal cancer TCR marker CDR3 sequence set by immunomap analysis:
1、采样及免疫图谱分析1. Sampling and immunographic analysis
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)、101名大肠癌患者(100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30000；Collect 1301 control groups (including healthy people and non-tumor disease patients, 1300 people are used for model building, 1 healthy person is used for validation), 101 colorectal cancer patients (100 people are used for model building, 1 person is used for validation) And the peripheral blood (10mL per person) of a subject with unknown health status, the TCR antigenic determinant 3 (CDR3) amino acid sequence of the subject and the control group was obtained by high-throughput sequencing to ensure the functionality of each sample The total number of CDR3 sequences of TCR is not less than 30000;
2、对每个样本的TCR的CDR3序列进行随机不放回抽样，使每个样本的CDR3序列数量总和均为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 CDR3 sequences of the TCR of each sample, so that the total number of CDR3 sequences in each sample is 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，确定大肠癌TCR标志物CDR3序列：3. Determine the CDR3 sequence of colorectal cancer 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 CDR3 sequences of the 1300 control group samples used to build the model were aggregated and deduplicated, and set as a control sequence set;
b)将100名用于建立模型的大肠癌样本的所有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，设为大肠癌特征序列集。如图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大肠癌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) All CDR3 sequences of the 100 colorectal cancer samples used to build the model were aggregated and deduplicated, and then all sequences containing sequence duplication with the control sequence set were removed, and set as a colorectal cancer characteristic sequence set. 1A, wherein the abscissa represents a particular combination of amino acid sequences are added to the control sequence of CDR3 sequences or a set of signature sequences colorectal set, the ordinate represents the sequence is repeated the number of times C X appears in a sample numerical value.
c)按照同样作图方法，将1名健康人、1名大肠癌患者和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大肠癌特征序列集合进行作图，如图1B-D。从图中可见，大肠癌患者的免疫图谱中，含有较多种类且较高重复出现次数的大肠癌特征序列；健康人的免疫图谱中，只有极少量大肠癌特征序列；而未知健康状况受试者，有高于健康人的大肠癌特征序列，说明此人有较高风险罹患大肠癌。c) According to the same mapping method, the immune profiles of 1 healthy person, 1 colorectal cancer patient and 1 subject with unknown health status are mapped with reference to the control sequence set and the colorectal cancer characteristic sequence set, as shown in Figure 1B- D. It can be seen from the figure that the immune maps of patients with colorectal cancer contain more types of colorectal cancer feature sequences with high repetitions; the immune maps of healthy people contain only a very small number of colorectal cancer feature sequences; while subjects with unknown health status , have a higher colorectal cancer characteristic sequence than healthy people, indicating that this person has a higher risk of colorectal cancer.
d)将大肠癌特征序列集中，将所有出现在两个及以上参与建模大肠癌样本里的CDR3序列，按“所有参与建模大肠癌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大肠癌样本数”从高到低排序，排名前100者即为大肠癌TCR标志物CDR3序列。
d) Set the colorectal cancer feature sequences, and put all the CDR3 sequences that appear in two or more colorectal cancer samples participating in the modeling, and press the "sum of the number of repeated occurrences C X in a single sample of this sequence in all the colorectal cancer samples participating in the modeling." ×Number of colorectal cancer samples that contain the sequence involved in modeling” is sorted from high to low, and the top 100 is the colorectal cancer TCR marker CDR3 sequence.
实施例2Example 2
验证大肠癌TCR标志物CDR3序列集的特异性：Validation of the specificity of the colorectal cancer TCR marker CDR3 sequence set:
1、采样及免疫图谱分析1. Sampling and immunographic analysis
采集358名非大肠癌的肿瘤患者、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30000；对每个样本的TCR的CDR3序列进行随机不放回抽样，使每个样本的CDR3序列数量总和均为30000。The peripheral blood (10 mL per person) of 358 non-colorectal cancer tumor patients and 3 subjects with unknown health status was collected, and the antigenic determinant 3 (CDR3) amino acids of the TCR of the subjects and the control group were obtained by high-throughput sequencing Sequence, to ensure that the total number of CDR3 sequences of functional TCRs of each sample is not less than 30,000; the CDR3 sequences of TCR of each sample are randomly sampled without replacement, so that the total number of CDR3 sequences of each sample is 30,000.
2、从实施例1的对照组中随机挑选100名健康人及45名非肿瘤疾病病人。2. 100 healthy people and 45 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、45名非肿瘤疾病病人、100名大肠癌患者，以及实施例2新获取的358名非大肠癌肿瘤患者、3名未知健康状况受试者的免疫图谱，分析其大肠癌特征性指数。3. According to 100 healthy people, 45 non-tumor disease patients, 100 colorectal cancer patients from Example 1, and 358 non-colorectal cancer tumor patients and 3 subjects with unknown health conditions newly obtained in Example 2. The immune profile was analyzed to analyze the characteristic index of colorectal cancer.
其中大肠癌特征性指数定义为：某个样本中，属于大肠癌特征序列集的所有CDR3序列在该样本内重复出现次数C
X的总和。
The colorectal cancer characteristic index is defined as: in a certain sample, the sum of the repetition times C X of all CDR3 sequences belonging to the colorectal cancer characteristic sequence set in the sample.
分析结果见下表2及图2。大肠癌组与健康人(p＝6.2E-120)、非肿瘤疾病(p＝1.6E-73)、其它肿瘤(p＝3.2E-275)都有显著差异，这证明了大肠癌特征序列集的特异性。The analysis results are shown in Table 2 and Figure 2 below. The colorectal cancer group was significantly different from healthy people (p=6.2E-120), non-tumor diseases (p=1.6E-73), and other tumors (p=3.2E-275), which proved that the colorectal cancer feature sequence set specificity.
表2 不同样本组的大肠癌特征性指数Table 2 Colorectal cancer characteristic index of different sample groups
4、分析各组的大肠癌特征指数(下表3)，未知健康状况受试者(检测样本)中，3人的大肠癌特征指数都高“其它肿瘤”组的平均值+2×SD(54+2×95＝243)，此3人具有较高风险罹患大肠癌。与临床体检结果对照后，3人确为大肠癌患者。此实施例证明了利用大肠癌特征序列集及大肠癌特征性指数，预测受试者罹患大肠癌风险的可行性。4. Analyze the colorectal cancer characteristic index of each group (Table 3 below). Among the subjects with unknown health status (test samples), the colorectal cancer characteristic index of 3 people is higher than the average value of the "other tumors" group + 2 × SD ( 54+2×95=243), these 3 people have a higher risk of colorectal cancer. After comparing with the results of clinical physical examination, 3 people were confirmed to be patients with colorectal cancer. This example demonstrates the feasibility of predicting the risk of colorectal cancer in a subject by using the colorectal cancer feature sequence set and the colorectal cancer feature index.
表3 大肠癌特征性指数分析表Table 3 Colorectal cancer characteristic index analysis table
综上所述，本发明所述大肠癌TCR标志物CDR3序列，确实具有显著的大肠癌特异性，不仅可以用于大肠癌预测受试者罹患大肠癌风险，未来还可用于大肠癌的生物免疫治疗。To sum up, the colorectal cancer TCR marker CDR3 sequence of the present invention does have significant colorectal cancer specificity, and can not only be used for colorectal cancer to predict the risk of colorectal cancer in subjects, but also can be used for colorectal cancer biological immunity in the future. treat.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.
Claims (7)
- 一种大肠癌的外周血TCR标志物，其特征在于，包括序列SpecSeq1-100所示蛋白中的至少一种。A peripheral blood TCR marker for colorectal cancer, characterized by comprising at least one of the proteins represented by the sequence SpecSeq1-100.
- 根据权利要求1所述的大肠癌的外周血TCR标志物，其特征在于，所述标志物的蛋白序列为SpecSeq1-100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 colorectal cancer peripheral blood TCR marker according to claim 1, wherein the protein sequence of the marker is the sequence shown in SpecSeq1-100 after substitution, deletion and/or replacement of one or more bases , which can express proteins with the same function.
- 权利要求1或2所述的标志物在制备检测或治疗大肠癌的制剂中的应用。The application of the marker according to claim 1 or 2 in the preparation of a preparation for detecting or treating colorectal cancer.
- 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细胞受体的质粒、病毒载体或核酸片段。The use according to claim 3, wherein the preparation comprises a plasmid, a viral vector or a nucleic acid fragment containing the T cell receptor of the marker.
- 一种用于大肠癌检测的试剂盒，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 colorectal cancer detection, characterized in that it comprises an antibody that can specifically bind to the marker of claim 1.
- 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。A preparation, characterized in that it comprises an antibody that can specifically bind to the marker of claim 1.
- 一种检测大肠癌的蛋白质芯片，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 colorectal cancer, characterized in that the protein chip comprises a substrate and a specific antibody spotted on the substrate, and the specific antibody is capable of specifically binding to the marker of claim 1 antibody.
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