CN111693702A - 一种黑色素瘤的外周血tcr标志物及其检测试剂盒和应用 - Google Patents
一种黑色素瘤的外周血tcr标志物及其检测试剂盒和应用 Download PDFInfo
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Abstract
本发明公开了一种黑色素瘤的外周血TCR标志物及其检测试剂盒和应用。该标志物包括序列为SEQ ID NO.1～100所示的蛋白中的至少一种。本发明基于高通量测序方法，只需要采取少量外周血，提取RNA，通过对样本的处理建立免疫图谱文库，再经过高通量测序和TCR数据分析，首先确定黑色素瘤外周血中特征性TCR序列，然后将待测样本测试结果与该特征性TCR序列比对，从而确定是否患有黑色素瘤。本发明能够同时比较巨大数量的黑色素瘤特异性TCR序列，相比单独检测一种或几种标记物，具有更高的特异性和准确性，提高了诊断效率。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种黑色素瘤的外周血TCR标志物及其检测试剂盒和应用。
背景技术
黑色素瘤又称恶性黑色素瘤，是一种与骨伤科有关的皮肤肿瘤。良性黑色素瘤又称色素痣，多不引起注意，但黑色素瘤可以一开始即为恶性，通常由交界痣恶变而来。黑色素瘤在我国虽然是少见恶性肿瘤，但病死率高，发病率也在逐年增加。我国黑色素瘤与欧美白种人差异较大，两者在发病机制、生物学行为、组织学形态、治疗方法以及预后等方面差异较大。在亚洲人和其他有色人种中，原发于肢端的黑色素瘤约占50％，常见的原发部位多见于足底、足趾、手指末端及甲下等肢端部位，原发于黏膜，如直肠、肛门、外阴、眼、口鼻咽部位的黑色素瘤约占20％～30％；而对于白种人来说，原发于皮肤的黑色素瘤约占90％，原发部位常见于背部、胸腹部和下肢皮肤；原发于黏膜的黑色素瘤只占1％～5％。
在我国，皮肤黑色素瘤的高危人群主要包括严重的日光晒伤史，皮肤癌病史，肢端皮肤有色素痣、慢性炎症，及其不恰当的处理，如盐腌、切割、针挑、绳勒等。黏膜黑色素瘤的高危因素尚不明确。因此色素瘤高危人群的筛查，有助于早期发现、早期诊断、早期治疗，同时也是提高黑色素瘤疗效的关键，也是社会的迫切需求。
目前针对黑色素瘤的诊断手段主要有：
1.临床症状判断
黑色素瘤好发于皮肤，因此视诊是早期诊断的最简便手段。原发病变、受累部位和区域淋巴结的视诊和触诊是黑色素瘤初步诊断的常用手段。
皮肤黑色素瘤多由痣发展而来，痣的早期恶变症状可总结为以下ABCDE法则：
A、非对称(asymmetry)：色素斑的一半与另一半看起来不对称；B、边缘不规则(border irregularity)：边缘不整或有切迹、锯齿等，不像正常色素痣那样具有光滑的圆形或椭圆形轮廓；C、颜色改变(color variation)：正常色素痣通常为单色，而黑色素瘤主要表现为污浊的黑色，也可有褐、棕、棕黑、蓝、粉、黑甚至白色等多种不同颜色；D、直径(diameter)：色素痣直径＞5～6mm或色素痣明显长大时要注意，黑色素瘤通常比普通痣大，对直径＞1cm的色素痣最好做活检评估；E、隆起(elevation)：一些早期的黑色素瘤，整个瘤体会有轻微的隆起。
ABCDE法则的唯一不足在于它没有将黑色素瘤的发展速度考虑在内，如几周或几个月内发生显著变化的趋势。皮肤镜可以弥补肉眼观察的不足，同时可以检测和对比可疑黑色素瘤的变化，其应用可显著提高黑色素瘤早期诊断的准确度。黑色素瘤进一步发展可出现卫星灶、溃疡、反复不愈、区域淋巴结转移和移行转移。晚期黑色素瘤根据不同的转移部位症状不一，容易转移的部位为肺、肝、骨、脑。眼和直肠来源的黑色素瘤容易发生肝转移。
2.影像学诊断
1)超声检查(Ultrasonography,US)
超声检查因操作简便、灵活直观、无创便携等特点，是临床上最常用的影像学检查方法。黑色素瘤的超声检查主要用于区域淋巴结、皮下结节性质的判定，为临床治疗方法的选择及手术方案的制定提供重要信息。实时超声造影技术可以揭示转移灶的血流动力学改变，特别是帮助鉴别和诊断小的肝转移、淋巴结转移等方面具有优势。
2)X线计算机断层成像(Computed Tomography,CT)
常规采用平扫增强扫描方式(常用碘对比剂)。目前除应用于黑色素瘤临床诊断及分期外，也常应用于黑色素瘤的疗效评价，肿瘤体积测量、肺和骨等其他脏器转移评价，临床应用广泛。
3)磁共振成像(Magnetic Resonance Imaging,MRI)
常规采用平扫增强扫描方式(常用对比剂Gd-DTPA)，因其具有无辐射影响，组织分辨率高，可以多方位、多序列参数成像，并具有形态结合功能(包括弥散加权成像、灌注加权成像和波谱分析)综合成像技术能力，成为临床黑色素瘤诊断和疗效评价的常用影像技术。
4)正电子发射计算机断层成像(Positron Emission Tomography-CT,PET-CT)
氟-18-脱氧葡萄糖(18F-FDG)PET-CT全身显像的优势在于：①对肿瘤进行分期，通过一次检查能够全面评价淋巴结转移及远处器官的转移；②再分期，因PET功能影像不受解剖结构的影响，可准确显示解剖结构发生变化后或者是解剖结构复杂部位的复发转移灶；③疗效评价，对于抑制肿瘤活性的靶向药物，疗效评价更加敏感、准确；④指导放疗生物靶区的勾画和肿瘤病灶活跃区域的穿刺活检；⑤评价肿瘤的恶性程度和预后。常规CT对于皮肤或者皮下转移的诊断灵敏度较差，而PET-CT可弥补其不足。
综上所述，影像学检查应根据当地实际情况和患者经济情况决定，造成检测成本过高，必查项目包括区域淋巴结(颈部、腋窝、腹股沟、腘窝等)超声，胸部CT，腹盆部超声、CT或MRI，全身骨扫描及头颅检查(CT或MRI)。经济情况好的患者可行全身PET-CT检查，特别是原发灶不明的患者。
3.实验室检查
血常规、肝肾功能和乳酸脱氢酶(LDH)，这些指标主要为后续治疗做准备，同时了解预后情况。尽管LDH并非检测转移的敏感指标，但能指导预后。黑色素瘤尚无特异的血清肿瘤标志物，目前不推荐肿瘤标志物检查。
4.病灶活检
皮肤黑色素瘤的活检方式包括切除活检、切取活检和环钻活检，一般不采取削刮活检。常规推荐切除活检，切缘0.3～0.5cm，切口应沿皮纹走行方向(如肢体一般选择沿长轴的切口)。避免直接的扩大切除，以免改变区域淋巴回流影响以后前哨淋巴结活检的质量。部分切取活检不利于组织学诊断和厚度测量，增加了误诊和错误分期风险。切取活检和环钻活检一般仅用于大范围病变或特殊部位的诊断性活检，比如在颜面部、手掌、足底、耳、手指、足趾或甲下等部位的病灶，或巨大的病灶，完整切除活检无法实现时，可考虑进行切取活检或者环钻活检，但是会对人体造成不可避免的创伤。
5.病理学诊断
组织病理学是黑色素瘤确诊的最主要手段，免疫组织化学染色是鉴别黑色素瘤的主要辅助手段。无论黑色素瘤体表病灶或者转移灶活检或手术切除组织标本，均需经病理组织学诊断。病理诊断须与临床证据相结合，全面了解患者的病史和影像学检查等信息，对于诊断困难的病例，还应该多请专家会诊，并且多种指标的综合评估，对最终检测结果的判读造成了困难。
发明内容
针对现有技术中的上述不足，本发明提供一种黑色素瘤的外周血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黑色素瘤风险患者。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种黑色素瘤的外周血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。
表1标志物序列
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。
进一步地，标志物为外周血TCR CDR3序列。
上述标志物在制备治疗黑色素瘤的制剂中的应用。
进一步地，制剂中包括含有该标志物的T细胞受体，或能表达产生该标志物的T细胞受体的质粒、病毒载体或核酸片段。
一种用于黑色素瘤检测的试剂盒，包括能与上述标志物发生特异性结合的抗体。
一种制剂，包括能与上述标志物发生特异性结合的抗体；所述制剂可用于对黑色素瘤进行诊断、预测、检测或筛查。
一种检测黑色素瘤的蛋白质芯片，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。
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1018，TCR多样性的理论值是1014。BCR与TCR序列中，抗原决定簇3(CDR3)是决定其抗原特异性最重要的部分，因此CDR3的序列被认为可以代表BCR、TCR序列的特性。
在各种疾病中，随着不同的抗原刺激，BCR和TCR的多样性或者表达水平都会发生改变。因此，利用BCR或者TCR高通量测序结果可以追踪疾病的发生、发展。人体内细胞中，衰老蛋白质降解后，其片段会被运输到细胞表面，通过组织相容性抗原II(MCHII)呈递给免疫系统中的T细胞。正常细胞呈递的抗原片段，由于免疫耐受的关系，不会引起免疫反应。一旦当正常细胞出现癌变后，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。
本发明的有益效果为：
1、本发明中，首先利用1300个非黑色素瘤的对照组样本、和20个黑色素瘤病人的TCR高通量测序数据建立了人工智能分析模型，通过和这些黑色素瘤特异性TCR序列对比，可以清楚的判断待测样本中是否有较高黑色素瘤风险者。
2、通过高通量测序分析TCR变化可以发现很早期的黑色素瘤，利用黑色素瘤特有的TCR CDR3序列分析人的免疫系统中的T细胞对黑色素瘤的反应，是一种新型的检测方法。
3、本发明通过采用高通量测序技术同时比较数量巨大的特异性TCR序列，比起单独检测一种或几种标记物，具有更高的特异性和准确性。
4、本发明中使用的高通量测序仪器成本低于大型影像学设备，且可向第三方外包，此外，采样、处理的人力成本低于同时检测多种标记物，也低于大量细胞学检测，因此，本发明大大降低了检测成本。
5、本发明只需要采取少量外周血，采样简便、安全。
6、本发明中所述TCR CDR3序列，可用于黑色素瘤的免疫治疗。
附图说明
图1为本发明中，对照组CDR3序列及黑色素瘤特征序列。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黑色素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CX的对数值；黑色素瘤患者的免疫图谱具有多个种类且重复次数较高的黑色素瘤特征序列，健康人极少黑色素瘤特征序列，而未知受试者的黑色素瘤特征比较明显，说明罹患黑色素瘤风险较高。
图2为本发明中，针对黑色素瘤特征序列集，计算健康人、非肿瘤病人、非黑色素瘤肿瘤患者和黑色素瘤患者的黑色素瘤特征性指数，健康人、非肿瘤病人、非黑色素瘤肿瘤患者的黑色素瘤特征性指数均与黑色素瘤患者具有显著差异，证明了黑色素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黑色素瘤。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1通过免疫图谱分析，获得黑色素瘤TCR标志物CDR3序列集
1、采样及免疫图谱分析(方法参考专利ZL201910300069.9)
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)、21名黑色素瘤患者(20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；
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CX；
3、通过分析TCR CDR3数据，确定黑色素瘤TCR标志物CDR3序列：
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；
b)将20名用于建立模型的黑色素瘤样本的所有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，设为黑色素瘤特征序列集。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黑色素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CX的对数值。
c)按照同样作图方法，将1名健康人、1名黑色素瘤患者和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黑色素瘤特征序列集合进行作图，见附图1B-D。从图中可见，黑色素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黑色素瘤特征序列；健康人的免疫图谱中，只有极少量黑色素瘤特征序列；而未知健康状况受试者，有高于健康人的黑色素瘤特征序列，说明此人有较高风险罹患黑色素瘤。
d)将黑色素瘤特征序列集中，将所有出现在两个及以上参与建模黑色素瘤样本里的CDR3序列，按“所有参与建模黑色素瘤样本里该序列单样本中重复出现次数CX的总和×包含该序列的参与建模黑色素瘤样本数”从高到低排序，排名前100者即为黑色素瘤TCR标志物CDR3序列，具体序列如SEQ ID NO.1～100所示。
实施例2验证黑色素瘤TCR标志物CDR3序列集的特异性
1、采样及免疫图谱分析(方法参考专利ZL201910300069.9)
采集325名非黑色素瘤的肿瘤患者、5名未知健康状况受试者的外周血(每人10mL)，通过高通量测序得到受试者和对照组的TCR的抗原决定簇3(CDR3)氨基酸序列，保证每个样本的功能性TCR的CDR3序列总数综合不低于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。
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。
3、根据来自实施例1的100名健康人、45名非肿瘤疾病病人、20名黑色素瘤患者，以及实施例2新获取的325名非黑色素瘤肿瘤患者、5名未知健康状况受试者的免疫图谱，分析其黑色素瘤特征性指数。
其中黑色素瘤特征性指数定义为：某个样本中，属于黑色素瘤特征序列集的所有CDR3序列在该样本内重复出现次数CX的总和。分析结果见下表2及附图2。黑色素瘤组与健康人(p＝2.9E-69)、非肿瘤疾病(p＝1.3E-35)、其它肿瘤(p＝7.1E-208)都有显著差异，这证明了黑色素瘤特征序列集的特异性。
表2不同样本组的黑色素瘤特征性指数
4、分析各组的黑色素瘤特征指数(表3)，未知健康状况受试者(检测样本)中，前3人的黑色素瘤特征指数高于“其它肿瘤”组的平均值+2×SD(10.3+2×5.4＝21.1)，此3人具有较高风险罹患黑色素瘤；后2人黑色素瘤特征指数接近健康人或非肿瘤疾病组平均值，罹患黑色素瘤风险低。与临床体检结果对照后，前3人确为黑色素瘤患者，而后2人为健康人。此实施例证明了利用黑色素瘤特征序列集及黑色素瘤特征性指数，预测受试者罹患黑色素瘤风险的可行性。
表3黑色素瘤特征性指数分析
| 健康人 | 非肿瘤疾病 | 其他肿瘤 | 黑色素瘤 | 检测样本 | |
| Mean | 22.73 | 10.78 | 10.31 | 5463.60 | 1646.80 |
| SD | 69.35 | 6.93 | 5.41 | 1409.10 | 1683.45 |
| mean+2SD | 161.44 | 24.64 | 21.12 |
综上所述，本发明所述黑色素瘤TCR标志物CDR3序列，确实具有显著的黑色素瘤特异性，不仅可以用于黑色素瘤预测受试者罹患黑色素瘤风险，未来还可用于黑色素瘤的生物免疫治疗。
序列表
<110> 成都益安博生物技术有限公司
<120> 一种黑色素瘤的外周血TCR标志物及其检测试剂盒和应用
<160> 100
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ser Val Glu Leu Pro Gly Ser Leu Gly Thr Tyr Glu Gln Tyr
1 5 10
<210> 2
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ala Ser Ser Glu Glu Gly Trp Asp Ala Asp Thr Gln Tyr
1 5 10
<210> 3
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Ser Ser Gly Tyr Gly Gly Gly Thr Asp Thr Gln Tyr
1 5 10
<210> 4
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ala Ser Ser Gln Gly Val Arg Thr Ser Tyr Glu Gln Tyr
1 5 10
<210> 5
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Ser Ser Leu Ser Arg Leu Ser Ala Thr Asp Thr Gln Tyr
1 5 10
<210> 6
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ala Ser Ser Gln Ser Thr Val His Glu Gln Tyr
1 5 10
<210> 7
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gln Gln Pro Arg Asn Gly Val Trp Leu His
1 5 10
<210> 8
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Ala Ser Ser Pro Lys Gly Arg Asp Ser Asn Gln Pro Gln His
1 5 10
<210> 9
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ala Thr Ser Ala Arg Gln Gly Leu Ala Phe
1 5 10
<210> 10
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Ala Ser Lys Arg Gly Val Ala Gly Val Arg Ser Ser Tyr Asn Glu Gln
1 5 10 15
Phe
<210> 11
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Ala Ser Ser Pro Arg Thr Gly Gly Phe Thr Lys Asn Ile Gln Tyr
1 5 10 15
<210> 12
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ser Ala Arg Asp Arg Glu Asn Asn Gly Arg Ala Gly Thr Gln Tyr
1 5 10 15
<210> 13
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Ala Ser Asp Ala Lys Gly Gln Gly Asn Thr Glu Ala Phe
1 5 10
<210> 14
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Ala Ser Ser Asp Gly Thr Ala Ile Asn Glu Lys Leu Phe
1 5 10
<210> 15
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Ala Ser Ala Thr Pro Gly Ser Thr Gly Glu Leu Phe
1 5 10
<210> 16
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Ala Ser Ser Leu Ala Ala Ser Arg Ser Glu Gln Phe
1 5 10
<210> 17
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Ser Val Leu Leu Glu Thr Ser Ala Tyr Glu Gln Tyr
1 5 10
<210> 18
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Ala Ser Ser Ser Gln Gln Gly Phe Gly Asp Ile Gln Tyr
1 5 10
<210> 19
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Ala Ser His Pro Pro Gly Leu Ala Gly Thr Gly Glu Leu Phe
1 5 10
<210> 20
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Ala Trp Ala Lys Gly Gln Leu Asp Gly Tyr Thr
1 5 10
<210> 21
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Ala Ser Ser Gly Asn Thr Lys Gly Ser Tyr Glu Gln Tyr
1 5 10
<210> 22
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Ala Ser Ser Gly Gly Asp Thr Gln Pro Gln His
1 5 10
<210> 23
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Ala Ser Ser Glu Ala Thr Glu Ile Glu Gln Phe
1 5 10
<210> 24
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Ala Thr Ser Glu Ser Glu Ala Gln Pro Gln His
1 5 10
<210> 25
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Ala Thr Ser Arg Ala Gly Gln Gly Phe Glu Gln Phe
1 5 10
<210> 26
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Ala Ser Ser Leu Ala Gly Gln Thr Ser Tyr Asp
1 5 10
<210> 27
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Ala Ser Thr Ser Gly Gly Val Pro Tyr Gly Tyr Thr
1 5 10
<210> 28
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Ala Ser Ser Gln Ser Pro Thr Ala Asp Asn Glu Gln Phe
1 5 10
<210> 29
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Ala Ser Asn Gln Arg Gly Gly Ala Tyr Asn Glu Gln Phe
1 5 10
<210> 30
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Ser Ala Arg Leu Ser Ala Gly Leu Asp Tyr Asn Glu Gln Phe
1 5 10
<210> 31
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Ala Ser Ser Pro Gln Gly Val Gly Ala Gly Asn Thr Gln Tyr
1 5 10
<210> 32
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Ala Ser Val Lys Gln Gly Ser Tyr Glu Gln Tyr
1 5 10
<210> 33
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Ala Ser Ser Leu Ser Glu Arg Met Asp Glu Gln Phe
1 5 10
<210> 34
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Ala Ser Ser Gln Gly Pro Gly Thr Asp Glu Ala Phe
1 5 10
<210> 35
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Ala Ser Ser Leu Ala Gly Glu Arg Gly Tyr Thr
1 5 10
<210> 36
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Ala Ala Ser Met Gly Gln Val Tyr Glu Gln Tyr
1 5 10
<210> 37
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Ala Ser Ser Leu Leu Pro Ser Val Gln Ala Ala Tyr Asn Glu Gln Phe
1 5 10 15
<210> 38
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Ala Ser Ser Arg Thr Leu Gly Thr Gly Arg Gly Tyr Thr
1 5 10
<210> 39
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Ala Thr Ser Ile Thr Glu Arg Arg Glu Leu Phe
1 5 10
<210> 40
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Ala Thr Ser Arg Gly Tyr Asp Ile Pro Ser Asn Gln Pro Gln His
1 5 10 15
<210> 41
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Ala Ser Ser Glu Ser Thr Arg Asn Glu Gln Phe
1 5 10
<210> 42
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Ala Ser Ser Asp Leu Val Ser Arg Asp Ala Gly Asp Thr Gln Tyr
1 5 10 15
<210> 43
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Ala Thr Gly Pro Ala Glu Gly Lys Pro Gln His
1 5 10
<210> 44
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Ala Ser Ser Tyr Gln Gly Ala Leu Arg Ala Phe
1 5 10
<210> 45
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
Ser Ala Ala Ala Gly Val Ser Ser Thr Asp Thr Gln Tyr
1 5 10
<210> 46
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
Ala Ser Ser Phe Val Thr Gly Ser Gly Glu Leu Phe
1 5 10
<210> 47
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
Ser Val Val Pro Ser Thr Val Ala Thr Tyr Glu Gln Tyr
1 5 10
<210> 48
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Ala Ser Ser Leu Ser Gly Val Lys Gly Gln Phe
1 5 10
<210> 49
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
Ala Ser Ser Tyr Asn Gln Gly Val Gln Val Glu Gln Tyr
1 5 10
<210> 50
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
Ala Ser Ser Leu Leu Gly Gln Gly Val Gly Gly Glu Leu Phe
1 5 10
<210> 51
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 51
Ala Ser Arg Pro Thr Gly Arg Gly Gly Gly Tyr Thr
1 5 10
<210> 52
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 52
Ala Ser Ser Lys Tyr Pro Asp Gly Ser Ser Thr Asp Thr Gln Tyr
1 5 10 15
<210> 53
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 53
Ser Ala Arg Val Gly Arg Ala Ser Ala Ser Thr Asp Thr Gln Tyr
1 5 10 15
<210> 54
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 54
Ser Val Gly Thr Ser Gly Ser Ile Gly Tyr
1 5 10
<210> 55
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 55
Ala Thr Ser Arg Asp Pro Gly Pro Tyr Ser Asn Gln Pro Gln His
1 5 10 15
<210> 56
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 56
Ser Gly Glu Ala Gly Glu Gly Asn Glu Gln Phe
1 5 10
<210> 57
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 57
Ala Ser Leu Ser Gly Arg Glu Tyr Asn Glu Gln Phe
1 5 10
<210> 58
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 58
Ala Ser Ser Pro Glu Ser Gly Ala Gly Arg Ser Thr Gly Glu Leu Phe
1 5 10 15
<210> 59
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 59
Ala Ser Ser Leu Gly Pro Ala Gly Pro Thr His Asn Glu Gln Phe
1 5 10 15
<210> 60
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 60
Ala Ser Ser Phe Trp Asp Gly Ile Ser Glu Ala Phe
1 5 10
<210> 61
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 61
Ala Ser Ser Leu Gly Arg Thr Ala Phe Asn Glu Lys Leu Phe
1 5 10
<210> 62
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 62
Ala Thr Thr Gly Thr Gly Lys Val Ser Ser Tyr Asn Glu Gln Phe
1 5 10 15
<210> 63
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 63
Ala Ser Ser Leu Glu Gln Val Leu Arg Thr Asp Thr Gln Tyr
1 5 10
<210> 64
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 64
Ala Ser Ser Tyr Gly Pro Ala Thr Glu Gln Tyr
1 5 10
<210> 65
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 65
Ala Ser Ser Glu Trp Ser Glu Ser Tyr Asn Glu Gln Phe
1 5 10
<210> 66
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 66
Ala Ser Arg Phe Gly Gln Gly Ser Asn Glu Lys Leu Phe
1 5 10
<210> 67
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 67
Ala Ser Ser Asp Tyr Phe Ile Val Thr Glu Ala Phe
1 5 10
<210> 68
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 68
Ala Trp Ser Val Tyr Glu Gly Pro Gly Phe Ala Tyr Asn Glu Gln Phe
1 5 10 15
<210> 69
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 69
Ala Ser Ser Ser Thr Leu Lys Gly Gly Leu Gln Glu Thr Gln Tyr
1 5 10 15
<210> 70
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 70
Ala Thr Ser Ser Arg Thr Ser Gly Ser Ser Glu Gln Tyr
1 5 10
<210> 71
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 71
Ala Ser Ser Thr Gly Leu Ala Gly Thr Val
1 5 10
<210> 72
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 72
Ser Ala Lys Ser Ser Ile His Tyr Gly Tyr Thr
1 5 10
<210> 73
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 73
Ala Ser Ser Ala Gln Gln Val Ala Thr Gly Ala Ser Thr Asp Thr Gln
1 5 10 15
Tyr
<210> 74
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 74
Ala Ser Ser Ala Arg Phe Glu Gln Phe
1 5
<210> 75
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 75
Ala Ser Ser Arg Asp Glu Thr Gly Gly Asp Gly Tyr Thr
1 5 10
<210> 76
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 76
Ala Ser Ile Arg Pro Gly Leu Ala Gly Asp Glu Gln Phe
1 5 10
<210> 77
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 77
Ala Ser Ser Ala Arg Glu Gly Glu Thr Asp Thr Gln Tyr
1 5 10
<210> 78
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 78
Ala Ser Ser Glu Gly Gly Gly Ser Gly Gly Thr Glu Ala Phe
1 5 10
<210> 79
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 79
Ala Ser Ser Phe Arg Gln Phe Gly Tyr Glu Gln Tyr
1 5 10
<210> 80
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 80
Ala Ser Ser Gln Val Gly Gly Arg Gly Ile Gly Glu Gln Tyr
1 5 10
<210> 81
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 81
Gln Gln Trp Val Trp Gly Arg His Arg Tyr Ala Val
1 5 10
<210> 82
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 82
Ala Ser Ser Glu Arg Trp Glu Gly Leu Asp Glu Gln Phe
1 5 10
<210> 83
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 83
Ala Ser Ser Leu Ala Ala Gly Val Thr Gly Thr Glu Gln Tyr
1 5 10
<210> 84
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 84
Ala Ser Ser Ala Glu Leu Phe Gln Glu Thr Gln Tyr
1 5 10
<210> 85
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 85
Ala Ser Ser Thr Arg Phe Ala Gln Tyr
1 5
<210> 86
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 86
Ala Thr Ser Arg Asp Gln Gly Tyr Asp Thr Gly Glu Leu Phe
1 5 10
<210> 87
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 87
Ser Val Glu Leu Ala Gly Glu His Glu Gln Tyr
1 5 10
<210> 88
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 88
Pro Ala Val Thr Gly Pro Val Ser Ala Ser Ser Thr
1 5 10
<210> 89
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 89
Ala Ser Gln Thr Thr Ser Ser Thr Asp Thr Gln Tyr
1 5 10
<210> 90
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 90
Ala Ser Ser Pro Ala Thr Gly Glu Leu Asp Thr Gln Tyr
1 5 10
<210> 91
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 91
Ala Trp Ser Pro Pro Leu Val Gly Thr Leu Glu Ala Phe
1 5 10
<210> 92
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 92
Ala Ser Ala Arg Asp Arg Asp Gly Glu Leu Phe
1 5 10
<210> 93
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 93
Ala Ser Ser Gly Arg Thr Gly Thr Ala Gly Glu Leu Phe
1 5 10
<210> 94
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 94
Ala Ser Ser Leu Ala Gln Pro Leu Asn Thr Glu Ala Phe
1 5 10
<210> 95
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 95
Ala Ser Ser Pro Gly Glu Arg Ile Ser Gly Asn Thr Ile Tyr
1 5 10
<210> 96
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 96
Ala Ser Ser Tyr Arg Glu Asp Gln Ile Asn Glu Gln Phe
1 5 10
<210> 97
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 97
Ala Ser Thr Ser Arg Thr Ser Thr Asn Asn Glu Gln Phe
1 5 10
<210> 98
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 98
Ala Ser Arg Arg Thr Thr Val Asp Tyr Gly Tyr Thr
1 5 10
<210> 99
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 99
Ser Ala Phe Asp Ala Ser Gly Tyr Asn Glu Gln Phe
1 5 10
<210> 100
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 100
Ala Ser Arg His Arg Asp Ala Phe Thr Asp Thr Gln Tyr
1 5 10
Claims (7)
1.一种黑色素瘤的外周血TCR标志物，其特征在于，所述标志物包括序列为SEQ IDNO.1～100所示的蛋白中的至少一种。
2.根据权利要求1所述的黑色素瘤的外周血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。
3.权利要求1所述的标志物在制备治疗黑色素瘤的制剂中的应用。
4.根据权利要求3所述的应用，其特征在于，所述制剂包括含有该标志物的T细胞受体，或能表达产生该标志物的T细胞受体的质粒、病毒载体或核酸片段。
5.一种用于黑色素瘤检测的试剂盒，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。
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。
7.一种检测黑色素瘤的蛋白质芯片，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。
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| PCT/CN2021/101697 WO2022012284A1 (zh) | 2020-07-11 | 2021-06-23 | 一种黑色素瘤的外周血tcr标志物及其检测试剂盒和应用 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113567682A (zh) * | 2021-07-23 | 2021-10-29 | 成都益安博生物技术有限公司 | 一种阿尔茨海默病的外周血tcr标志物及其检测试剂盒和应用 |
| WO2022012284A1 (zh) * | 2020-07-11 | 2022-01-20 | 成都益安博生物技术有限公司 | 一种黑色素瘤的外周血tcr标志物及其检测试剂盒和应用 |
| WO2022194033A1 (zh) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | 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 |
| WO2022194036A1 (zh) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | 一种经典霍奇金淋巴瘤的外周血tcr标志物及其检测试剂盒和应用 |
| WO2022194039A1 (zh) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | 一种急性b淋巴细胞白血病的外周血tcr标志物及其检测试剂盒和应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022194033A1 (zh) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | 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 |
| WO2022194036A1 (zh) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | 一种经典霍奇金淋巴瘤的外周血tcr标志物及其检测试剂盒和应用 |
| WO2022194039A1 (zh) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | 一种急性b淋巴细胞白血病的外周血tcr标志物及其检测试剂盒和应用 |
| CN113567682A (zh) * | 2021-07-23 | 2021-10-29 | 成都益安博生物技术有限公司 | 一种阿尔茨海默病的外周血tcr标志物及其检测试剂盒和应用 |
| WO2023000688A1 (zh) * | 2021-07-23 | 2023-01-26 | 成都益安博生物技术有限公司 | 一种阿尔茨海默病的外周血tcr标志物及其检测试剂盒和应用 |
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