CN113621617B - cDNA、mRNA、蛋白及评价胶质瘤预后性的试剂盒和系统 - Google Patents

cDNA、mRNA、蛋白及评价胶质瘤预后性的试剂盒和系统 Download PDF

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CN113621617B
CN113621617B CN202010318367.3A CN202010318367A CN113621617B CN 113621617 B CN113621617 B CN 113621617B CN 202010318367 A CN202010318367 A CN 202010318367A CN 113621617 B CN113621617 B CN 113621617B
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江涛
赵征
柴睿超
陈婧
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Beijing Rencheng Neurotumor Biotechnology Engineering Research Center Co ltd
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Abstract

本公开涉及一种cDNA、mRNA、蛋白及评价胶质瘤预后性的试剂盒和系统，其中，该cDNA的序列中含有SEQ ID NO:1的片段，或者，该cDNA的序列中含有SEQ ID NO:1的片段的互补片段；其中，所述SEQ ID NO:1的片段至少含有SEQ ID NO:1的第2635‑2652位、第2623‑2640位或者第2627‑2645位的序列。通过上述技术方案，本公开有效地提高了胶质瘤预后性评价的准确性。

Description

cDNA、mRNA、蛋白及评价胶质瘤预后性的试剂盒和系统
技术领域
本公开涉及生物技术领域，具体地，涉及一种与胶质瘤预后性相关的cDNA、一种由该cDNA转录得到的mRNA、一种由该mRNA编码得到的蛋白、一种用于评价胶质瘤预后性的试剂盒以及一种用于评价胶质瘤预后性的系统。
背景技术
胶质瘤是源自神经上皮的肿瘤的统称，占颅脑肿瘤的40％-50％，是最常见的原发性颅内肿瘤。胶质瘤难以根治，手术后往往会复发，而且预后性不佳。世界卫生组织分级系统根据胶质瘤的组织学特征将胶质瘤分为Ⅰ-Ⅳ级，不同级别胶质瘤的预后性各不相同。
准确评价胶质瘤患者的预后性具有重要的临床、科研及社会价值。在临床工作中，准确的预后性评估可指导医生针对高风险患者制定个性化的检查及治疗方案，帮助医生制定合理的复查及随访计划，进而提高医疗服务的质量。在科研中，准确评估患者预后性的风险层级，可以为研发针对高风险病人的有效治疗方案提供重要依据，并且可以成为检验新型治疗效果的重要参考。从社会角度来说，准确评估患者预后性，可为病患及家属提供科学的生存预期，指引病人依从治疗计划，避免过度医疗，减轻家庭经济压力，有助于改善医患关系。
目前，主要依据胶质瘤的病理级别进行胶质瘤的预后性评价，但是同一病理级别的不同胶质瘤患者的预后性也会存在较大差异，仅依据胶质瘤病理级别进行胶质瘤的预后性评价仍然不够准确。
发明内容
本公开的目的是提供一种与胶质瘤预后性相关的cDNA、一种由该cDNA转录得到的mRNA、一种由该mRNA编码得到的蛋白、一种用于评价胶质瘤预后性的试剂盒以及一种用于评价胶质瘤预后性的系统，能够进一步提高胶质瘤预后性评价的准确性。
第一方面，本公开提供一种cDNA，该cDNA的序列中含有SEQ ID NO:1的片段，或者，该cDNA的序列中含有SEQ ID NO:1的片段的互补片段；其中，所述SEQ ID NO:1的片段至少含有SEQ ID NO:1的第2635-2652位、第2623-2640位或者第2627-2645位的序列；
优选地，所述SEQ ID NO:1的片段至少含有SEQ ID NO:1的第2588-2687位的序列。
可选地，所述cDNA与人MET基因相对应，且所述cDNA的序列中不含有人MET基因的19号外显子。
可选地，所述cDNA为单链DNA，且所述cDNA的序列为SEQ ID NO:1所示的序列，或者所述cDNA的序列为SEQ ID NO:1所示的序列的互补序列。
可选地，所述cDNA为双链DNA，且所述cDNA的其中一条单链的序列为SEQ ID NO:1所示的序列，另一条单链的序列为SEQ ID NO:1所示的序列的互补序列。
第二方面，本公开提供一种mRNA，该mRNA是第一方面中任意一项所述的cDNA的逆转录模板。
可选地，所述mRNA的序列中含有SEQ ID NO:2的片段，所述SEQ ID NO:2的片段至少含有SEQ ID NO:2的第3820-3837位、第3832-3849位或者第3827-3845位的序列；
优选地，所述SEQ ID NO:2的片段至少含有SEQ ID NO:2的第3785-3884位的序列。
可选地，所述mRNA的序列为SEQ ID NO:2所示的序列。
第三方面，本公开提供一种蛋白，该蛋白由第二方面中任意一项所述的mRNA编码。
可选地，所述蛋白的氨基酸序列中含有SEQ ID NO:3的片段，所述SEQ ID NO:3的片段至少含有SEQ ID NO:3的第1207-1220位的序列；
优选地，所述SEQ ID NO:3的片段至少含有SEQ ID NO:3的第1175-1220位的序列。
可选地，所述蛋白的氨基酸序列为SEQ ID NO:3所示的序列。
第四方面，本公开提供一种评价胶质瘤预后性的试剂盒，该试剂盒包括能够特异性地扩增第一方面中任意一项所述的cDNA的第一引物对，和/或，能够与第一方面中任意一项所述的cDNA特异性杂交的第一探针；和/或，
该试剂盒包括能够特异性地扩增第二方面中任意一项所述的mRNA的第二引物对，和/或，能够与第二方面中任意一项所述的mRNA特异性杂交的第二探针；和/或，
该试剂盒包括抗第三方面中任意一项所述的蛋白的抗体以及第三方面中任意一项所述的蛋白。
可选地，所述第一引物对含有如SEQ ID NO:4所示的第一引物和如SEQ ID NO:5所示的第二引物，所述第一探针的序列中含有如SEQ ID NO:6所示的序列；
所述第二引物对含有如SEQ ID NO:7所示的第三引物和如SEQ ID NO:8所示的第四引物，所述第二探针的序列中含有如SEQ ID NO:9所示的序列。
第五方面，本公开提供一种分子试剂在制备评价胶质瘤预后性的试剂盒中的用途，所述分子试剂包括如下(1)-(8)中的至少一种：
(1)第一方面中任意一项所述的cDNA；
(2)能够特异性地扩增第一方面中任意一项所述的cDNA的第一引物对；
(3)能够与第一方面中任意一项所述的cDNA特异性杂交的第一探针；
(4)第二方面中任意一项所述的mRNA；
(5)能够特异性地扩增第二方面中任意一项所述的mRNA的第二引物对；
(6)能够与第二方面中任意一项所述的mRNA特异性杂交的第二探针；
(7)第三方面中任意一项所述的蛋白；
(8)抗第三方面中任意一项所述的蛋白的抗体。
第六方面，本公开提供一种评价胶质瘤预后性的系统，该系统包括扩增装置、测序装置、计算装置和输出装置；
所述扩增装置含有采集单元和扩增单元，所述采集单元用于采集模板核酸片段和扩增引物，所述扩增单元用于利用所述扩增引物对所述模板核酸片段进行扩增，得到扩增产物；
所述测序装置用于对所述扩增产物进行核酸序列测序，得到扩增产物序列；
所述计算装置包括存储器和处理器，所述存储器中存储有计算机程序，所述处理器被配置为执行所述存储器中存储的计算机程序，以实现如下判别：
若所述扩增产物序列中含有如第一方面中任意一项所述的cDNA的序列和/或如第二方面中任意一项所述的mRNA的序列，则判定所述模板核酸片段对应的胶质瘤预后性较差；
所述输出装置用于输出所述计算装置的判定结果。
第七方面，本公开提供一种评价胶质瘤预后性的方法，该方法包括检测待测胶质瘤样本中是否含有如上所述的cDNA和/或mRNA和/或蛋白的步骤，如果待测胶质瘤样本中含有如上所述的cDNA和/或mRNA和/或蛋白，则指示该待测胶质瘤样本对应的胶质瘤患者的预后性更差。
通过上述技术方案，本公开有效地提高了胶质瘤预后性评价的准确性。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本公开的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本公开，但并不构成对本公开的限制。在附图中：
图1是本公开实施例提供的cDNA琼脂糖凝胶核酸电泳图；
图2是本公开实施例提供的cDNASanger测序结果图；
图3是本公开实施例提供的全级别胶质瘤样本患者生存曲线图；
图4是本公开实施例提供的IDH突变的全级别胶质瘤样本患者生存曲线图；
图5是本公开实施例提供的继发胶质母细胞瘤样本患者生存曲线图；
图6是本公开实施例提供的IDH突变的继发胶质母细胞瘤样本患者生存曲线图。
具体实施方式
以下结合附图对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
本公开的第一方面提供一种cDNA，该cDNA的序列中含有SEQ ID NO:1的片段，或者，该cDNA的序列中含有SEQ ID NO:1的片段的互补片段；其中，所述SEQ ID NO:1的片段至少含有SEQ ID NO:1的第2635-2652位、第2623-2640位或者第2627-2645位的序列；优选地，所述SEQ ID NO:1的片段至少含有SEQ ID NO:1的第2588-2687位的序列。
优选地，所述cDNA与人MET基因相对应，且所述cDNA的序列中不含有人MET基因的19号外显子。
根据本公开，所述cDNA可以为单链DNA，且所述cDNA的序列可以为SEQ ID NO:1所示的序列，或者所述cDNA的序列可以为SEQ ID NO:1所示的序列的互补序列。
根据本公开，所述cDNA可以为双链DNA，且所述cDNA的其中一条单链的序列可以为SEQ ID NO:1所示的序列，另一条单链的序列可以为SEQ ID NO:1所示的序列的互补序列。
上述cDNA是19号外显子缺失的人MET基因对应的cDNA，本公开的发明人发现，当胶质瘤细胞的mRNA可逆转录出上述cDNA时，胶质瘤的恶性程度更高，抗药性更强，患者的预后性更差。其中，所述SEQ ID NO:1的片段中含有上述19号外显子的缺失位点，SEQ ID NO:1所示的序列中含有人MET基因的除19号外显子外的所有外显子，SEQ ID NO:1所示的序列如下所示：
tacaagatgttgcatcactttactttaattgcatgatttatcagaacaactattaacatacgaagtaccattcagttcagctgcaggtataggcagtgacaagtatctaattcttagaagaatcacttactcccacaatctgtccagacacattaatctaaggacaagtttataaatagcaaacgtgattttcacattgcagtgttctcaagaatgtatatacaagtgtgtagtcctgttgatgggatgtttccccgagttctttctattgatgcgttcatgctcttgaccctggtagagacagttctttctttccacagagcagattttcttttgtcatccaccatttacaatactctgtgaattagtattacttattacatttatgaaacggcaatatttggattgtaatagctcttcagtacaattccttgtgtcttctggtaagtctctgcactatcagagagcttttagttataatcattttcctgcaacaacagccaaactcaactattgagtttcagtgtgacacaccctctttgtagcttgctgggttaacccttggcttcaagtcctgatgatgtaatgagggtggggtgtattggcaatcagtacatttccttatcgcaatttacagtcattgaaaatcatgctgtcattaatcccagtctgacataccttttctaaaatgttcacagtgcagtgtttttgtggcctaacaaaatttttctcatatcattaaaaataaacatttttataaaaaatataacactttaaatgtttacgtcgacaaaaccagttagagtaacctacaccacatgcactatacagtagcaagcacaaaattccacagaatgaagcatcacaaagttctgctcagggtggctattccatctaggtgaaatagctgggattttcaattgcctttttcatttgtttctaaagtatgttttgcttaacataaaacacaccctaatgcaaaataaaactccccaaaagttttgtttccaattgcttgcgaggtgggaacctgccaccgagacagaggctaatcttttcaatccatccaccctttctttgctctacctatgagctgtgattggaaccaatgaaccttttagtaaaatgtatcctgctttacaaacatgctgagttatctttaaaaatatttatcaacaaattacttgtcttattttgagttttcatttaaaaaaatacacacaaaacatctacatgttcacattcattagatcagagtagcatcattctcaaacagtgggtttttatatgacaactaaatatttcatgatgactaaattatttcatacaaattttcttaatgtcatctatgtacattacatcctgttcatttataaacatgttttgtgtgatgctattttgaaactttgactgtattgcctataaatattcttccaaccaaaggaatgtctatacaaaaatttataagggctgggcgcagtggctcatgcctgtaatcctagcactttgggaggctgaggtgggtggatttcttgagtccaggagtttgaaaccagccttggcaacatggcaaaaccccgtctctacaaaaaatacaaaaattagccggggatggtggtgtgcgcctgtagtcccagctattcgggaggcttgagcctgggaggtggaggttgcagtgagctatgatcacaccactgcactacagccctggcaacagagtgagatcctgtctcaaaaaacaaacaaacatttaaaagggaatgtttacctaatgggtgaatgacaccatcagtaatgaaaaatgttattgaccatacaacacacaaaagtgttttgaaacactagaattctaaatgagtggcctgttctggggctgccgctcctgtcctgagcattactatctcttctgagttttctgtgatcaagaagccctcaatatttcctgcttcaatttcccatatgaaatcaagtggtacctgatttttaaaaaattcaacccagaattcaagttttggactccaatatgacaggagtgttgtcacggctgcaggccattggtccgtggcctgtgggaccaagcctctggttctgatgctctgtcagataagaaattccttagaatccagtaatttaaataacaatacaagtcctataatagtgcaattttggcaagagcaaagaatatcgatggccttttaaaggtcaggcagtgaaaaaaccattggacaaagtgtggactgttgctttgacatagtactagcactatgatgtctcccagaaggaggctggtcgtgtgtccacctcatcatcagcgttatcttctgatgacaacagagaaggatacggagcgacacattttacgttcacataagtagcgttcacatggacatagtgctccccaatgaaagtagagaagatcgctgatatccgggacaccagttcagaaaaggatgggcgcatttcggctttagggtgccagcattttagcattacttcatataaggggtctgggcagtattcgggttgtaggagtcttctcccttgcaacaagtaaacagttatatcaaaggtgtttacgtcaggataaggtggggctcctcttgtcatcagctcccagaggagcacgccaaaggaccaatacagtttcttgcagccaagtctctgtggacaaactttttgcttgcaagatatttcatgcctttggctacttgaagaccaaagccaataagatcttttacagttggattatgagtctcatttcgaatgaaatttcgaagatctccatgtttcatgtatggtaggaccaccagcggagacccttcacttcgcaggcagattcccaggagcgagaggacattgggatgactaaaatctttcatgatgattccctcggtcagaaattgggaaacttctcctatgtcagtgattctgttcaaggatttcacagcacagtgaattttcttgccatcattgtccaacaaagtcccatgatatacacaaccaaaatgccctcttcctatgacttcattgaaatgcacaatcaggctactgggcccaatcactacatgctgcactgcctggaccagctctggatttagagcactgaggtcaatgtggacagtattttgcagtaatggactggatatatcagagtccccactagttaggatgggggacatgtctgtcagaggatactgcacttgtcggcatgaaccgttctgagatgaattaggaaactgatcttctggaaaagtagctcggtagtctacagattcatttgaaaccatttctgtagttgggcttacacttcgggcacttacaagcctatccaaatgaggagtgtgtactcttgcatcgtagcgaactaattcactgcccagatctttaatttgctttctctttttcagccacaggaaaaacccaagtagtaataacagtgctgttgatattgagacaacaccagcaatcaatcctgtgaaattctgatctggttgaactattacttttccaaggacggttgaagaaattgcttgcttccactctatatttagctcgctgttcaatttcagcaggtcattggggaccgtgcataaaacggcttcagaatgtaagtgtatattctcacagctcttatttccaacttttaacacttcacctttaactgcttcagggtcaatatcatttcccttaatttccagtacattttcattgcccattgagatcatcactggcttttcaaaaggcttaaacacaggattatgtacataaatgagatcaaagtatttggaaaggatcccatctaacatgaaaaaggctttggttttcagggggagttgcagattcagctgttgcagggaaggagtggtacaacagattatctctgaattagagcgatgttgacatgccactgtaaagttccttcctgcttcatgcacatttatgaccattctcgggacactaactgaattcaggtttttcccaacacctgttattgtgctcccaccactaataaaagatttggttggatgaatttcatagacaatgggatcttcacggtaactgaagatgcttgtctctcggttggctaagtcaattttcaatttaacagcaaactcagttgaaatggtttgggctggggtataacattcaagaatactgtttgacacactttttaaagtacatgtttttccaccaattgaaatgtgtctagaattcccactgtttaggtaatttccagttaaagtaagtaaagtgccaccagccataggaccgtatttcggcgaaatacttgttattacaggatccacataggagaatgtactgtattgtgttgtcccgtggccatttgaaataattatggacatattgaaatgcttattcatggcaggaccaactgtgcatttcaatgtattcatcgtgctctcacttaaagtcaaggtgcagctctcatttccaaggagaactctagttttctttaaatcaaatttattattcctccgaaatccaaagtcccagccacatatggtcagccttgtccctccttcaaggggtgcactatttgggaaaaccttgtagattgcaggcagacagatctgttgagtccatgtcccgctcaggcattcctccgatcgcacacatttgtcgtggcaccagccacactgaacaaagggtggggcagagaggcattgactgcaggactggaaatgtctgcagcccaagccattcaatgggatcttcgtgatcttcttcccagtgataaccagtgtgtagccattttggtttaatgtatgctccacaatcacttctggagacactggatgggagtccaggagaaaattcacatgaggggttgatggtcctgatcgagaaaccacaacctgcatgaagcgaccctctgatgtcccaagattagctatggtgaggtctcctttaatgaaggtggatatagatgttaagaggacttcgctgaattgacccatgaataagtcaacgcgctgcaaagctgtggtaaactctgttcgatattcatcacggcgcgcttcacagcctgatgaatttctcagaagtgtcctattaaagcagtgctcatgattgggtccgtaaaaatgctggagacatctcacattgtttttgttgacgatcttgttgaagaagtcgttgacatatttgatagggaatgcacacatggcagatcgatccattggttcggcagaatctggcttgctttgtgcgaacaccccgaaaagaatgtcatcattcaggctggctcctatttgtctagcaagctgggccccaggcttgctgacatacgcagcctgaagtatattaaacacttccttctttgtggatctcttttttctcttttctgtgagaatacactccagaggcatttccatgtaggaatgcaatccagagtttatggaacagaacctgattattcttgtgtgaaaagtctgagcatctagagtttccctttggaccgtcaagaagtaaataaaattgttgctttcaaaggcatggacatacttaatggggtaagaatctctgaactcaggtaaaacatcaatgtaggactggtccgtcaaaaacataaaaccatctttcgtttcctttagccttctcactgatatcgaatgcaatggatgatctgggaaataagaagaatttatggtattgcctacaaagaagttgatgaaccggtcctttacagatgaaaggactttggctcccagggcgctcaccacacagtcaggacactggctgggctcttctatctgtggggagaatatgcagtgaacctccgactgtatgtcagcagtatgattgtggggaaagacatgtcgctggcaggtccctctgttgacgctgccacagctaatgagttgatcatcatagtaggtgtcgacaactagagccatgttgatgttatctttccaaacacctcctgataaattggctttgctgctgcagtcctgacatgggaaacaatctgggtgttccagcacaggcccagtcttgtactcagcaaccttctgaaggtcttcctcatttaaaacataaatgtagttagtggcaccaaggaaaatgtgatgctcatgtagaatgacattctggatgggtgtttccgcggtgaagttgggaagctgatacttcatattcacattcatctcggactttgctagtgcctctttacactccccattgctcctctgcaccaaggtaaacaggagcacgaggatgccaggtgcaagcacagcgggggccttcattatgagaggtttatctttcggtgcccaggaaccagtggagaagtcagcggcgcaaggaccacacgcgcgctccgcgcctccccgcctcctctcagcaagtcagctgtcgccccgcatctggctcgcgccctccactcggctccgcatctgctcacaaagcgctcggggcgccgcgggcggcgagggcctccgggtcacctgc。
其中，SEQ ID NO:1的第2635-2652位的序列为ccaatacagtttcttgca；SEQ ID NO:1的第2623-2640位的序列为cacgccaaaggaccaata；SEQ ID NO:1的第2627-2645位的序列为ccaaaggaccaatacagtt。
本公开的第二方面提供一种mRNA，该mRNA是第一方面中任意一项所述的cDNA的逆转录模板。
优选地，所述mRNA的序列中含有SEQ ID NO:2的片段，所述SEQ ID NO:2的片段至少含有SEQ ID NO:2的第3820-3837位、第3832-3849位或者第3827-3845位的序列；优选地，所述SEQ ID NO:2的片段至少含有SEQ ID NO:2的第3785-3884位的序列。
优选地，所述mRNA的序列为SEQ ID NO:2所示的序列。
其中，SEQ ID NO:2所示的序列为与SEQ ID NO:1所示的序列互补的RNA序列，SEQID NO:2所示的序列如下所示：
gcaggugacccggaggcccucgccgcccgcggcgccccgagcgcuuugugagcagaugcggagccgaguggagggcgcgagccagaugcggggcgacagcugacuugcugagaggaggcggggaggcgcggagcgcgcgugugguccuugcgccgcugacuucuccacugguuccugggcaccgaaagauaaaccucucauaaugaaggcccccgcugugcuugcaccuggcauccucgugcuccuguuuaccuuggugcagaggagcaauggggaguguaaagaggcacuagcaaaguccgagaugaaugugaauaugaaguaucagcuucccaacuucaccgcggaaacacccauccagaaugucauucuacaugagcaucacauuuuccuuggugccacuaacuacauuuauguuuuaaaugaggaagaccuucagaagguugcugaguacaagacugggccugugcuggaacacccagauuguuucccaugucaggacugcagcagcaaagccaauuuaucaggagguguuuggaaagauaacaucaacauggcucuaguugucgacaccuacuaugaugaucaacucauuagcuguggcagcgucaacagagggaccugccagcgacaugucuuuccccacaaucauacugcugacauacagucggagguucacugcauauucuccccacagauagaagagcccagccaguguccugacuguguggugagcgcccugggagccaaaguccuuucaucuguaaaggaccgguucaucaacuucuuuguaggcaauaccauaaauucuucuuauuucccagaucauccauugcauucgauaucagugagaaggcuaaaggaaacgaaagaugguuuuauguuuuugacggaccaguccuacauugauguuuuaccugaguucagagauucuuaccccauuaaguauguccaugccuuugaaagcaacaauuuuauuuacuucuugacgguccaaagggaaacucuagaugcucagacuuuucacacaagaauaaucagguucuguuccauaaacucuggauugcauuccuacauggaaaugccucuggaguguauucucacagaaaagagaaaaaagagauccacaaagaaggaaguguuuaauauacuucaggcugcguaugucagcaagccuggggcccagcuugcuagacaaauaggagccagccugaaugaugacauucuuuucgggguguucgcacaaagcaagccagauucugccgaaccaauggaucgaucugccaugugugcauucccuaucaaauaugucaacgacuucuucaacaagaucgucaacaaaaacaaugugagaugucuccagcauuuuuacggacccaaucaugagcacugcuuuaauaggacacuucugagaaauucaucaggcugugaagcgcgccgugaugaauaucgaacagaguuuaccacagcuuugcagcgcguugacuuauucaugggucaauucagcgaaguccucuuaacaucuauauccaccuucauuaaaggagaccucaccauagcuaaucuugggacaucagagggucgcuucaugcagguugugguuucucgaucaggaccaucaaccccucaugugaauuuucuccuggacucccauccagugucuccagaagugauuguggagcauacauuaaaccaaaauggcuacacacugguuaucacugggaagaagaucacgaagaucccauugaauggcuugggcugcagacauuuccaguccugcagucaaugccucucugccccacccuuuguucaguguggcuggugccacgacaaaugugugcgaucggaggaaugccugagcgggacauggacucaacagaucugucugccugcaaucuacaagguuuucccaaauagugcaccccuugaaggagggacaaggcugaccauauguggcugggacuuuggauuucggaggaauaauaaauuugauuuaaagaaaacuagaguucuccuuggaaaugagagcugcaccuugacuuuaagugagagcacgaugaauacauugaaaugcacaguugguccugccaugaauaagcauuucaauauguccauaauuauuucaaauggccacgggacaacacaauacaguacauucuccuauguggauccuguaauaacaaguauuucgccgaaauacgguccuauggcugguggcacuuuacuuacuuuaacuggaaauuaccuaaacagugggaauucuagacacauuucaauugguggaaaaacauguacuuuaaaaagugugucaaacaguauucuugaauguuauaccccagcccaaaccauuucaacugaguuugcuguuaaauugaaaauugacuuagccaaccgagagacaagcaucuucaguuaccgugaagaucccauugucuaugaaauucauccaaccaaaucuuuuauuaguggugggagcacaauaacagguguugggaaaaaccugaauucaguuagugucccgagaauggucauaaaugugcaugaagcaggaaggaacuuuacaguggcaugucaacaucgcucuaauucagagauaaucuguuguaccacuccuucccugcaacagcugaaucugcaacucccccugaaaaccaaagccuuuuucauguuagaugggauccuuuccaaauacuuugaucucauuuauguacauaauccuguguuuaagccuuuugaaaagccagugaugaucucaaugggcaaugaaaauguacuggaaauuaagggaaaugauauugacccugaagcaguuaaaggugaaguguuaaaaguuggaaauaagagcugugagaauauacacuuacauucugaagccguuuuaugcacgguccccaaugaccugcugaaauugaacagcgagcuaaauauagaguggaagcaagcaauuucuucaaccguccuuggaaaaguaauaguucaaccagaucagaauuucacaggauugauugcugguguugucucaauaucaacagcacuguuauuacuacuuggguuuuuccuguggcugaaaaagagaaagcaaauuaaagaucugggcagugaauuaguucgcuacgaugcaagaguacacacuccucauuuggauaggcuuguaagugcccgaaguguaagcccaacuacagaaaugguuucaaaugaaucuguagacuaccgagcuacuuuuccagaagaucaguuuccuaauucaucucagaacgguucaugccgacaagugcaguauccucugacagacaugucccccauccuaacuaguggggacucugauauauccaguccauuacugcaaaauacuguccacauugaccucagugcucuaaauccagagcugguccaggcagugcagcauguagugauugggcccaguagccugauugugcauuucaaugaagucauaggaagagggcauuuugguuguguauaucaugggacuuuguuggacaaugauggcaagaaaauucacugugcugugaaauccuugaacagaaucacugacauaggagaaguuucccaauuucugaccgagggaaucaucaugaaagauuuuagucaucccaauguccucucgcuccugggaaucugccugcgaagugaagggucuccgcuggugguccuaccauacaugaaacauggagaucuucgaaauuucauucgaaaugagacucauaauccaacuguaaaagaucuuauuggcuuuggucuucaaguagccaaaggcaugaaauaucuugcaagcaaaaaguuuguccacagagacuuggcugcaagaaacuguauugguccuuuggcgugcuccucugggagcugaugacaagaggagccccaccuuauccugacguaaacaccuuugauauaacuguuuacuuguugcaagggagaagacuccuacaacccgaauacugcccagaccccuuauaugaaguaaugcuaaaaugcuggcacccuaaagccgaaaugcgcccauccuuuucugaacuggugucccggauaucagcgaucuucucuacuuucauuggggagcacuauguccaugugaacgcuacuuaugugaacguaaaaugugucgcuccguauccuucucuguugucaucagaagauaacgcugaugaugagguggacacacgaccagccuccuucugggagacaucauagugcuaguacuaugucaaagcaacaguccacacuuuguccaaugguuuuuucacugccugaccuuuaaaaggccaucgauauucuuugcucuugccaaaauugcacuauuauaggacuuguauuguuauuuaaauuacuggauucuaaggaauuucuuaucugacagagcaucagaaccagaggcuuggucccacaggccacggaccaauggccugcagccgugacaacacuccugucauauuggaguccaaaacuugaauucuggguugaauuuuuuaaaaaucagguaccacuugauuucauaugggaaauugaagcaggaaauauugagggcuucuugaucacagaaaacucagaagagauaguaaugcucaggacaggagcggcagccccagaacaggccacucauuuagaauucuaguguuucaaaacacuuuuguguguuguauggucaauaacauuuuucauuacugauggugucauucacccauuagguaaacauucccuuuuaaauguuuguuuguuuuuugagacaggaucucacucuguugccagggcuguagugcaguggugugaucauagcucacugcaaccuccaccucccaggcucaagccucccgaauagcugggacuacaggcgcacaccaccauccccggcuaauuuuuguauuuuuuguagagacgggguuuugccauguugccaaggcugguuucaaacuccuggacucaagaaauccacccaccucagccucccaaagugcuaggauuacaggcaugagccacugcgcccagcccuuauaaauuuuuguauagacauuccuuugguuggaagaauauuuauaggcaauacagucaaaguuucaaaauagcaucacacaaaacauguuuauaaaugaacaggauguaauguacauagaugacauuaagaaaauuuguaugaaauaauuuagucaucaugaaauauuuaguugucauauaaaaacccacuguuugagaaugaugcuacucugaucuaaugaaugugaacauguagauguuuuguguguauuuuuuuaaaugaaaacucaaaauaagacaaguaauuuguugauaaauauuuuuaaagauaacucagcauguuuguaaagcaggauacauuuuacuaaaagguucauugguuccaaucacagcucauagguagagcaaagaaaggguggauggauugaaaagauuagccucugucucgguggcagguucccaccucgcaagcaauuggaaacaaaacuuuuggggaguuuuauuuugcauuaggguguguuuuauguuaagcaaaacauacuuuagaaacaaaugaaaaaggcaauugaaaaucccagcuauuucaccuagauggaauagccacccugagcagaacuuugugaugcuucauucuguggaauuuugugcuugcuacuguauagugcaugugguguagguuacucuaacugguuuugucgacguaaacauuuaaaguguuauauuuuuuauaaaaauguuuauuuuuaaugauaugagaaaaauuuuguuaggccacaaaaacacugcacugugaacauuuuagaaaagguaugucagacugggauuaaugacagcaugauuuucaaugacuguaaauugcgauaaggaaauguacugauugccaauacaccccacccucauuacaucaucaggacuugaagccaaggguuaacccagcaagcuacaaagagggugugucacacugaaacucaauaguugaguuuggcuguuguugcaggaaaaugauuauaacuaaaagcucucugauagugcagagacuuaccagaagacacaaggaauuguacugaagagcuauuacaauccaaauauugccguuucauaaauguaauaaguaauacuaauucacagaguauuguaaaugguggaugacaaaagaaaaucugcucuguggaaagaaagaacugucucuaccagggucaagagcaugaacgcaucaauagaaagaacucggggaaacaucccaucaacaggacuacacacuuguauauacauucuugagaacacugcaaugugaaaaucacguuugcuauuuauaaacuuguccuuagauuaaugugucuggacagauugugggaguaagugauucuucuaagaauuagauacuugucacugccuauaccugcagcugaacugaaugguacuucguauguuaauaguuguucugauaaaucaugcaauuaaaguaaagugaugcaacaucuugua。
其中，SEQ ID NO:2的第3820-3837位的序列为ugcaagaaacuguauugg；SEQ ID NO:2的第3832-3849位的序列为uauugguccuuuggcgug；SEQ ID NO:2的第3827-3845位的序列为aacuguauugguccuuugg。
本公开的第三方面提供一种蛋白，该蛋白由第二方面中任意一项所述的mRNA编码。
优选地，所述蛋白的氨基酸序列中含有SEQ ID NO:3的片段，所述SEQ ID NO:3的片段至少含有SEQ ID NO:3的第1207-1220位的序列；
优选地，所述SEQ ID NO:3的片段至少含有SEQ ID NO:3的第1175-1220位的序列。
优选地，所述蛋白的氨基酸序列为SEQ ID NO:3所示的序列。
其中，SEQ ID NO:3所示的序列为由SEQ ID NO:2所示的RNA序列编码的氨基酸序列，SEQ ID NO:3所示的氨基酸序列如下所示：
MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCIGPLACSSGS。
其中，SEQ ID NO:3的第1207-1220位的序列为ARNCIGPLACSSGS。
SEQ ID NO:3的第1175-1220位的序列为NPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCIGPLACSSGS。
本公开的第四方面提供一种评价胶质瘤预后性的试剂盒，该试剂盒包括能够特异性地扩增第一方面中任意一项所述的cDNA的第一引物对，和/或，能够与第一方面中任意一项所述的cDNA特异性杂交的第一探针；和/或，该试剂盒包括能够特异性地扩增第二方面中任意一项所述的mRNA的第二引物对，和/或，能够与第二方面中任意一项所述的mRNA特异性杂交的第二探针；和/或，该试剂盒包括抗第三方面中任意一项所述的蛋白的抗体以及第三方面中任意一项所述的蛋白。
其中，所述第一引物对只要能特异性地扩增如第一方面任意一项所述的cDNA即可，优选地，所述第一引物对含有如SEQ ID NO:4所示的第一引物和SEQ ID NO:5所示的第二引物。第一探针只要能与如第一方面任意一项所述的cDNA特异性杂交即可，优选地，所述第一探针的序列中含有如SEQ ID NO:6所示的序列。所述第二引物对只要能特异性地扩增如第二方面中任意一项所述的mRNA即可，优选地，所述第二引物对含有SEQ ID NO:7所示的第三引物和如SEQ ID NO:8所示的第四引物。第二探针只要能与如第二方面中任意一项所述的mRNA特异性杂交即可，优选地，所述第二探针的序列中含有如SEQ ID NO:9所示的序列。
其中，SEQ ID NO:4所示的序列为：aatttctgaccgagggaatcat；SEQ ID NO:5所示的序列为：acgccaaaggaccaatacagtttc；SEQ ID NO:6所示的序列为：gccaaaggaccaatacagtttctt；SEQ ID NO:7所示的序列为：taatacgactcactataggggagcacgccaaaggaccaataca；SEQ ID NO:8所示的序列为：tgaaatatcttgcaagcaaaaagtt；SEQ ID NO:9所示的序列为：agcacgccaaaggaccaatacagt。
其中，抗第三方面任意一项所述的蛋白的抗体可以为单克隆抗体和/或多克隆抗体。
本公开的第五方面提供一种分子试剂在制备评价胶质瘤预后性的试剂盒中的用途，所述分子试剂包括如下(1)-(8)中的至少一种：
(1)第一方面中任意一项所述的cDNA；(2)能够特异性地扩增第一方面中任意一项所述的cDNA的第一引物对；(3)能够与第一方面中任意一项所述的cDNA特异性杂交的第一探针；(4)第二方面中任意一项所述的mRNA；(5)能够特异性地扩增第二方面中任意一项所述的mRNA的第二引物对；(6)能够与第二方面中任意一项所述的mRNA特异性杂交的第二探针；(7)第三方面中任意一项所述的蛋白；(8)抗第三方面中任意一项所述的蛋白的抗体。
本公开的第六方面提供一种评价胶质瘤预后性的系统，该系统包括扩增装置、测序装置、计算装置和输出装置；所述扩增装置含有采集单元和扩增单元，所述采集单元用于采集模板核酸片段和扩增引物，所述扩增单元用于利用所述扩增引物对所述模板核酸片段进行扩增，得到扩增产物；所述测序装置用于对所述扩增产物进行核酸序列测序，得到扩增产物序列；所述计算装置包括存储器和处理器，所述存储器中存储有计算机程序，所述处理器被配置为执行所述存储器中存储的计算机程序，以实现如下判别：若所述扩增产物序列中含有如第一方面中任意一项所述的cDNA的序列和/或如第二方面中任意一项所述的mRNA的序列，则判定所述模板核酸片段对应的胶质瘤预后性较差；所述输出装置用于输出所述计算装置的判定结果。
本公开的第七方面提供一种评价胶质瘤预后性的方法，该方法包括检测待测胶质瘤样本中是否含有如上所述的cDNA和/或mRNA和/或蛋白的步骤，如果待测胶质瘤样本中含有如上所述的cDNA和/或mRNA和/或蛋白，则指示该待测胶质瘤样本对应的胶质瘤患者的预后性更差。其中，可以通过PCR、NASBA、TMA、RT-RPA、核酸杂交或者高通量测序的方法检测上述cDNA和/或mRNA，可以通过免疫杂交检测上述蛋白。
以下通过实施例进一步详细说明本公开。
制备实施例
本制备实施例用于获取胶质瘤样本，并获取其中的总RNA和总cDNA。
使用符合医学伦理委员会标准的操作，收集胶质瘤的样本1211例，并判断每一例胶质瘤样本的病理学特征。其中，收集样本的每一位病人都在收集样本之前得到本人及其治疗专家的同意，并具有书面的证明材料。其中，利用病理学诊断方法诊断胶质瘤，并根据胶质瘤样本对应患者的总生存期来评估胶质瘤样本的预后性，患者总生存期越长，则胶质瘤的预后性越好。胶质瘤样本对应患者的性别、年龄、病理学分级、肿瘤类别、以及总生存期等特征见表1。
表1
Figure BDA0002460349850000101
使用DNA提取试剂盒(购自Qiagen)，按照其使用说明书提取上述胶质瘤样本的总RNA。该总RNA经完整性分析仪检测，确认其RNA完整性指数(RNA Integrity Number,RIN)大于7.0。使用反转录试剂盒(购自Invitrogen)按照其使用说明书以该总RNA为模板合成双链cDNA。
实施例1
本实施例对制备实施例合成得到的胶质瘤样本的cDNA进行PCR验证。
PCR验证所用的引物为如SEQ ID NO:4所示的第一引物和如SEQ ID NO:5所示的第二引物。PCR的操作按照合成引物和PCR试剂盒的说明书进行。PCR的产物经过琼脂糖凝胶核酸电泳展示扩增条带的有无，如图1所示，所出现的扩增条带使用DNA凝胶回收试剂盒(QIAquick PCR purification kit，购自Qiagen)进行回收，然后进行Sanger测序，测序结果见图2。
由图1和图2可知，胶质瘤样品中存在19号外显子缺失的MET基因。
实施例2
本实施例对制备实施例收集得到的胶质瘤样本的RNA进行测序。
将各个样本的RNA使用RNA文库构建试剂盒(购自Illumina)构建RNA文库，然后使用测序平台(IlluminaHiSeq 2000)对RNA文库进行RNA测序，并筛选出RNA测序结果中含有如SEQ ID NO:2所示的mRNA的胶质瘤样本，结果见表2。
表2
Figure BDA0002460349850000121
由表2可以看出，检测到含有如SEQ ID NO:2所示的mRNA的胶质瘤样本有12例，其中位生存期为284天，未检测到如SEQ ID NO:2所示的mRNA的胶质瘤样本有1199例，其中位生存期为866天，说明含有本公开的SEQ ID NO:2所示的mRNA的胶质瘤预后较差，由此可以用于评价胶质瘤的预后性。
此外，分别在全级别胶质瘤、IDH突变的全级别胶质瘤、继发胶质母细胞瘤和IDH突变的胶质母细胞瘤中，通过生存曲线(Kaplan–Meiercurve)对含有如SEQ ID NO:2所示的mRNA的胶质瘤样本和不含有如SEQ ID NO:2所示的mRNA的胶质瘤样本的整体生存情况进行了比较，结果均显示含有如SEQ ID NO:2所示的mRNA的胶质瘤样本整体生存较差，见图3-6。
以上结合附图详细描述了本公开的优选实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。
序列表
<110> 北京市神经外科研究所
<120> cDNA、mRNA、蛋白及评价胶质瘤预后性的试剂盒和系统
<130> 16124BJNI-CRC
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6471
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tacaagatgt tgcatcactt tactttaatt gcatgattta tcagaacaac tattaacata 60
cgaagtacca ttcagttcag ctgcaggtat aggcagtgac aagtatctaa ttcttagaag 120
aatcacttac tcccacaatc tgtccagaca cattaatcta aggacaagtt tataaatagc 180
aaacgtgatt ttcacattgc agtgttctca agaatgtata tacaagtgtg tagtcctgtt 240
gatgggatgt ttccccgagt tctttctatt gatgcgttca tgctcttgac cctggtagag 300
acagttcttt ctttccacag agcagatttt cttttgtcat ccaccattta caatactctg 360
tgaattagta ttacttatta catttatgaa acggcaatat ttggattgta atagctcttc 420
agtacaattc cttgtgtctt ctggtaagtc tctgcactat cagagagctt ttagttataa 480
tcattttcct gcaacaacag ccaaactcaa ctattgagtt tcagtgtgac acaccctctt 540
tgtagcttgc tgggttaacc cttggcttca agtcctgatg atgtaatgag ggtggggtgt 600
attggcaatc agtacatttc cttatcgcaa tttacagtca ttgaaaatca tgctgtcatt 660
aatcccagtc tgacatacct tttctaaaat gttcacagtg cagtgttttt gtggcctaac 720
aaaatttttc tcatatcatt aaaaataaac atttttataa aaaatataac actttaaatg 780
tttacgtcga caaaaccagt tagagtaacc tacaccacat gcactataca gtagcaagca 840
caaaattcca cagaatgaag catcacaaag ttctgctcag ggtggctatt ccatctaggt 900
gaaatagctg ggattttcaa ttgccttttt catttgtttc taaagtatgt tttgcttaac 960
ataaaacaca ccctaatgca aaataaaact ccccaaaagt tttgtttcca attgcttgcg 1020
aggtgggaac ctgccaccga gacagaggct aatcttttca atccatccac cctttctttg 1080
ctctacctat gagctgtgat tggaaccaat gaacctttta gtaaaatgta tcctgcttta 1140
caaacatgct gagttatctt taaaaatatt tatcaacaaa ttacttgtct tattttgagt 1200
tttcatttaa aaaaatacac acaaaacatc tacatgttca cattcattag atcagagtag 1260
catcattctc aaacagtggg tttttatatg acaactaaat atttcatgat gactaaatta 1320
tttcatacaa attttcttaa tgtcatctat gtacattaca tcctgttcat ttataaacat 1380
gttttgtgtg atgctatttt gaaactttga ctgtattgcc tataaatatt cttccaacca 1440
aaggaatgtc tatacaaaaa tttataaggg ctgggcgcag tggctcatgc ctgtaatcct 1500
agcactttgg gaggctgagg tgggtggatt tcttgagtcc aggagtttga aaccagcctt 1560
ggcaacatgg caaaaccccg tctctacaaa aaatacaaaa attagccggg gatggtggtg 1620
tgcgcctgta gtcccagcta ttcgggaggc ttgagcctgg gaggtggagg ttgcagtgag 1680
ctatgatcac accactgcac tacagccctg gcaacagagt gagatcctgt ctcaaaaaac 1740
aaacaaacat ttaaaaggga atgtttacct aatgggtgaa tgacaccatc agtaatgaaa 1800
aatgttattg accatacaac acacaaaagt gttttgaaac actagaattc taaatgagtg 1860
gcctgttctg gggctgccgc tcctgtcctg agcattacta tctcttctga gttttctgtg 1920
atcaagaagc cctcaatatt tcctgcttca atttcccata tgaaatcaag tggtacctga 1980
tttttaaaaa attcaaccca gaattcaagt tttggactcc aatatgacag gagtgttgtc 2040
acggctgcag gccattggtc cgtggcctgt gggaccaagc ctctggttct gatgctctgt 2100
cagataagaa attccttaga atccagtaat ttaaataaca atacaagtcc tataatagtg 2160
caattttggc aagagcaaag aatatcgatg gccttttaaa ggtcaggcag tgaaaaaacc 2220
attggacaaa gtgtggactg ttgctttgac atagtactag cactatgatg tctcccagaa 2280
ggaggctggt cgtgtgtcca cctcatcatc agcgttatct tctgatgaca acagagaagg 2340
atacggagcg acacatttta cgttcacata agtagcgttc acatggacat agtgctcccc 2400
aatgaaagta gagaagatcg ctgatatccg ggacaccagt tcagaaaagg atgggcgcat 2460
ttcggcttta gggtgccagc attttagcat tacttcatat aaggggtctg ggcagtattc 2520
gggttgtagg agtcttctcc cttgcaacaa gtaaacagtt atatcaaagg tgtttacgtc 2580
aggataaggt ggggctcctc ttgtcatcag ctcccagagg agcacgccaa aggaccaata 2640
cagtttcttg cagccaagtc tctgtggaca aactttttgc ttgcaagata tttcatgcct 2700
ttggctactt gaagaccaaa gccaataaga tcttttacag ttggattatg agtctcattt 2760
cgaatgaaat ttcgaagatc tccatgtttc atgtatggta ggaccaccag cggagaccct 2820
tcacttcgca ggcagattcc caggagcgag aggacattgg gatgactaaa atctttcatg 2880
atgattccct cggtcagaaa ttgggaaact tctcctatgt cagtgattct gttcaaggat 2940
ttcacagcac agtgaatttt cttgccatca ttgtccaaca aagtcccatg atatacacaa 3000
ccaaaatgcc ctcttcctat gacttcattg aaatgcacaa tcaggctact gggcccaatc 3060
actacatgct gcactgcctg gaccagctct ggatttagag cactgaggtc aatgtggaca 3120
gtattttgca gtaatggact ggatatatca gagtccccac tagttaggat gggggacatg 3180
tctgtcagag gatactgcac ttgtcggcat gaaccgttct gagatgaatt aggaaactga 3240
tcttctggaa aagtagctcg gtagtctaca gattcatttg aaaccatttc tgtagttggg 3300
cttacacttc gggcacttac aagcctatcc aaatgaggag tgtgtactct tgcatcgtag 3360
cgaactaatt cactgcccag atctttaatt tgctttctct ttttcagcca caggaaaaac 3420
ccaagtagta ataacagtgc tgttgatatt gagacaacac cagcaatcaa tcctgtgaaa 3480
ttctgatctg gttgaactat tacttttcca aggacggttg aagaaattgc ttgcttccac 3540
tctatattta gctcgctgtt caatttcagc aggtcattgg ggaccgtgca taaaacggct 3600
tcagaatgta agtgtatatt ctcacagctc ttatttccaa cttttaacac ttcaccttta 3660
actgcttcag ggtcaatatc atttccctta atttccagta cattttcatt gcccattgag 3720
atcatcactg gcttttcaaa aggcttaaac acaggattat gtacataaat gagatcaaag 3780
tatttggaaa ggatcccatc taacatgaaa aaggctttgg ttttcagggg gagttgcaga 3840
ttcagctgtt gcagggaagg agtggtacaa cagattatct ctgaattaga gcgatgttga 3900
catgccactg taaagttcct tcctgcttca tgcacattta tgaccattct cgggacacta 3960
actgaattca ggtttttccc aacacctgtt attgtgctcc caccactaat aaaagatttg 4020
gttggatgaa tttcatagac aatgggatct tcacggtaac tgaagatgct tgtctctcgg 4080
ttggctaagt caattttcaa tttaacagca aactcagttg aaatggtttg ggctggggta 4140
taacattcaa gaatactgtt tgacacactt tttaaagtac atgtttttcc accaattgaa 4200
atgtgtctag aattcccact gtttaggtaa tttccagtta aagtaagtaa agtgccacca 4260
gccataggac cgtatttcgg cgaaatactt gttattacag gatccacata ggagaatgta 4320
ctgtattgtg ttgtcccgtg gccatttgaa ataattatgg acatattgaa atgcttattc 4380
atggcaggac caactgtgca tttcaatgta ttcatcgtgc tctcacttaa agtcaaggtg 4440
cagctctcat ttccaaggag aactctagtt ttctttaaat caaatttatt attcctccga 4500
aatccaaagt cccagccaca tatggtcagc cttgtccctc cttcaagggg tgcactattt 4560
gggaaaacct tgtagattgc aggcagacag atctgttgag tccatgtccc gctcaggcat 4620
tcctccgatc gcacacattt gtcgtggcac cagccacact gaacaaaggg tggggcagag 4680
aggcattgac tgcaggactg gaaatgtctg cagcccaagc cattcaatgg gatcttcgtg 4740
atcttcttcc cagtgataac cagtgtgtag ccattttggt ttaatgtatg ctccacaatc 4800
acttctggag acactggatg ggagtccagg agaaaattca catgaggggt tgatggtcct 4860
gatcgagaaa ccacaacctg catgaagcga ccctctgatg tcccaagatt agctatggtg 4920
aggtctcctt taatgaaggt ggatatagat gttaagagga cttcgctgaa ttgacccatg 4980
aataagtcaa cgcgctgcaa agctgtggta aactctgttc gatattcatc acggcgcgct 5040
tcacagcctg atgaatttct cagaagtgtc ctattaaagc agtgctcatg attgggtccg 5100
taaaaatgct ggagacatct cacattgttt ttgttgacga tcttgttgaa gaagtcgttg 5160
acatatttga tagggaatgc acacatggca gatcgatcca ttggttcggc agaatctggc 5220
ttgctttgtg cgaacacccc gaaaagaatg tcatcattca ggctggctcc tatttgtcta 5280
gcaagctggg ccccaggctt gctgacatac gcagcctgaa gtatattaaa cacttccttc 5340
tttgtggatc tcttttttct cttttctgtg agaatacact ccagaggcat ttccatgtag 5400
gaatgcaatc cagagtttat ggaacagaac ctgattattc ttgtgtgaaa agtctgagca 5460
tctagagttt ccctttggac cgtcaagaag taaataaaat tgttgctttc aaaggcatgg 5520
acatacttaa tggggtaaga atctctgaac tcaggtaaaa catcaatgta ggactggtcc 5580
gtcaaaaaca taaaaccatc tttcgtttcc tttagccttc tcactgatat cgaatgcaat 5640
ggatgatctg ggaaataaga agaatttatg gtattgccta caaagaagtt gatgaaccgg 5700
tcctttacag atgaaaggac tttggctccc agggcgctca ccacacagtc aggacactgg 5760
ctgggctctt ctatctgtgg ggagaatatg cagtgaacct ccgactgtat gtcagcagta 5820
tgattgtggg gaaagacatg tcgctggcag gtccctctgt tgacgctgcc acagctaatg 5880
agttgatcat catagtaggt gtcgacaact agagccatgt tgatgttatc tttccaaaca 5940
cctcctgata aattggcttt gctgctgcag tcctgacatg ggaaacaatc tgggtgttcc 6000
agcacaggcc cagtcttgta ctcagcaacc ttctgaaggt cttcctcatt taaaacataa 6060
atgtagttag tggcaccaag gaaaatgtga tgctcatgta gaatgacatt ctggatgggt 6120
gtttccgcgg tgaagttggg aagctgatac ttcatattca cattcatctc ggactttgct 6180
agtgcctctt tacactcccc attgctcctc tgcaccaagg taaacaggag cacgaggatg 6240
ccaggtgcaa gcacagcggg ggccttcatt atgagaggtt tatctttcgg tgcccaggaa 6300
ccagtggaga agtcagcggc gcaaggacca cacgcgcgct ccgcgcctcc ccgcctcctc 6360
tcagcaagtc agctgtcgcc ccgcatctgg ctcgcgccct ccactcggct ccgcatctgc 6420
tcacaaagcg ctcggggcgc cgcgggcggc gagggcctcc gggtcacctg c 6471
<210> 2
<211> 6471
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
gcaggugacc cggaggcccu cgccgcccgc ggcgccccga gcgcuuugug agcagaugcg 60
gagccgagug gagggcgcga gccagaugcg gggcgacagc ugacuugcug agaggaggcg 120
gggaggcgcg gagcgcgcgu gugguccuug cgccgcugac uucuccacug guuccugggc 180
accgaaagau aaaccucuca uaaugaaggc ccccgcugug cuugcaccug gcauccucgu 240
gcuccuguuu accuuggugc agaggagcaa uggggagugu aaagaggcac uagcaaaguc 300
cgagaugaau gugaauauga aguaucagcu ucccaacuuc accgcggaaa cacccaucca 360
gaaugucauu cuacaugagc aucacauuuu ccuuggugcc acuaacuaca uuuauguuuu 420
aaaugaggaa gaccuucaga agguugcuga guacaagacu gggccugugc uggaacaccc 480
agauuguuuc ccaugucagg acugcagcag caaagccaau uuaucaggag guguuuggaa 540
agauaacauc aacauggcuc uaguugucga caccuacuau gaugaucaac ucauuagcug 600
uggcagcguc aacagaggga ccugccagcg acaugucuuu ccccacaauc auacugcuga 660
cauacagucg gagguucacu gcauauucuc cccacagaua gaagagccca gccagugucc 720
ugacugugug gugagcgccc ugggagccaa aguccuuuca ucuguaaagg accgguucau 780
caacuucuuu guaggcaaua ccauaaauuc uucuuauuuc ccagaucauc cauugcauuc 840
gauaucagug agaaggcuaa aggaaacgaa agaugguuuu auguuuuuga cggaccaguc 900
cuacauugau guuuuaccug aguucagaga uucuuacccc auuaaguaug uccaugccuu 960
ugaaagcaac aauuuuauuu acuucuugac gguccaaagg gaaacucuag augcucagac 1020
uuuucacaca agaauaauca gguucuguuc cauaaacucu ggauugcauu ccuacaugga 1080
aaugccucug gaguguauuc ucacagaaaa gagaaaaaag agauccacaa agaaggaagu 1140
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<210> 3
<211> 1220
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu Val Leu Leu Phe
1 5 10 15
Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys
20 25 30
Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala
35 40 45
Glu Thr Pro Ile Gln Asn Val Ile Leu His Glu His His Ile Phe Leu
50 55 60
Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys
65 70 75 80
Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe
85 90 95
Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp
100 105 110
Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp
115 120 125
Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His
130 135 140
Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys
145 150 155 160
Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val
165 170 175
Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe
180 185 190
Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp
195 200 205
His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp
210 215 220
Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu
225 230 235 240
Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser Asn
245 250 255
Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala Gln
260 265 270
Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu
275 280 285
His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg
290 295 300
Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala
305 310 315 320
Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser
325 330 335
Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp
340 345 350
Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile Lys
355 360 365
Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg
370 375 380
Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg
385 390 395 400
Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr
405 410 415
Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly
420 425 430
Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly
435 440 445
Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln
450 455 460
Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu
465 470 475 480
Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu His Thr Leu
485 490 495
Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys
500 505 510
Ile Pro Leu Asn Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln
515 520 525
Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys
530 535 540
Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile
545 550 555 560
Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu
565 570 575
Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg
580 585 590
Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu
595 600 605
Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys
610 615 620
Thr Val Gly Pro Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile
625 630 635 640
Ser Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp
645 650 655
Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly
660 665 670
Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg
675 680 685
His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn
690 695 700
Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe
705 710 715 720
Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe
725 730 735
Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser
740 745 750
Phe Ile Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu Asn
755 760 765
Ser Val Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg
770 775 780
Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys
785 790 795 800
Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys
805 810 815
Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe Asp
820 825 830
Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys Pro Val
835 840 845
Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly Asn Asp
850 855 860
Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly Asn Lys
865 870 875 880
Ser Cys Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val
885 890 895
Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys
900 905 910
Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp
915 920 925
Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Thr Ala
930 935 940
Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg Lys Gln
945 950 955 960
Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg Val His
965 970 975
Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser Pro Thr
980 985 990
Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro
995 1000 1005
Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln Val
1010 1015 1020
Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly Asp Ser
1025 1030 1035 1040
Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile Asp Leu Ser
1045 1050 1055
Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln His Val Val Ile Gly
1060 1065 1070
Pro Ser Ser Leu Ile Val His Phe Asn Glu Val Ile Gly Arg Gly His
1075 1080 1085
Phe Gly Cys Val Tyr His Gly Thr Leu Leu Asp Asn Asp Gly Lys Lys
1090 1095 1100
Ile His Cys Ala Val Lys Ser Leu Asn Arg Ile Thr Asp Ile Gly Glu
1105 1110 1115 1120
Val Ser Gln Phe Leu Thr Glu Gly Ile Ile Met Lys Asp Phe Ser His
1125 1130 1135
Pro Asn Val Leu Ser Leu Leu Gly Ile Cys Leu Arg Ser Glu Gly Ser
1140 1145 1150
Pro Leu Val Val Leu Pro Tyr Met Lys His Gly Asp Leu Arg Asn Phe
1155 1160 1165
Ile Arg Asn Glu Thr His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe
1170 1175 1180
Gly Leu Gln Val Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe
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Ser Ser Gly Ser
1220
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aatttctgac cgagggaatc at 22
<210> 5
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acgccaaagg accaatacag tttc 24
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gccaaaggac caatacagtt tctt 24
<210> 7
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
taatacgact cactataggg gagcacgcca aaggaccaat aca 43
<210> 8
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tgaaatatct tgcaagcaaa aagtt 25
<210> 9
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agcacgccaa aggaccaata cagt 24

Claims (9)

1.一种cDNA，其特征在于，所述cDNA与人MET基因相对应，且所述cDNA的序列中不含有人MET基因的19号外显子；
所述cDNA为单链DNA，且所述cDNA的序列为SEQ ID NO:1所示的序列，或者所述cDNA的序列为SEQ ID NO:1所示的序列的互补序列；或者，
所述cDNA为双链DNA，且所述cDNA的其中一条单链的序列为SEQ ID NO:1所示的序列，另一条单链的序列为SEQ ID NO:1所示的序列的互补序列。
2.一种mRNA，其特征在于，该mRNA为权利要求1所述的cDNA的逆转录模板。
3.根据权利要求2所述的mRNA，其特征在于，所述mRNA的序列为SEQ ID NO:2所示的序列。
4.一种蛋白，其特征在于，该蛋白由权利要求2-3中任意一项所述的mRNA编码。
5.根据权利要求4所述的蛋白，其特征在于，所述蛋白的氨基酸序列为SEQ ID NO:3所示的序列。
6.一种评价胶质瘤预后性的试剂盒，其特征在于，该试剂盒包括能够特异性地扩增权利要求1所述的cDNA的第一引物对，能够与权利要求1所述的cDNA特异性杂交的第一探针；
该试剂盒包括能够特异性地扩增权利要求2-3中任意一项所述的mRNA的第二引物对，能够与权利要求2-3中任意一项所述的mRNA特异性杂交的第二探针；
该试剂盒包括抗权利要求4-5中任意一项所述的蛋白的抗体以及权利要求4-5中任意一项所述的蛋白。
7.根据权利要求6所述的试剂盒，其特征在于，所述第一引物对含有如SEQ ID NO:4所示的第一引物和如SEQ ID NO:5所示的第二引物，所述第一探针的序列中含有如SEQ IDNO:6所示的序列；
所述第二引物对含有如SEQ ID NO:7所示的第三引物和如SEQ ID NO:8所示的第四引物，所述第二探针的序列中含有如SEQ ID NO:9所示的序列。
8.一种分子试剂在制备评价胶质瘤预后性的试剂盒中的用途，其特征在于，所述分子试剂包括如下(1)-(8)中的至少一种：
(1)权利要求1所述的cDNA；
(2)能够特异性地扩增权利要求1所述的cDNA的第一引物对；
(3)能够与权利要求1所述的cDNA特异性杂交的第一探针；
(4)权利要求2-3中任意一项所述的mRNA；
(5)能够特异性地扩增权利要求2-3中任意一项所述的mRNA的第二引物对；
(6)能够与权利要求2-3中任意一项所述的mRNA特异性杂交的第二探针；
(7)权利要求4-5中任意一项所述的蛋白；
(8)抗权利要求4-5中任意一项所述的蛋白的抗体。
9.一种评价胶质瘤预后性的系统，其特征在于，该系统包括扩增装置、测序装置、计算装置和输出装置；
所述扩增装置含有采集单元和扩增单元，所述采集单元用于采集模板核酸片段和扩增引物，所述扩增单元用于利用所述扩增引物对所述模板核酸片段进行扩增，得到扩增产物；
所述测序装置用于对所述扩增产物进行核酸序列测序，得到扩增产物序列；
所述计算装置包括存储器和处理器，所述存储器中存储有计算机程序，所述处理器被配置为执行所述存储器中存储的计算机程序，以实现如下判别：
若所述扩增产物序列中含有如权利要求1所述的cDNA的序列和/或如权利要求2-3中任意一项所述的mRNA的序列，则判定所述模板核酸片段对应的胶质瘤预后性较差；
所述输出装置用于输出所述计算装置的判定结果。
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Citations (3)

* Cited by examiner, † Cited by third party
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