CN114107490A - 评价患者对met抑制剂敏感性的试剂盒 - Google Patents

评价患者对met抑制剂敏感性的试剂盒 Download PDF

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CN114107490A
CN114107490A CN202010872686.9A CN202010872686A CN114107490A CN 114107490 A CN114107490 A CN 114107490A CN 202010872686 A CN202010872686 A CN 202010872686A CN 114107490 A CN114107490 A CN 114107490A
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江涛
柴睿超
赵征
陈婧
张克难
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Beijing Rencheng Neurotumor Biotechnology Engineering Research Center Co ltd
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Beijing Neurosurgical Institute
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Abstract

本公开涉及一种评价患者对MET抑制剂敏感性的试剂盒，该试剂盒包括能够特异性地扩增第一核苷酸序列的第一引物对，和/或，能够与所述第一核苷酸序列特异性结合的第一探针，其中，所述第一核苷酸序列如SEQ ID NO.1所示；和/或，该试剂盒包括能够特异性地扩增第二核苷酸序列的第二引物对，和/或，能够与所述第二核苷酸序列特异性结合的第二探针，其中，所述第二核苷酸序列如SEQ ID NO.4所示。利用本公开提供的试剂盒能够快速筛选出对MET抑制剂敏感的患者，使患者得到有效治疗。

Description

评价患者对MET抑制剂敏感性的试剂盒
技术领域
本公开涉及生物技术领域，具体地，涉及一种评价患者对MET抑制剂敏感性的试剂盒。
背景技术
间质上皮转化因子(MET)基因位于人7号染色体长臂，含有21个外显子，是肝细胞生长因子(HGF)的受体络氨酸激酶，MET与HGF结合可以激活参与肿瘤发生和转移的RAS-MAPK和PI3K-AKT信号传导途径。C-MET是MET基因编码产生的具有自主磷酸活性的跨膜受体，是胚胎发育、器官再生以及伤口愈合的关键因素。MET基因异常形式有突变、扩增、重排和过表达，MET基因异常是导致多种肿瘤的重要因素。
MET抑制剂能够有效治疗MET基因异常导致的多种肿瘤。但是，不同患者对MET抑制剂的敏感性不同会导致治疗效果因人而异，因此，亟需筛选出对MET抑制剂敏感性较好的患者进行治疗以取得较好的治疗效果。
发明内容
本公开的目的是提供一种评价患者对MET抑制剂敏感性的试剂盒，利用该试剂盒能够筛选出对MET抑制剂敏感性较好的患者，使患者得到有效治疗。
为了实现上述目的，第一方面，本公开提供一种评价患者对MET抑制剂敏感性的试剂盒，该试剂盒包括能够特异性地扩增第一核苷酸序列的第一引物对，和/或，能够与所述第一核苷酸序列特异性结合的第一探针，其中，所述第一核苷酸序列如SEQ ID NO.1所示；和/或，
该试剂盒包括能够特异性地扩增第二核苷酸序列的第二引物对，和/或，能够与所述第二核苷酸序列特异性结合的第二探针，其中，所述第二核苷酸序列如SEQ ID NO.4所示。
可选地，所述第一核苷酸序列如SEQ ID NO.2所示；优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；
所述第二核苷酸序列如SEQ ID NO.5所示；优选地，所述第二核苷酸序列如SEQ IDNO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列。
可选地，所述患者包括胶质瘤患者和/或胃腺癌患者。
第二方面，本公开提供一种评价患者对MET抑制剂敏感性的试剂盒，该试剂盒包括能够特异性地扩增第三核苷酸序列的第三引物对，和/或，能够与所述第三核苷酸序列特异性结合的第三探针，其中，所述第三核苷酸序列如SEQ ID NO.7所示；和/或，
该试剂盒包括能够特异性地扩增第四核苷酸序列的第四引物对，和/或，能够与所述第四核苷酸序列特异性结合的第四探针，其中，所述第四核苷酸序列如SEQ ID NO.10所示。
可选地，所述第三核苷酸序列如SEQ ID NO.8所示；优选地，所述第三核苷酸序列如SEQ ID NO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；
所述第四核苷酸序列如SEQ ID NO.11所示；优选地，所述第四核苷酸序列如SEQID NO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列。
第三方面，本公开提供一种评价患者对MET抑制剂敏感性的试剂盒，该试剂盒包括抗第一氨基酸序列的第一抗体，其中，所述第一氨基酸序列如SEQ ID NO.13所示；和/或，
该试剂盒包括抗第二氨基酸序列的第二抗体，其中，所述第二氨基酸序列如SEQID NO.16所示。
可选地，所述第一氨基酸序列如SEQ ID NO.14所示；优选地，所述第一氨基酸序列如SEQ ID NO.15所示，其中，SEQ ID NO.15所示的氨基酸序列由SEQ ID NO.9所示的mRNA编码得到；
所述第二氨基酸序列如SEQ ID NO.17所示；优选地，所述第二氨基酸序列如SEQID NO.18所示，其中，SEQ ID NO.18所示的氨基酸序列由SEQ ID NO.12所示的mRNA编码得到。
第四方面，本公开提供一种分子试剂在制备评价患者对MET抑制剂敏感性的试剂盒中的用途，所述分子试剂包括如下(1)～(10)中的至少一种：
(1)能够特异性扩增第一核苷酸序列的第一引物对；
(2)能够与所述第一核苷酸序列特异性结合的第一探针；
其中，所述第一核苷酸序列如SEQ ID NO.1所示；
(3)能够特异性扩增第二核苷酸序列的第二引物对；
(4)能够与所述第二核苷酸序列特异性结合的第二探针；
其中，所述第二核苷酸序列如SEQ ID NO.4所示；
(5)能够特异性扩增第三核苷酸序列的第三引物对；
(6)能够与所述第三核苷酸序列特异性结合的第三探针；
其中，所述第三核苷酸序列如SEQ ID NO.7所示；
(7)能够特异性扩增第四核苷酸序列的第四引物对；
(8)能够与所述第四核苷酸序列特异性结合的第四探针；
其中，所述第四核苷酸序列如SEQ ID NO.10所示；
(9)抗第一氨基酸序列的第一抗体；
其中，所述第一氨基酸序列如SEQ ID NO.13所示；
(10)抗第二氨基酸序列的第二抗体；
其中，所述第二氨基酸序列如SEQ ID NO.16所示。
可选地，所述第一核苷酸序列如SEQ ID NO.2所示；优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；
所述第二核苷酸序列如SEQ ID NO.5所示；优选地，所述第二核苷酸序列如SEQ IDNO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列；
所述第三核苷酸序列如SEQ ID NO.8所示；优选地，所述第三核苷酸序列如SEQ IDNO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；
所述第四核苷酸序列如SEQ ID NO.11所示；优选地，所述第四核苷酸序列如SEQID NO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列；
所述第一氨基酸序列如SEQ ID NO.14所示；优选地，所述第一氨基酸序列如SEQID NO.15所示，其中，SEQ ID NO.15所示的氨基酸序列由SEQ ID NO.9所示的mRNA编码得到；
所述第二氨基酸序列如SEQ ID NO.17所示；优选地，所述第二氨基酸序列如SEQID NO.18所示，其中，SEQ ID NO.18所示的氨基酸序列由SEQ ID NO.12所示的mRNA编码得到。
第五方面，本公开提供一种评价患者对MET抑制剂敏感性的系统，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第一核苷酸序列、第二核苷酸序列、第三核苷酸序列或第四核苷酸序列中的至少一种，则判定患者对MET抑制剂敏感；
其中，所述第一核苷酸序列如SEQ ID NO.1所示；优选地，所述第一核苷酸序列如SEQ ID NO.2所示；更优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；
所述第二核苷酸序列如SEQ ID NO.4所示；优选地，所述第二核苷酸序列如SEQ IDNO.5所示；更优选地，所述第二核苷酸序列如SEQ ID NO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列；
所述第三核苷酸序列如SEQ ID NO.7所示；优选地，所述第三核苷酸序列如SEQ IDNO.8所示；更优选地，所述第三核苷酸序列如SEQ ID NO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；
所述第四核苷酸序列如SEQ ID NO.10所示；优选地，所述第四核苷酸序列如SEQID NO.11所示；更优选地，所述第四核苷酸序列如SEQ ID NO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列。
第六方面，本公开提供一种评价患者对MET抑制剂敏感性的方法，该方法包括检测肿瘤样本细胞中是否含有如上所述的第一核苷酸序列、第二核苷酸序列、第三核苷酸序列、第四核苷酸序列、第一氨基酸序列或者第二氨基酸序列的步骤，如果检测到检测肿瘤样本细胞中含有如上所述的第一核苷酸序列、第二核苷酸序列、第三核苷酸序列、第四核苷酸序列、第一氨基酸序列或者第二氨基酸序列中的至少一种，则判定所述肿瘤样本细胞对应的患者对MET抑制剂敏感。
通过上述技术方案，利用本公开提供的试剂盒能够快速筛选出对MET抑制剂敏感的患者，使患者得到有效治疗。
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为本公开实施例提供的胶质母细胞瘤PDC样本A的MET基因9～11号外显子读段数示意图；
图2为本公开实施例提供的胶质母细胞瘤PDC样本B的MET基因9～11号外显子读段数示意图；
图3为本公开实施例提供的胶质母细胞瘤PDC样本C的MET基因9～11号外显子读段数示意图；
图4为本公开实施例提供的胶质母细胞瘤PDC样本D的MET基因9～11号外显子读段数示意图；
图5为本公开实施例提供的胶质母细胞瘤PDC样本A的MET基因18～20号外显子读段数示意图；
图6为本公开实施例提供的胶质母细胞瘤PDC样本B的MET基因18～20号外显子读段数示意图；
图7为本公开实施例提供的胶质母细胞瘤PDC样本C的MET基因18～20号外显子读段数示意图；
图8为本公开实施例提供的胶质母细胞瘤PDC样本D的MET基因18～20号外显子读段数示意图；
图9为本公开实施例提供的胶质母细胞瘤PDC样本A、B、C和D的PCR扩增产物的琼脂糖凝胶电泳图；
图10为本公开实施例提供的又一胶质母细胞瘤PDC样本A、B、C和D的PCR扩增产物的琼脂糖凝胶电泳图；
图11为本公开实施例提供的不同浓度TMZ处理后的各胶质母细胞瘤PDC样本的细胞相对存活率曲线图；
图12为本公开实施例提供的不同浓度克唑替尼处理后的各胶质母细胞瘤PDC样本的细胞相对存活率曲线图。
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评价患者对MET抑制剂敏感性的试剂盒，该试剂盒包括能够特异性地扩增第一核苷酸序列的第一引物对，和/或，能够与所述第一核苷酸序列特异性结合的第一探针，其中，所述第一核苷酸序列如SEQ ID NO.1所示；和/或，该试剂盒包括能够特异性地扩增第二核苷酸序列的第二引物对，和/或，能够与所述第二核苷酸序列特异性结合的第二探针，其中，所述第二核苷酸序列如SEQ ID NO.4所示。
可选地，所述第一核苷酸序列如SEQ ID NO.2所示；优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；所述第二核苷酸序列如SEQ ID NO.5所示；优选地，所述第二核苷酸序列如SEQID NO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列。
可选地，所述患者包括胶质瘤患者和/或胃腺癌患者。
上述技术方案中，SEQ ID NO.1所示的序列为包含人MET基因10号外显子缺失位点的cDNA片段，SEQ ID NO.2所示的序列为包含SEQ ID NO.1所示的序列的cDNA片段；SEQ IDNO.4所示的序列为包含人MET基因19号外显子缺失位点的cDNA片段，SEQ ID NO.5所示序列为包含SEQ ID NO.4所示的序列的cDNA片段。
本公开的发明人发现，当肿瘤细胞的mRNA可逆转录出具有10号外显子和/或19号外显子缺失位点的MET基因cDNA时，肿瘤细胞对MET抑制剂的敏感性更强，因此，可以通过检测肿瘤细胞中MET基因cDNA的10号外显子和19号外显子的缺失状态，来确定肿瘤细胞对MET抑制剂的敏感性，并以此来筛选出适合使用MET抑制剂的患者，由此得到本公开。
其中，能特异性扩增含SEQ ID NO.1所示的核苷酸序列的第一引物对均可用于本公开，例如，所述第一引物对可以含有如SEQ ID NO.19所示的上游引物和SEQ ID NO.20所示的下游引物。能与SEQ ID NO.1所示的核苷酸序列特异性结合的第一探针均可用于本公开，例如，所述第一探针的序列中可以含有如SEQ ID NO.21所示的序列。
能特异性扩增含SEQ ID NO.4所示的核苷酸序列的第二引物对均可用于本公开，例如，所述第二引物对可以含有如SEQ ID NO.22所示的上游引物和SEQ ID NO.23所示的下游引物。能与SEQ ID NO.4所示的核苷酸序列特异性结合的第二探针均可用于本公开，例如，所述第二探针的序列中可以含有如SEQ ID NO.24所示的序列。
本公开的第二方面提供一种评价患者对MET抑制剂敏感性的试剂盒，该试剂盒包括能够特异性地扩增第三核苷酸序列的第三引物对，和/或，能够与所述第三核苷酸序列特异性结合的第三探针，其中，所述第三核苷酸序列如SEQ ID NO.7所示；和/或，该试剂盒包括能够特异性地扩增第四核苷酸序列的第四引物对，和/或，能够与所述第四核苷酸序列特异性结合的第四探针，其中，所述第四核苷酸序列如SEQ ID NO.10所示。
可选地，所述第三核苷酸序列如SEQ ID NO.8所示；优选地，所述第三核苷酸序列如SEQ ID NO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；所述第四核苷酸序列如SEQ ID NO.11所示；优选地，所述第四核苷酸序列如SEQID NO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列。
上述技术方案中，SEQ ID NO.7所示的序列为包含人MET基因10号外显子缺失位点的mRNA片段，SEQ ID NO.8所示的序列为包含SEQ ID NO.7所示的序列的mRNA片段；SEQ IDNO.10所示的序列为包含人MET基因19号外显子缺失位点的mRNA片段，SEQ ID NO.11所示序列为包含SEQ ID NO.10所示的序列的mRNA片段。
其中，能特异性扩增SEQ ID NO.7所示的核苷酸序列的第三引物对均可用于本公开，例如，所述第三引物对可以含有如SEQ ID NO.25所示的上游引物和SEQ ID NO.26所示的下游引物。能与SEQ ID NO.7所示的核苷酸序列特异性结合的第三探针均可用于本公开，例如，所述第三探针的序列中可以含有如SEQ ID NO.27所示的序列。
能特异性扩增SEQ ID NO.10所示的核苷酸序列的第四引物对均可用于本公开，例如，所述第四引物对可以含有如SEQ ID NO.28所示的上游引物和SEQ ID NO.29所示的下游引物。能与SEQ ID NO.10所示的核苷酸序列特异性结合的第四探针均可用于本公开，例如，所述第四探针的序列中可以含有如SEQ ID NO.30所示的序列。
本公开的第三方面提供一种评价患者对MET抑制剂敏感性的试剂盒，该试剂盒包括抗第一氨基酸序列的第一抗体，其中，所述第一氨基酸序列如SEQ ID NO.13所示；和/或，该试剂盒包括抗第二氨基酸序列的第二抗体，其中，所述第二氨基酸序列如SEQ ID NO.16所示。
可选地，所述第一氨基酸序列如SEQ ID NO.14所示；优选地，所述第一氨基酸序列如SEQ ID NO.15所示，其中，SEQ ID NO.15所示的氨基酸序列由SEQ ID NO.9所示的mRNA编码得到；所述第二氨基酸序列如SEQ ID NO.17所示；优选地，所述第二氨基酸序列如SEQ IDNO.18所示，其中，SEQ ID NO.18所示的氨基酸序列由SEQ ID NO.12所示的mRNA编码得到。
其中，抗第一氨基酸序列和第二氨基酸序列的抗体可以外单克隆抗体和/或多克隆抗体。
本公开的第四方面提供一种分子试剂在制备评价患者对MET抑制剂敏感性的试剂盒中的用途，所述分子试剂包括如下(1)～(10)中的至少一种：(1)能够特异性扩增第一核苷酸序列的第一引物对；(2)能够与所述第一核苷酸序列特异性结合的第一探针；其中，所述第一核苷酸序列如SEQ ID NO.1所示；(3)能够特异性扩增第二核苷酸序列的第二引物对；(4)能够与所述第二核苷酸序列特异性结合的第二探针；其中，所述第二核苷酸序列如SEQ ID NO.4所示；(5)能够特异性扩增第三核苷酸序列的第三引物对；(6)能够与所述第三核苷酸序列特异性结合的第三探针；其中，所述第三核苷酸序列如SEQ ID NO.7所示；(7)能够特异性扩增第四核苷酸序列的第四引物对；(8)能够与所述第四核苷酸序列特异性结合的第四探针；其中，所述第四核苷酸序列如SEQ ID NO.10所示；(9)抗第一氨基酸序列的第一抗体；其中，所述第一氨基酸序列如SEQ ID NO.13所示；(10)抗第二氨基酸序列的第二抗体；其中，所述第二氨基酸序列如SEQ ID NO.16所示。
可选地，所述第一核苷酸序列如SEQ ID NO.2所示；优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列。所述第二核苷酸序列如SEQ ID NO.5所示；优选地，所述第二核苷酸序列如SEQID NO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列。所述第三核苷酸序列如SEQ ID NO.8所示；优选地，所述第三核苷酸序列如SEQ ID NO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列。所述第四核苷酸序列如SEQ ID NO.11所示；优选地，所述第四核苷酸序列如SEQ ID NO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列。所述第一氨基酸序列如SEQ ID NO.14所示；优选地，所述第一氨基酸序列如SEQ ID NO.15所示，其中，SEQ ID NO.15所示的氨基酸序列由SEQ ID NO.9所示的mRNA编码得到。所述第二氨基酸序列如SEQ ID NO.17所示；优选地，所述第二氨基酸序列如SEQ ID NO.18所示，其中，SEQID NO.18所示的氨基酸序列由SEQ ID NO.12所示的mRNA编码得到。
本公开的第五方面提供一种评价患者对MET抑制剂敏感性的系统，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第一核苷酸序列、第二核苷酸序列、第三核苷酸序列或第四核苷酸序列中的至少一种，则判定患者对MET抑制剂敏感；其中，所述第一核苷酸序列如SEQ ID NO.1所示；优选地，所述第一核苷酸序列如SEQ ID NO.2所示；更优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；所述第二核苷酸序列如SEQ ID NO.4所示；优选地，所述第二核苷酸序列如SEQID NO.5所示；更优选地，所述第二核苷酸序列如SEQ ID NO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列；所述第三核苷酸序列如SEQ IDNO.7所示；优选地，所述第三核苷酸序列如SEQ ID NO.8所示；更优选地，所述第三核苷酸序列如SEQ ID NO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；所述第四核苷酸序列如SEQ ID NO.10所示；优选地，所述第四核苷酸序列如SEQ ID NO.11所示；更优选地，所述第四核苷酸序列如SEQ ID NO.12所示，其中，SEQ IDNO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列。
本公开的第六方面提供一种评价患者对MET抑制剂敏感性的方法，该方法包括检测肿瘤样本细胞中是否含有如上所述的第一核苷酸序列、第二核苷酸序列、第三核苷酸序列、第四核苷酸序列、第一氨基酸序列或者第二氨基酸序列的步骤，如果检测到检测肿瘤样本细胞中含有如上所述的第一核苷酸序列、第二核苷酸序列、第三核苷酸序列、第四核苷酸序列、第一氨基酸序列或者第二氨基酸序列中的至少一种，则判定所述肿瘤样本细胞对应的患者对MET抑制剂敏感。其中，可以通过PCR、RT-RPA、核酸杂交或者高通量测序的方法检测上述第一核苷酸序列、第二核苷酸序列、第三核苷酸序列和第四核苷酸序列，可以通过免疫杂交检测上述第一氨基酸序列和第二氨基酸序列。
下面通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
制备实施例
本制备实施例用于获取胶质母细胞瘤的PDC(病人来源细胞)样本及总RNA和总cDNA。
使用符合医学伦理委员会标准的操作，收集胶质母细胞瘤样本4例，其中，收集样本的每一位患者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。
对上述收集到的4例胶质母细胞瘤样本分别进行酶解，得到胶质母细胞瘤PDC样本A、B、C和D。
使用RNA提取试剂盒(购自Qiagen)，按照其使用说明书分别提取上述胶质母细胞瘤PDC样本A、B、C和D的总RNA。提取到的各总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)按照其使用说明书分别以各总RNA为模板合成各双链cDNA。
实施例1
本实施例对制备实施例合成得到的各胶质母细胞瘤PDC样本的总cDNA进行验证。
(1)利用二代测序技术验证10号外显子缺失的cDNA
利用二代测序技术分别对胶质母细胞瘤PDC样本A、B、C和D的总cDNA进行测序，然后利用IGV(Integrative Genomics Viewer)软件识别支持MET基因9～10号外显子、10～11号外显子及9～11号外显子的读段数，若读段数大于等于3，说明存在10号外显子缺失的MET基因cDNA。测试见过见图1～4。
其中，图1为本公开实施例提供的胶质母细胞瘤PDC样本A的MET基因9～11号外显子读段数示意图；图2为本公开实施例提供的胶质母细胞瘤PDC样本B的MET基因9～11号外显子读段数示意图；图3为本公开实施例提供的胶质母细胞瘤PDC样本C的MET基因9～11号外显子读段数示意图；图4为本公开实施例提供的胶质母细胞瘤PDC样本D的MET基因9～11号外显子读段数示意图。
由图1～4可知，胶质母细胞瘤PDC样本A和胶质母细胞瘤PDC样本B的总cDNA中均存在10号外显子缺失的MET基因cDNA。
(2)利用二代测序技术验证19号外显子缺失的cDNA
利用二代测序技术分别对胶质母细胞瘤PDC样本A、B、C和D的总cDNA进行测序，然后利用IGV(Integrative Genomics Viewer)软件识别支持MET基因18～19号外显子、19～20号外显子及18～20号外显子的读段数，若读段数大于等于3，说明存在19号外显子缺失的MET基因cDNA。测试见过见图5～8。
其中，图5为本公开实施例提供的胶质母细胞瘤PDC样本A的MET基因18～20号外显子读段数示意图；图6为本公开实施例提供的胶质母细胞瘤PDC样本B的MET基因18～20号外显子读段数示意图；图7为本公开实施例提供的胶质母细胞瘤PDC样本C的MET基因18～20号外显子读段数示意图；图8为本公开实施例提供的胶质母细胞瘤PDC样本D的MET基因18～20号外显子读段数示意图。
由图5～8可知，胶质母细胞瘤PDC样本A的总cDNA中存在19号外显子缺失的MET基因cDNA。
(3)利用PCR技术验证10号外显子缺失的cDNA
PCR验证所用的引物为如SEQ ID NO.19所示的上游引物和如SEQ ID NO.20所示的下游引物。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，扩增结果如图9所示。
图9为本公开实施例提供的胶质母细胞瘤PDC样本A、B、C和D的PCR扩增产物的琼脂糖凝胶电泳图。
由图9可知，胶质母细胞瘤PDC样本A和胶质母细胞瘤PDC样本B的总cDNA中均存在10号外显子缺失的MET基因cDNA。
(4)利用PCR技术验证19号外显子缺失的cDNA验证
PCR验证所用的引物为如SEQ ID NO.22所示的上游引物和如SEQ ID NO.23所示的下游引物。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，扩增结果如图9所示。
图10为本公开实施例提供的胶质母细胞瘤PDC样本A、B、C和D的PCR扩增产物的琼脂糖凝胶电泳图。
由图10可知，胶质母细胞瘤PDC样本A的总cDNA中存在19号外显子缺失的MET基因cDNA。
实施例2
本实施例用于验证制备实施例制备得到的各胶质母细胞瘤的PDC样本对抗肿瘤药物替莫唑胺(TMZ)和MET抑制剂克唑替尼的敏感性。
取上述胶质母细胞瘤PDC样本A、B、C和D，使用专用培养基重悬，然后分别接种到96孔细胞培养板中，使得每个培养孔中含有3000个细胞。接种完成后，将96孔细胞培养板置于含有5％CO2的37℃培养箱中培养24小时。培养结束后，在各培养孔中加入不同浓度的TMZ或克唑替尼，每个浓度梯度设10个重复孔。加药后继续培养72小时，然后使用CCK-8测量每个培养孔中的活细胞数，结果见表1和表2，图11和图12。
表1不同浓度TMZ处理后的活细胞数
Figure BDA0002651621250000081
表2不同浓度克唑替尼处理后的活细胞数
Figure BDA0002651621250000091
由表1和图11可以看出，胶质母细胞瘤PDC样本A和B对抗肿瘤药物替莫唑胺(TMZ)不敏感，胶质母细胞瘤PDC样本C和D对抗肿瘤药物替莫唑胺(TMZ)较敏感。
由表2和图12可以看出，胶质母细胞瘤PDC样本A、B、C和D均对MET抑制剂克唑替尼敏感；而且，胶质母细胞瘤PDC样本A和B对克唑替尼的敏感性强于胶质母细胞瘤PDC样本C和D；此外，胶质母细胞瘤PDC样本A对克唑替尼的敏感性强于胶质母细胞瘤PDC样本B。
由此可见，当肿瘤细胞中存在MET基因的10号外显子缺失和19号外显子缺失时，肿瘤细胞对MET抑制剂具有较强的敏感性，而且，当肿瘤细胞中同时存在MET基因的10号外显子缺失和19号外显子缺失时，肿瘤细胞对MET抑制剂的敏感性最强。
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，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
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> 评价患者对MET抑制剂敏感性的试剂盒
<130> 17347BJNI-CRC
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gttgacatgc ctaataaaag atttggttgg atgaatttc 39
<210> 2
<211> 381
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cttaatttcc agtacatttt cattgcccat tgagatcatc actggctttt caaaaggctt 60
aaacacagga ttatgtacat aaatgagatc aaagtatttg gaaaggatcc catctaacat 120
gaaaaaggct ttggttttca gggggagttg cagattcagc tgttgcaggg aaggagtggt 180
acaacagatt atctctgaat tagagcgatg ttgacatgcc taataaaaga tttggttgga 240
tgaatttcat agacaatggg atcttcacgg taactgaaga tgcttgtctc tcggttggct 300
aagtcaattt tcaatttaac agcaaactca gttgaaatgg tttgggctgg ggtataacat 360
tcaagaatac tgtttgacac a 381
<210> 3
<211> 6537
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
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 aggaccacac 2640
atctgacttg gtggtaaact tttgagtttg cagactttcc aaagccatcc acttcactgg 2700
cagctttgca cctgttttgt tgtgtacact atagtattct ttatcataca tgtctctggc 2760
aagaccaaaa tcagcaacct tgactgtgaa tttttcatcc agcatacagt ttcttgcagc 2820
caagtctctg tggacaaact ttttgcttgc aagatatttc atgcctttgg ctacttgaag 2880
accaaagcca ataagatctt ttacagttgg attatgagtc tcatttcgaa tgaaatttcg 2940
aagatctcca tgtttcatgt atggtaggac caccagcgga gacccttcac ttcgcaggca 3000
gattcccagg agcgagagga cattgggatg actaaaatct ttcatgatga ttccctcggt 3060
cagaaattgg gaaacttctc ctatgtcagt gattctgttc aaggatttca cagcacagtg 3120
aattttcttg ccatcattgt ccaacaaagt cccatgatat acacaaccaa aatgccctct 3180
tcctatgact tcattgaaat gcacaatcag gctactgggc ccaatcacta catgctgcac 3240
tgcctggacc agctctggat ttagagcact gaggtcaatg tggacagtat tttgcagtaa 3300
tggactggat atatcagagt ccccactagt taggatgggg gacatgtctg tcagaggata 3360
ctgcacttgt cggcatgaac cgttctgaga tgaattagga aactgatctt ctggaaaagt 3420
agctcggtag tctacagatt catttgaaac catttctgta gttgggctta cacttcgggc 3480
acttacaagc ctatccaaat gaggagtgtg tactcttgca tcgtagcgaa ctaattcact 3540
gcccagatct ttaatttgct ttctcttttt cagccacagg aaaaacccaa gtagtaataa 3600
cagtgctgtt gatattgaga caacaccagc aatcaatcct gtgaaattct gatctggttg 3660
aactattact tttccaagga cggttgaaga aattgcttgc ttccactcta tatttagctc 3720
gctgttcaat ttcagcaggt cattggggac cgtgcataaa acggcttcag aatgtaagtg 3780
tatattctca cagctcttat ttccaacttt taacacttca cctttaactg cttcagggtc 3840
aatatcattt cccttaattt ccagtacatt ttcattgccc attgagatca tcactggctt 3900
ttcaaaaggc ttaaacacag gattatgtac ataaatgaga tcaaagtatt tggaaaggat 3960
cccatctaac atgaaaaagg ctttggtttt cagggggagt tgcagattca gctgttgcag 4020
ggaaggagtg gtacaacaga ttatctctga attagagcga tgttgacatg cctaataaaa 4080
gatttggttg gatgaatttc atagacaatg ggatcttcac ggtaactgaa gatgcttgtc 4140
tctcggttgg ctaagtcaat tttcaattta acagcaaact cagttgaaat ggtttgggct 4200
ggggtataac attcaagaat actgtttgac acacttttta aagtacatgt ttttccacca 4260
attgaaatgt gtctagaatt cccactgttt aggtaatttc cagttaaagt aagtaaagtg 4320
ccaccagcca taggaccgta tttcggcgaa atacttgtta ttacaggatc cacataggag 4380
aatgtactgt attgtgttgt cccgtggcca tttgaaataa ttatggacat attgaaatgc 4440
ttattcatgg caggaccaac tgtgcatttc aatgtattca tcgtgctctc acttaaagtc 4500
aaggtgcagc tctcatttcc aaggagaact ctagttttct ttaaatcaaa tttattattc 4560
ctccgaaatc caaagtccca gccacatatg gtcagccttg tccctccttc aaggggtgca 4620
ctatttggga aaaccttgta gattgcaggc agacagatct gttgagtcca tgtcccgctc 4680
aggcattcct ccgatcgcac acatttgtcg tggcaccagc cacactgaac aaagggtggg 4740
gcagagaggc attgactgca ggactggaaa tgtctgcagc ccaagccatt caatgggatc 4800
ttcgtgatct tcttcccagt gataaccagt gtgtagccat tttggtttaa tgtatgctcc 4860
acaatcactt ctggagacac tggatgggag tccaggagaa aattcacatg aggggttgat 4920
ggtcctgatc gagaaaccac aacctgcatg aagcgaccct ctgatgtccc aagattagct 4980
atggtgaggt ctcctttaat gaaggtggat atagatgtta agaggacttc gctgaattga 5040
cccatgaata agtcaacgcg ctgcaaagct gtggtaaact ctgttcgata ttcatcacgg 5100
cgcgcttcac agcctgatga atttctcaga agtgtcctat taaagcagtg ctcatgattg 5160
ggtccgtaaa aatgctggag acatctcaca ttgtttttgt tgacgatctt gttgaagaag 5220
tcgttgacat atttgatagg gaatgcacac atggcagatc gatccattgg ttcggcagaa 5280
tctggcttgc tttgtgcgaa caccccgaaa agaatgtcat cattcaggct ggctcctatt 5340
tgtctagcaa gctgggcccc aggcttgctg acatacgcag cctgaagtat attaaacact 5400
tccttctttg tggatctctt ttttctcttt tctgtgagaa tacactccag aggcatttcc 5460
atgtaggaat gcaatccaga gtttatggaa cagaacctga ttattcttgt gtgaaaagtc 5520
tgagcatcta gagtttccct ttggaccgtc aagaagtaaa taaaattgtt gctttcaaag 5580
gcatggacat acttaatggg gtaagaatct ctgaactcag gtaaaacatc aatgtaggac 5640
tggtccgtca aaaacataaa accatctttc gtttccttta gccttctcac tgatatcgaa 5700
tgcaatggat gatctgggaa ataagaagaa tttatggtat tgcctacaaa gaagttgatg 5760
aaccggtcct ttacagatga aaggactttg gctcccaggg cgctcaccac acagtcagga 5820
cactggctgg gctcttctat ctgtggggag aatatgcagt gaacctccga ctgtatgtca 5880
gcagtatgat tgtggggaaa gacatgtcgc tggcaggtcc ctctgttgac gctgccacag 5940
ctaatgagtt gatcatcata gtaggtgtcg acaactagag ccatgttgat gttatctttc 6000
caaacacctc ctgataaatt ggctttgctg ctgcagtcct gacatgggaa acaatctggg 6060
tgttccagca caggcccagt cttgtactca gcaaccttct gaaggtcttc ctcatttaaa 6120
acataaatgt agttagtggc accaaggaaa atgtgatgct catgtagaat gacattctgg 6180
atgggtgttt ccgcggtgaa gttgggaagc tgatacttca tattcacatt catctcggac 6240
tttgctagtg cctctttaca ctccccattg ctcctctgca ccaaggtaaa caggagcacg 6300
aggatgccag gtgcaagcac agcgggggcc ttcattatga gaggtttatc tttcggtgcc 6360
caggaaccag tggagaagtc agcggcgcaa ggaccacacg cgcgctccgc gcctccccgc 6420
ctcctctcag caagtcagct gtcgccccgc atctggctcg cgccctccac tcggctccgc 6480
atctgctcac aaagcgctcg gggcgccgcg ggcggcgagg gcctccgggt cacctgc 6537
<210> 4
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tcagctccca gaggagcacg ccaaaggacc aatacagttt cttgcagcca agtctctgtg 60
<210> 5
<211> 247
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aaggggtctg ggcagtattc gggttgtagg agtcttctcc cttgcaacaa gtaaacagtt 60
atatcaaagg tgtttacgtc aggataaggt ggggctcctc ttgtcatcag ctcccagagg 120
agcacgccaa aggaccaata cagtttcttg cagccaagtc tctgtggaca aactttttgc 180
ttgcaagata tttcatgcct ttggctactt gaagaccaaa gccaataaga tcttttacag 240
ttggatt 247
<210> 6
<211> 6471
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
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> 7
<211> 59
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaaauucauc caaccaaauc uuuuauuagg caugucaaca ucgcucuaau ucagagaua 59
<210> 8
<211> 381
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 8
ugugucaaac aguauucuug aauguuauac cccagcccaa accauuucaa cugaguuugc 60
uguuaaauug aaaauugacu uagccaaccg agagacaagc aucuucaguu accgugaaga 120
ucccauuguc uaugaaauuc auccaaccaa aucuuuuauu aggcauguca acaucgcucu 180
aauucagaga uaaucuguug uaccacuccu ucccugcaac agcugaaucu gcaacucccc 240
cugaaaacca aagccuuuuu cauguuagau gggauccuuu ccaaauacuu ugaucucauu 300
uauguacaua auccuguguu uaagccuuuu gaaaagccag ugaugaucuc aaugggcaau 360
gaaaauguac uggaaauuaa g 381
<210> 9
<211> 6537
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 9
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
guuuaauaua cuucaggcug cguaugucag caagccuggg gcccagcuug cuagacaaau 1200
aggagccagc cugaaugaug acauucuuuu cgggguguuc gcacaaagca agccagauuc 1260
ugccgaacca auggaucgau cugccaugug ugcauucccu aucaaauaug ucaacgacuu 1320
cuucaacaag aucgucaaca aaaacaaugu gagaugucuc cagcauuuuu acggacccaa 1380
ucaugagcac ugcuuuaaua ggacacuucu gagaaauuca ucaggcugug aagcgcgccg 1440
ugaugaauau cgaacagagu uuaccacagc uuugcagcgc guugacuuau ucauggguca 1500
auucagcgaa guccucuuaa caucuauauc caccuucauu aaaggagacc ucaccauagc 1560
uaaucuuggg acaucagagg gucgcuucau gcagguugug guuucucgau caggaccauc 1620
aaccccucau gugaauuuuc uccuggacuc ccauccagug ucuccagaag ugauugugga 1680
gcauacauua aaccaaaaug gcuacacacu gguuaucacu gggaagaaga ucacgaagau 1740
cccauugaau ggcuugggcu gcagacauuu ccaguccugc agucaaugcc ucucugcccc 1800
acccuuuguu caguguggcu ggugccacga caaaugugug cgaucggagg aaugccugag 1860
cgggacaugg acucaacaga ucugucugcc ugcaaucuac aagguuuucc caaauagugc 1920
accccuugaa ggagggacaa ggcugaccau auguggcugg gacuuuggau uucggaggaa 1980
uaauaaauuu gauuuaaaga aaacuagagu ucuccuugga aaugagagcu gcaccuugac 2040
uuuaagugag agcacgauga auacauugaa augcacaguu gguccugcca ugaauaagca 2100
uuucaauaug uccauaauua uuucaaaugg ccacgggaca acacaauaca guacauucuc 2160
cuauguggau ccuguaauaa caaguauuuc gccgaaauac gguccuaugg cugguggcac 2220
uuuacuuacu uuaacuggaa auuaccuaaa cagugggaau ucuagacaca uuucaauugg 2280
uggaaaaaca uguacuuuaa aaaguguguc aaacaguauu cuugaauguu auaccccagc 2340
ccaaaccauu ucaacugagu uugcuguuaa auugaaaauu gacuuagcca accgagagac 2400
aagcaucuuc aguuaccgug aagaucccau ugucuaugaa auucauccaa ccaaaucuuu 2460
uauuaggcau gucaacaucg cucuaauuca gagauaaucu guuguaccac uccuucccug 2520
caacagcuga aucugcaacu cccccugaaa accaaagccu uuuucauguu agaugggauc 2580
cuuuccaaau acuuugaucu cauuuaugua cauaauccug uguuuaagcc uuuugaaaag 2640
ccagugauga ucucaauggg caaugaaaau guacuggaaa uuaagggaaa ugauauugac 2700
ccugaagcag uuaaagguga aguguuaaaa guuggaaaua agagcuguga gaauauacac 2760
uuacauucug aagccguuuu augcacgguc cccaaugacc ugcugaaauu gaacagcgag 2820
cuaaauauag aguggaagca agcaauuucu ucaaccgucc uuggaaaagu aauaguucaa 2880
ccagaucaga auuucacagg auugauugcu gguguugucu caauaucaac agcacuguua 2940
uuacuacuug gguuuuuccu guggcugaaa aagagaaagc aaauuaaaga ucugggcagu 3000
gaauuaguuc gcuacgaugc aagaguacac acuccucauu uggauaggcu uguaagugcc 3060
cgaaguguaa gcccaacuac agaaaugguu ucaaaugaau cuguagacua ccgagcuacu 3120
uuuccagaag aucaguuucc uaauucaucu cagaacgguu caugccgaca agugcaguau 3180
ccucugacag acaugucccc cauccuaacu aguggggacu cugauauauc caguccauua 3240
cugcaaaaua cuguccacau ugaccucagu gcucuaaauc cagagcuggu ccaggcagug 3300
cagcauguag ugauugggcc caguagccug auugugcauu ucaaugaagu cauaggaaga 3360
gggcauuuug guuguguaua ucaugggacu uuguuggaca augauggcaa gaaaauucac 3420
ugugcuguga aauccuugaa cagaaucacu gacauaggag aaguuuccca auuucugacc 3480
gagggaauca ucaugaaaga uuuuagucau cccaaugucc ucucgcuccu gggaaucugc 3540
cugcgaagug aagggucucc gcuggugguc cuaccauaca ugaaacaugg agaucuucga 3600
aauuucauuc gaaaugagac ucauaaucca acuguaaaag aucuuauugg cuuuggucuu 3660
caaguagcca aaggcaugaa auaucuugca agcaaaaagu uuguccacag agacuuggcu 3720
gcaagaaacu guaugcugga ugaaaaauuc acagucaagg uugcugauuu uggucuugcc 3780
agagacaugu augauaaaga auacuauagu guacacaaca aaacaggugc aaagcugcca 3840
gugaagugga uggcuuugga aagucugcaa acucaaaagu uuaccaccaa gucagaugug 3900
ugguccuuug gcgugcuccu cugggagcug augacaagag gagccccacc uuauccugac 3960
guaaacaccu uugauauaac uguuuacuug uugcaaggga gaagacuccu acaacccgaa 4020
uacugcccag accccuuaua ugaaguaaug cuaaaaugcu ggcacccuaa agccgaaaug 4080
cgcccauccu uuucugaacu ggugucccgg auaucagcga ucuucucuac uuucauuggg 4140
gagcacuaug uccaugugaa cgcuacuuau gugaacguaa aaugugucgc uccguauccu 4200
ucucuguugu caucagaaga uaacgcugau gaugaggugg acacacgacc agccuccuuc 4260
ugggagacau cauagugcua guacuauguc aaagcaacag uccacacuuu guccaauggu 4320
uuuuucacug ccugaccuuu aaaaggccau cgauauucuu ugcucuugcc aaaauugcac 4380
uauuauagga cuuguauugu uauuuaaauu acuggauucu aaggaauuuc uuaucugaca 4440
gagcaucaga accagaggcu uggucccaca ggccacggac caauggccug cagccgugac 4500
aacacuccug ucauauugga guccaaaacu ugaauucugg guugaauuuu uuaaaaauca 4560
gguaccacuu gauuucauau gggaaauuga agcaggaaau auugagggcu ucuugaucac 4620
agaaaacuca gaagagauag uaaugcucag gacaggagcg gcagccccag aacaggccac 4680
ucauuuagaa uucuaguguu ucaaaacacu uuuguguguu guauggucaa uaacauuuuu 4740
cauuacugau ggugucauuc acccauuagg uaaacauucc cuuuuaaaug uuuguuuguu 4800
uuuugagaca ggaucucacu cuguugccag ggcuguagug caguggugug aucauagcuc 4860
acugcaaccu ccaccuccca ggcucaagcc ucccgaauag cugggacuac aggcgcacac 4920
caccaucccc ggcuaauuuu uguauuuuuu guagagacgg gguuuugcca uguugccaag 4980
gcugguuuca aacuccugga cucaagaaau ccacccaccu cagccuccca aagugcuagg 5040
auuacaggca ugagccacug cgcccagccc uuauaaauuu uuguauagac auuccuuugg 5100
uuggaagaau auuuauaggc aauacaguca aaguuucaaa auagcaucac acaaaacaug 5160
uuuauaaaug aacaggaugu aauguacaua gaugacauua agaaaauuug uaugaaauaa 5220
uuuagucauc augaaauauu uaguugucau auaaaaaccc acuguuugag aaugaugcua 5280
cucugaucua augaauguga acauguagau guuuugugug uauuuuuuua aaugaaaacu 5340
caaaauaaga caaguaauuu guugauaaau auuuuuaaag auaacucagc auguuuguaa 5400
agcaggauac auuuuacuaa aagguucauu gguuccaauc acagcucaua gguagagcaa 5460
agaaagggug gauggauuga aaagauuagc cucugucucg guggcagguu cccaccucgc 5520
aagcaauugg aaacaaaacu uuuggggagu uuuauuuugc auuagggugu guuuuauguu 5580
aagcaaaaca uacuuuagaa acaaaugaaa aaggcaauug aaaaucccag cuauuucacc 5640
uagauggaau agccacccug agcagaacuu ugugaugcuu cauucugugg aauuuugugc 5700
uugcuacugu auagugcaug ugguguaggu uacucuaacu gguuuugucg acguaaacau 5760
uuaaaguguu auauuuuuua uaaaaauguu uauuuuuaau gauaugagaa aaauuuuguu 5820
aggccacaaa aacacugcac ugugaacauu uuagaaaagg uaugucagac ugggauuaau 5880
gacagcauga uuuucaauga cuguaaauug cgauaaggaa auguacugau ugccaauaca 5940
ccccacccuc auuacaucau caggacuuga agccaagggu uaacccagca agcuacaaag 6000
aggguguguc acacugaaac ucaauaguug aguuuggcug uuguugcagg aaaaugauua 6060
uaacuaaaag cucucugaua gugcagagac uuaccagaag acacaaggaa uuguacugaa 6120
gagcuauuac aauccaaaua uugccguuuc auaaauguaa uaaguaauac uaauucacag 6180
aguauuguaa augguggaug acaaaagaaa aucugcucug uggaaagaaa gaacugucuc 6240
uaccaggguc aagagcauga acgcaucaau agaaagaacu cggggaaaca ucccaucaac 6300
aggacuacac acuuguauau acauucuuga gaacacugca augugaaaau cacguuugcu 6360
auuuauaaac uuguccuuag auuaaugugu cuggacagau ugugggagua agugauucuu 6420
cuaagaauua gauacuuguc acugccuaua ccugcagcug aacugaaugg uacuucguau 6480
guuaauaguu guucugauaa aucaugcaau uaaaguaaag ugaugcaaca ucuugua 6537
<210> 10
<211> 60
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 10
cacagagacu uggcugcaag aaacuguauu gguccuuugg cgugcuccuc ugggagcuga 60
<210> 11
<211> 247
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 11
aauccaacug uaaaagaucu uauuggcuuu ggucuucaag uagccaaagg caugaaauau 60
cuugcaagca aaaaguuugu ccacagagac uuggcugcaa gaaacuguau ugguccuuug 120
gcgugcuccu cugggagcug augacaagag gagccccacc uuauccugac guaaacaccu 180
uugauauaac uguuuacuug uugcaaggga gaagacuccu acaacccgaa uacugcccag 240
accccuu 247
<210> 12
<211> 6471
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 12
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
guuuaauaua cuucaggcug cguaugucag caagccuggg gcccagcuug cuagacaaau 1200
aggagccagc cugaaugaug acauucuuuu cgggguguuc gcacaaagca agccagauuc 1260
ugccgaacca auggaucgau cugccaugug ugcauucccu aucaaauaug ucaacgacuu 1320
cuucaacaag aucgucaaca aaaacaaugu gagaugucuc cagcauuuuu acggacccaa 1380
ucaugagcac ugcuuuaaua ggacacuucu gagaaauuca ucaggcugug aagcgcgccg 1440
ugaugaauau cgaacagagu uuaccacagc uuugcagcgc guugacuuau ucauggguca 1500
auucagcgaa guccucuuaa caucuauauc caccuucauu aaaggagacc ucaccauagc 1560
uaaucuuggg acaucagagg gucgcuucau gcagguugug guuucucgau caggaccauc 1620
aaccccucau gugaauuuuc uccuggacuc ccauccagug ucuccagaag ugauugugga 1680
gcauacauua aaccaaaaug gcuacacacu gguuaucacu gggaagaaga ucacgaagau 1740
cccauugaau ggcuugggcu gcagacauuu ccaguccugc agucaaugcc ucucugcccc 1800
acccuuuguu caguguggcu ggugccacga caaaugugug cgaucggagg aaugccugag 1860
cgggacaugg acucaacaga ucugucugcc ugcaaucuac aagguuuucc caaauagugc 1920
accccuugaa ggagggacaa ggcugaccau auguggcugg gacuuuggau uucggaggaa 1980
uaauaaauuu gauuuaaaga aaacuagagu ucuccuugga aaugagagcu gcaccuugac 2040
uuuaagugag agcacgauga auacauugaa augcacaguu gguccugcca ugaauaagca 2100
uuucaauaug uccauaauua uuucaaaugg ccacgggaca acacaauaca guacauucuc 2160
cuauguggau ccuguaauaa caaguauuuc gccgaaauac gguccuaugg cugguggcac 2220
uuuacuuacu uuaacuggaa auuaccuaaa cagugggaau ucuagacaca uuucaauugg 2280
uggaaaaaca uguacuuuaa aaaguguguc aaacaguauu cuugaauguu auaccccagc 2340
ccaaaccauu ucaacugagu uugcuguuaa auugaaaauu gacuuagcca accgagagac 2400
aagcaucuuc aguuaccgug aagaucccau ugucuaugaa auucauccaa ccaaaucuuu 2460
uauuaguggu gggagcacaa uaacaggugu ugggaaaaac cugaauucag uuaguguccc 2520
gagaaugguc auaaaugugc augaagcagg aaggaacuuu acaguggcau gucaacaucg 2580
cucuaauuca gagauaaucu guuguaccac uccuucccug caacagcuga aucugcaacu 2640
cccccugaaa accaaagccu uuuucauguu agaugggauc cuuuccaaau acuuugaucu 2700
cauuuaugua cauaauccug uguuuaagcc uuuugaaaag ccagugauga ucucaauggg 2760
caaugaaaau guacuggaaa uuaagggaaa ugauauugac ccugaagcag uuaaagguga 2820
aguguuaaaa guuggaaaua agagcuguga gaauauacac uuacauucug aagccguuuu 2880
augcacgguc cccaaugacc ugcugaaauu gaacagcgag cuaaauauag aguggaagca 2940
agcaauuucu ucaaccgucc uuggaaaagu aauaguucaa ccagaucaga auuucacagg 3000
auugauugcu gguguugucu caauaucaac agcacuguua uuacuacuug gguuuuuccu 3060
guggcugaaa aagagaaagc aaauuaaaga ucugggcagu gaauuaguuc gcuacgaugc 3120
aagaguacac acuccucauu uggauaggcu uguaagugcc cgaaguguaa gcccaacuac 3180
agaaaugguu ucaaaugaau cuguagacua ccgagcuacu uuuccagaag aucaguuucc 3240
uaauucaucu cagaacgguu caugccgaca agugcaguau ccucugacag acaugucccc 3300
cauccuaacu aguggggacu cugauauauc caguccauua cugcaaaaua cuguccacau 3360
ugaccucagu gcucuaaauc cagagcuggu ccaggcagug cagcauguag ugauugggcc 3420
caguagccug auugugcauu ucaaugaagu cauaggaaga gggcauuuug guuguguaua 3480
ucaugggacu uuguuggaca augauggcaa gaaaauucac ugugcuguga aauccuugaa 3540
cagaaucacu gacauaggag aaguuuccca auuucugacc gagggaauca ucaugaaaga 3600
uuuuagucau cccaaugucc ucucgcuccu gggaaucugc cugcgaagug aagggucucc 3660
gcuggugguc cuaccauaca ugaaacaugg agaucuucga aauuucauuc gaaaugagac 3720
ucauaaucca acuguaaaag aucuuauugg cuuuggucuu caaguagcca aaggcaugaa 3780
auaucuugca agcaaaaagu uuguccacag agacuuggcu gcaagaaacu guauuggucc 3840
uuuggcgugc uccucuggga gcugaugaca agaggagccc caccuuaucc ugacguaaac 3900
accuuugaua uaacuguuua cuuguugcaa gggagaagac uccuacaacc cgaauacugc 3960
ccagaccccu uauaugaagu aaugcuaaaa ugcuggcacc cuaaagccga aaugcgccca 4020
uccuuuucug aacugguguc ccggauauca gcgaucuucu cuacuuucau uggggagcac 4080
uauguccaug ugaacgcuac uuaugugaac guaaaaugug ucgcuccgua uccuucucug 4140
uugucaucag aagauaacgc ugaugaugag guggacacac gaccagccuc cuucugggag 4200
acaucauagu gcuaguacua ugucaaagca acaguccaca cuuuguccaa ugguuuuuuc 4260
acugccugac cuuuaaaagg ccaucgauau ucuuugcucu ugccaaaauu gcacuauuau 4320
aggacuugua uuguuauuua aauuacugga uucuaaggaa uuucuuaucu gacagagcau 4380
cagaaccaga ggcuuggucc cacaggccac ggaccaaugg ccugcagccg ugacaacacu 4440
ccugucauau uggaguccaa aacuugaauu cuggguugaa uuuuuuaaaa aucagguacc 4500
acuugauuuc auaugggaaa uugaagcagg aaauauugag ggcuucuuga ucacagaaaa 4560
cucagaagag auaguaaugc ucaggacagg agcggcagcc ccagaacagg ccacucauuu 4620
agaauucuag uguuucaaaa cacuuuugug uguuguaugg ucaauaacau uuuucauuac 4680
ugaugguguc auucacccau uagguaaaca uucccuuuua aauguuuguu uguuuuuuga 4740
gacaggaucu cacucuguug ccagggcugu agugcagugg ugugaucaua gcucacugca 4800
accuccaccu cccaggcuca agccucccga auagcuggga cuacaggcgc acaccaccau 4860
ccccggcuaa uuuuuguauu uuuuguagag acgggguuuu gccauguugc caaggcuggu 4920
uucaaacucc uggacucaag aaauccaccc accucagccu cccaaagugc uaggauuaca 4980
ggcaugagcc acugcgccca gcccuuauaa auuuuuguau agacauuccu uugguuggaa 5040
gaauauuuau aggcaauaca gucaaaguuu caaaauagca ucacacaaaa cauguuuaua 5100
aaugaacagg auguaaugua cauagaugac auuaagaaaa uuuguaugaa auaauuuagu 5160
caucaugaaa uauuuaguug ucauauaaaa acccacuguu ugagaaugau gcuacucuga 5220
ucuaaugaau gugaacaugu agauguuuug uguguauuuu uuuaaaugaa aacucaaaau 5280
aagacaagua auuuguugau aaauauuuuu aaagauaacu cagcauguuu guaaagcagg 5340
auacauuuua cuaaaagguu cauugguucc aaucacagcu cauagguaga gcaaagaaag 5400
gguggaugga uugaaaagau uagccucugu cucgguggca gguucccacc ucgcaagcaa 5460
uuggaaacaa aacuuuuggg gaguuuuauu uugcauuagg guguguuuua uguuaagcaa 5520
aacauacuuu agaaacaaau gaaaaaggca auugaaaauc ccagcuauuu caccuagaug 5580
gaauagccac ccugagcaga acuuugugau gcuucauucu guggaauuuu gugcuugcua 5640
cuguauagug cauguggugu agguuacucu aacugguuuu gucgacguaa acauuuaaag 5700
uguuauauuu uuuauaaaaa uguuuauuuu uaaugauaug agaaaaauuu uguuaggcca 5760
caaaaacacu gcacugugaa cauuuuagaa aagguauguc agacugggau uaaugacagc 5820
augauuuuca augacuguaa auugcgauaa ggaaauguac ugauugccaa uacaccccac 5880
ccucauuaca ucaucaggac uugaagccaa ggguuaaccc agcaagcuac aaagagggug 5940
ugucacacug aaacucaaua guugaguuug gcuguuguug caggaaaaug auuauaacua 6000
aaagcucucu gauagugcag agacuuacca gaagacacaa ggaauuguac ugaagagcua 6060
uuacaaucca aauauugccg uuucauaaau guaauaagua auacuaauuc acagaguauu 6120
guaaauggug gaugacaaaa gaaaaucugc ucuguggaaa gaaagaacug ucucuaccag 6180
ggucaagagc augaacgcau caauagaaag aacucgggga aacaucccau caacaggacu 6240
acacacuugu auauacauuc uugagaacac ugcaauguga aaaucacguu ugcuauuuau 6300
aaacuugucc uuagauuaau gugucuggac agauuguggg aguaagugau ucuucuaaga 6360
auuagauacu ugucacugcc uauaccugca gcugaacuga augguacuuc guauguuaau 6420
aguuguucug auaaaucaug caauuaaagu aaagugaugc aacaucuugu a 6471
<210> 13
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Val Tyr Glu Ile His Pro Thr Lys Ser Phe Ile Arg His Val Asn Ile
1 5 10 15
Ala Leu Ile Gln Arg
20
<210> 14
<211> 51
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Gln Thr Ile Ser Thr Glu Phe Ala Val Lys Leu Lys Ile Asp Leu Ala
1 5 10 15
Asn Arg Glu Thr Ser Ile Phe Ser Tyr Arg Glu Asp Pro Ile Val Tyr
20 25 30
Glu Ile His Pro Thr Lys Ser Phe Ile Arg His Val Asn Ile Ala Leu
35 40 45
Ile Gln Arg
50
<210> 15
<211> 764
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
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 Arg His Val Asn Ile Ala Leu Ile Gln Arg
755 760
<210> 16
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Val His Arg Asp Leu Ala Ala Arg Asn Cys Ile Gly Pro Leu Ala Cys
1 5 10 15
Ser Ser Gly Ser
20
<210> 17
<211> 50
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Asn Glu Thr His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu
1 5 10 15
Gln Val Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His
20 25 30
Arg Asp Leu Ala Ala Arg Asn Cys Ile Gly Pro Leu Ala Cys Ser Ser
35 40 45
Gly Ser
50
<210> 18
<211> 1220
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
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
1185 1190 1195 1200
Val His Arg Asp Leu Ala Ala Arg Asn Cys Ile Gly Pro Leu Ala Cys
1205 1210 1215
Ser Ser Gly Ser
1220
<210> 19
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atccaaccaa atcttttatt aggcatg 27
<210> 20
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cttctggaaa agtagctcgg tagtct 26
<210> 21
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
caaatctttt attaggcatg tcaacatc 28
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
aatttctgac cgagggaatc at 22
<210> 23
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
acgccaaagg accaatacag tttc 24
<210> 24
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gccaaaggac caatacagtt tctt 24
<210> 25
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
taatacgact cactataggg agcgatgttg acatgcctaa taaaaga 47
<210> 26
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gtgtgtcaaa cagtattctt g 21
<210> 27
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ttgacatgcc taataaaaga tttggttg 28
<210> 28
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
taatacgact cactataggg gagcacgcca aaggaccaat aca 43
<210> 29
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tgaaatatct tgcaagcaaa aagtt 25
<210> 30
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
agcacgccaa aggaccaata cagt 24

Claims (10)

1.一种评价患者对MET抑制剂敏感性的试剂盒，其特征在于，该试剂盒包括能够特异性地扩增第一核苷酸序列的第一引物对，和/或，能够与所述第一核苷酸序列特异性结合的第一探针，其中，所述第一核苷酸序列如SEQ ID NO.1所示；和/或，
该试剂盒包括能够特异性地扩增第二核苷酸序列的第二引物对，和/或，能够与所述第二核苷酸序列特异性结合的第二探针，其中，所述第二核苷酸序列如SEQ ID NO.4所示。
2.根据权利要求1所述的试剂盒，其特征在于，所述第一核苷酸序列如SEQ ID NO.2所示；优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；
所述第二核苷酸序列如SEQ ID NO.5所示；优选地，所述第二核苷酸序列如SEQ IDNO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列。
3.根据权利要求1或2所述的试剂盒，其特征在于，所述患者为胶质瘤患者。
4.一种评价患者对MET抑制剂敏感性的试剂盒，其特征在于，该试剂盒包括能够特异性地扩增第三核苷酸序列的第三引物对，和/或，能够与所述第三核苷酸序列特异性结合的第三探针，其中，所述第三核苷酸序列如SEQ ID NO.7所示；和/或，
该试剂盒包括能够特异性地扩增第四核苷酸序列的第四引物对，和/或，能够与所述第四核苷酸序列特异性结合的第四探针，其中，所述第四核苷酸序列如SEQ ID NO.10所示。
5.根据权利要求4所述的试剂盒，其特征在于，所述第三核苷酸序列如SEQ ID NO.8所示；优选地，所述第三核苷酸序列如SEQ ID NO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；
所述第四核苷酸序列如SEQ ID NO.11所示；优选地，所述第四核苷酸序列如SEQ IDNO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列。
6.一种评价患者对MET抑制剂敏感性的试剂盒，其特征在于，该试剂盒包括抗第一氨基酸序列的第一抗体，其中，所述第一氨基酸序列如SEQ ID NO.13所示；和/或，
该试剂盒包括抗第二氨基酸序列的第二抗体，其中，所述第二氨基酸序列如SEQ IDNO.16所示。
7.根据权利要求6所述的试剂盒，其特征在于，所述第一氨基酸序列如SEQ ID NO.14所示；优选地，所述第一氨基酸序列如SEQ ID NO.15所示，其中，SEQ ID NO.15所示的氨基酸序列由SEQ ID NO.9所示的mRNA编码得到；
所述第二氨基酸序列如SEQ ID NO.17所示；优选地，所述第二氨基酸序列如SEQ IDNO.18所示，其中，SEQ ID NO.18所示的氨基酸序列由SEQ ID NO.12所示的mRNA编码得到。
8.一种分子试剂在制备评价患者对MET抑制剂敏感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)～(10)中的至少一种：
(1)能够特异性扩增第一核苷酸序列的第一引物对；
(2)能够与所述第一核苷酸序列特异性结合的第一探针；
其中，所述第一核苷酸序列如SEQ ID NO.1所示；
(3)能够特异性扩增第二核苷酸序列的第二引物对；
(4)能够与所述第二核苷酸序列特异性结合的第二探针；
其中，所述第二核苷酸序列如SEQ ID NO.4所示；
(5)能够特异性扩增第三核苷酸序列的第三引物对；
(6)能够与所述第三核苷酸序列特异性结合的第三探针；
其中，所述第三核苷酸序列如SEQ ID NO.7所示；
(7)能够特异性扩增第四核苷酸序列的第四引物对；
(8)能够与所述第四核苷酸序列特异性结合的第四探针；
其中，所述第四核苷酸序列如SEQ ID NO.10所示；
(9)抗第一氨基酸序列的第一抗体；
其中，所述第一氨基酸序列如SEQ ID NO.13所示；
(10)抗第二氨基酸序列的第二抗体；
其中，所述第二氨基酸序列如SEQ ID NO.16所示。
9.根据权利要求8所述的用途，其特征在于，所述第一核苷酸序列如SEQ ID NO.2所示；优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；
所述第二核苷酸序列如SEQ ID NO.5所示；优选地，所述第二核苷酸序列如SEQ IDNO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列；
所述第三核苷酸序列如SEQ ID NO.8所示；优选地，所述第三核苷酸序列如SEQ IDNO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；
所述第四核苷酸序列如SEQ ID NO.11所示；优选地，所述第四核苷酸序列如SEQ IDNO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列；
所述第一氨基酸序列如SEQ ID NO.14所示；优选地，所述第一氨基酸序列如SEQ IDNO.15所示，其中，SEQ ID NO.15所示的氨基酸序列由SEQ ID NO.9所示的mRNA编码得到；
所述第二氨基酸序列如SEQ ID NO.17所示；优选地，所述第二氨基酸序列如SEQ IDNO.18所示，其中，SEQ ID NO.18所示的氨基酸序列由SEQ ID NO.12所示的mRNA编码得到。
10.一种评价患者对MET抑制剂敏感性的系统，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第一核苷酸序列、第二核苷酸序列、第三核苷酸序列或第四核苷酸序列中的至少一种，则判定患者对MET抑制剂敏感；
其中，所述第一核苷酸序列如SEQ ID NO.1所示；优选地，所述第一核苷酸序列如SEQID NO.2所示；更优选地，所述第一核苷酸序列如SEQ ID NO.3所示，其中，SEQ ID NO.3所示的核苷酸序列为10号外显子缺失的MET基因的cDNA序列；
所述第二核苷酸序列如SEQ ID NO.4所示；优选地，所述第二核苷酸序列如SEQ IDNO.5所示；更优选地，所述第二核苷酸序列如SEQ ID NO.6所示，其中，SEQ ID NO.6所示的核苷酸序列为19号外显子缺失的MET基因的cDNA序列；
所述第三核苷酸序列如SEQ ID NO.7所示；优选地，所述第三核苷酸序列如SEQ IDNO.8所示；更优选地，所述第三核苷酸序列如SEQ ID NO.9所示，其中，SEQ ID NO.9所示的核苷酸序列为10号外显子缺失的MET基因的mRNA序列；
所述第四核苷酸序列如SEQ ID NO.10所示；优选地，所述第四核苷酸序列如SEQ IDNO.11所示；更优选地，所述第四核苷酸序列如SEQ ID NO.12所示，其中，SEQ ID NO.12所示的核苷酸序列为19号外显子缺失的MET基因的mRNA序列。
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