CN113884684A - 一种活动性结核病多组学整合标志物、试剂盒及检测模型的构建方法 - Google Patents

一种活动性结核病多组学整合标志物、试剂盒及检测模型的构建方法 Download PDF

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CN113884684A
CN113884684A CN202111060620.0A CN202111060620A CN113884684A CN 113884684 A CN113884684 A CN 113884684A CN 202111060620 A CN202111060620 A CN 202111060620A CN 113884684 A CN113884684 A CN 113884684A
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张舒林
邹琛
徐润灏
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Abstract

本发明涉及生物医学技术领域，具体来说是一种活动性结核病多组学整合标志物、试剂盒及检测模型的构建方法，其特征在于，活动性结核病诊断多组学标志物为血液样本中的血浆蛋白标志物、氨基酸和胆汁酸，所述生物标志物包括：血浆蛋白APOA1、APOB、C3，氨基酸Orn、胆汁酸DCA和TCDCA的联合标志物；本发明首次采用血浆来源的APOA1、APOB、C3、Orn、DCA和TCDCA的联合标志物作为活动性肺结核检测的生物标记物，构建了活动性结核病检测的鉴别诊断模型，为结核病的临床诊断提供了新的方向，同时能够准确快速的对活动性肺结核进行检测，便于临床应用。

Description

一种活动性结核病多组学整合标志物、试剂盒及检测模型的 构建方法
技术领域
本发明涉及生物医学技术领域，具体来说涉及蛋白质组学技术、代谢组学技术、免疫比浊技术、质谱技术和机器学习算法，构建了涉及血浆蛋白和代谢分子标志物的活动性结核病检测模型。
背景技术
结核病(TB)目前仍是全球的重大公共卫生问题，对人类健康威胁极大。据世界卫生组织(WHO）统计，在2019年全球约1000万人新感染结核病，死亡人数近140万。结核病的高发病率和死亡率，以及通过空气传播，对患者和社会都产生了相当大的负担。因此，在早期及时而准确地发现结核感染，对于结核病患者的治疗及结核病扩散流行的控制是至关重要的。迄今为止，与结核有关的临床诊断方法均存在缺陷，如痰涂片镜检灵敏度较低，无法区分死菌活菌，且在多种环境中不适用；痰结核菌培养需要较长周期（6-8周），阳性率低（30%）；胸部X射线检查难以区分肺结核和其他肺部病变；基因检测的假阳性和假阴性率均高；结核菌素试验不能区分结核自然感染和卡介苗接种；血清结核抗体检测的灵敏度以及特异性目前还有局限。因此，仍需要发现更特异性和敏感的新的生物学标记物或者组合，为结核病的鉴别诊断提供快速、敏感、高效以及稳定的风险预测模型方法。
疾病状态下患者体内一些维持免疫平衡的蛋白异常表达，机体的生物转化模式亦发生变化，导致机体的免疫状态发生改变，体内的代谢物质随之改变。近些年来，检测机体的一些关键分子的异常表达预测疾病风险和治疗效果评判已有所报道，研究涉及前列腺癌、肝癌、乳腺癌、肝细胞癌、白血病以及卵巢癌等。结核菌与宿主的免疫防御系统的攻防转移是结核病发生发展的关键，如专利CN202110352760.9公开的活动性结核病标志物、试剂盒、检测方法及模型构建方法，通过在血浆蛋白中选用APOA4蛋白质、CFH蛋白质、CFHR5蛋白质、FGG蛋白质、MBL2蛋白质作为诊疗标志物，但在结核病发展过程中细菌和机体会通过调节相关蛋白的表达抑制或增强机体的免疫应答，而参与其中蛋白合成的氨基酸水平也将受到影响，结核病患者体内的微生物菌群的改变也将影响胆汁酸的代谢。筛选结核病发生和发展过程中关键蛋白分子、氨基酸与胆汁酸作为结核病诊疗标志物，是目前结核病的诊断以及鉴别诊断发展的新方向。
发明内容
本发明的目的在于解决现有结核病诊断技术存在的特异性不好，灵敏度不高的问题，提供一种基于多组学整合技术和机器学习算法的活动性结核病检测诊断模型及其构建方法，对结核病检测有良好的应用价值。
为了实现上述目的，本发明的第一方面公开了基于多组学整合技术和机器学习算法的活动性结核病检测诊断模型中用于诊断结核病的联合标志物包括APOA1、APOB、C3、Orn、DCA和TCDCA中的一种或几种的联合标志物。
其中，所述联合标志物中APOA1的蛋白质序列如SEQ ID NO.1所示；
所述联合标志物中APOB的蛋白质序列如SEQ ID NO.2所示；
所述联合标志物中C3的蛋白质序列如SEQ ID NO.3所示。
本发明的第二方面，公开了一种基于多组学整合联合标志物的活动性结核病检测试剂盒，包括用于从全血中分离血浆样本的试剂，血浆样本的蛋白质组学、氨基酸代谢组学和胆汁酸代谢组学定量分析相关试剂，用于检测APOA1、APOB、C3、Orn、DCA和TCDCA表达水平的试剂和包括APOA1、APOB、C3、Orn、DCA和TCDCA中一种或多种的标准品。
本发明的第三方面，公开了一种基于多组学整合联合标志物的活动性结核病检测试剂盒的检测方法，试剂盒中的检测试剂用于测定血浆样品中的血浆生物标志物的水平，包括以下步骤：
S1、测定受试者血浆样品中血浆蛋白标志物APOA1、APOB和C3的表达水平以及氨基酸标志物Orn和胆汁酸标志物DCA、TCDCA的表达水平；
S2、将测定的受试者血浆样品中的血浆生物和代谢分子标志物水平与正常受试者的血浆中标志物的水平进行对比；
S3、受试者的血浆样品中APOA1、APOB的表达水平相对于正常人受试者的水平的下降及C3的表达水平相对于正常人受试者的水平的上升，同时Orn和TCDCA在受试者体中升高，而DCA下降指示所述受试者存在结核病。
进一步地，TMT-LC/MS用于检测受试者的血浆样品中蛋白质的表达水平，其中所述受试者的血浆样品中APOA1、APOB的表达水平相对于正常人受试者的水平的下降及C3的表达水平相对于正常人受试者的水平的上升指示所述受试者存在结核病。
进一步地，使用免疫比浊方法检测APOA1、APOB、C3的表达水平。
进一步地，使用LCMS/MS方法检测Orn、DCA和TCDCA的表达水平。
进一步的，使用机器学习算法构建APOA1、APOB、C3、Orn、DCA和TCDCA的活动性结核病检测诊断模型。
本发明的最后一方面，公开了一种基于多组学整合联合标志物的活动性结核病快速诊断模型，采用检测算法构建APOA1、APOB、C3、Orn、DCA和TCDCA的活动性结核病检测诊断模型，所述检测算法包括以下步骤：
A、 从全血中分离血浆样本；
B、 测定血浆样本中血浆蛋白标志物APOA1、APOB、C3和氨基酸标志物Orn以及胆汁酸标志物DCA、TCDCA的表达水平；
C、 以80%的样本量作为训练集，20%的样本量作为测试集，对血浆蛋白标志物的表达进行整合分析，绘制诊断模型的ROC曲线，并根据ROC曲线构建得到活动性结核病检测模型，其中所述受试者的血浆样品中APOA1、APOB的表达水平相对于正常人受试者的水平的下降及C3的表达水平相对于正常人受试者的水平的上升，同时Orn和TCDCA在受试者体中升高，而DCA下降指示所述受试者存在结核病。
发明的有益效果
1. 本发明首次采用血浆来源的APOA1、APOB、C3、Orn、DCA和TCDCA的联合标志物作为活动性肺结核检测的生物标记物，构建了活动性结核病检测的检测诊断模型，为结核病的临床诊断提供了新的方向。因此，本发明克服了现有活动性肺结核缺乏明显典型症状，诊断检出率低、耗时长等缺点，具有特异性好、灵敏度高的特点，对活动性肺结核的辅助诊断有良好的临床应用价值。
2.本发明提供了利用血中血浆蛋白来源的APOA1、APOB、C3、以及代谢产物Orn、DCA和TCDCA作为活动性肺结核检测的生物标记物检测活动性肺结核的检测试剂以及试剂盒，能够准确快速的对活动性肺结核进行检测以及对活动性肺结核与其他肺部疾病进行区别检测，便于临床应用。
附图说明
图1为免疫比浊法对60例HC组、55例肺结核患者（TB组）、40例社区获得性肺炎（CAP组）和40例肺部肿瘤（LC组）的3个主要血浆蛋白标志物验证结果；
(A) APOA1 (B) APOB (C)C3。
图2为LCMS/MS方法对60例HC组、55例肺结核患者（TB组）、40例社区获得性肺炎（CAP组）和40例肺部肿瘤（LC组）的3个血浆代谢物质验证；
(A)Orn (B) DCA (C) TCDCA
图3为血浆多组学标志物检测诊断结核病的ROC曲线分析；
由APOA1、APOB、C3、Orn、DCA和TCDCA组成的活动性结核病检测诊断模型鉴别结核与非结核的ROC曲线分析。
具体实施方式
下面用实施例来进一步说明本发明，但本发明并不受其限制。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
1.简介
本发明提供了活动性结核病检测诊断模型的联合血浆多组学生物标志物为APOA1、APOB、C3、Orn、DCA和TCDCA，其指示结核病，且可以用于准确地鉴别诊断受试者中的结核病。
2.定义
在详细阐述本发明之前，提供要在本文中使用的某些术语的定义。除非另外定义，否则在本文中使用的所有技术和科学术语具有与本领域技术人员通常理解相同的含义。
术语“受试者”意图包括，能够直接地或间接地涉及结核病的任意障碍。受试者的例子包括哺乳动物，例如，人类、非人灵长类动物、狗、牛、马、猪、绵羊、山羊、猫、小鼠、兔、大鼠和转基因的非人动物。在某些实施方案中，所述受试者是人，例如，遭受结核病的人，处于遭受结核病及其相关的风险中的人，或者潜在地能够遭受结核病相关的痴呆的人。
术语“治疗”在本文中用于表示解除、减轻或缓解受试者中的疾病的至少一种征状。例如，关于结核病，术语“治疗”包括：解除、减轻或缓解认知损害(诸如记忆和/或定向的病损)或总体功能(所有功能，包括日常生活活动)的病损，和/或减慢或逆转总体或认知损害的进行性衰退。因此，术语“治疗”也包括：在疾病的临床表现或疾病的征状之前延迟或阻止发作，和/或减少疾病征状的发展或恶化的风险。
术语“约”或“大约”通常是指在给定的值或范围的5%内，或更优选1%内。
3.结核病的血浆多组学生物标志物
本发明涉及血浆多组学生物标志物：与“正常”受试者相比，其被发现在患有结核病的受试者的血浆生物样品中差别地存在。如果在样品中的一种血浆生物标志物的表达水平之间的差异被确定为统计上显著的，那么该血浆蛋白生物标志物在样品之间差别地存在。统计显著性的常见试验包括、但不限于：t-检验、ANOVA、Kniskal-Wallis、Wilcoxon、Mann-Whitney和比值比。单独的或组合的血浆蛋白生物标志物可以用于提供受试者罹患结核病的相对风险的量度。
4.测定样品中的血浆生物标志物的表达水平
通过任意合适的方法，可以测定生物样品中的血浆生物标志物的水平。可以使用任意可靠的用于测量样品中的血浆多组学标志物的水平或量的方法。通常，可以从生物样品检测和定量血浆蛋白，所述样品是采集受试者全血而分离得到的血浆样品，所述方法包括：蛋白定量方法(例如，串联质谱-液相色谱/质谱(TMT-LC/MS)、液相色谱-平行反应监测/质谱(LC-PRM/MS)等)、蛋白浓度测定方法(例如，酶联免疫吸附测定(ELISA)、免疫印迹法(WB)、蛋白芯片等）和模型构建算法(例如，逻辑回归算法、决策树、神经网络算法等）。其它示例性的技术包括聚合酶链式反应(PCR)、实时聚合酶链式反应(RT-PCR)等。
5.使用血浆多组学生物标志物鉴别诊断结核病
本发明描述的血浆生物标志物可以用于诊断试验中以评估受试者的结核病状态。疾病状态包括结核病的存在或不存在，结核病与其他肺部呼吸性疾病的区分。基于受试者的结核病状态，可以指示其它操作，包括、例如，其它诊断试验或治疗操作。
通常以测定的准确度、测定的灵敏度、测定的特异性或“曲线下面积”(AUC)（例如，在受试者操作特征(ROC)曲线下的面积）的方式，测量诊断试验的正确预测疾病状态的能力。本文中使用的准确度是错误分类的样品的比例的量度。可以将准确度计算为，正确分类的样品的总数除以样品的总数（例如在试验群体中）。灵敏度是通过试验预测为阳性的“真阳性”的量度，且可以计算为正确鉴别的结核病样品的数目除以结核病样品的总数。特异性是通过试验预测为阴性的“真阴性”的量度，且可以计算为正确鉴别的正常样品的数目除以正常样品的总数。AUC是受试者操作特征曲线下的面积的量度，所述曲线是灵敏度相对于假阳性率的图(1-特异性)。AUC越大，试验的预测值越有效。试验的实用性的其它有用的量度包括“阳性预测值”（它是试验为阳性的实际阳性的百分比）和“阴性预测值”（它是试验为阴性的实际阴性的百分比）。
下面用实施例来进一步说明本发明，但本发明并不受其限制。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
除非特别说明，以下实施例所用试剂和材料均于市购。
实施例1
从全血中分离血浆样本
用EDTA抗凝管采集活动性肺结核患者晨起空腹外周血全血，3000 rcf离心15min,6小时内分离血浆至新的1.5 mL离心管中。血浆样品保存于-80低温冰箱中。
实施例2
免疫比浊方法测定血浆样本中蛋白表达水平
样本收集：60例HC组、55例肺结核患者（TB组）、40例社区获得性肺炎（CAP组）和40例肺部肿瘤（LC组）。
按标准操作使用人APOA1、APOB 免疫比浊试剂盒(Mike, SiChuan, CN)和人C3免疫比浊试剂盒(Siemens, German)检测血浆蛋白标志物的表达水平。使用SPSS 22.0和MedCalc 15.0进行统计分析。正态性分布检验方法为K-S检验。呈正态分布的数据以x±s表示，使用独立样本t检验进行组间比较；呈非正态分布的计量资料以M(Q1,Q3)表示，组间比较采用Mann-Whitney U检验。将差异表达的蛋白进行受试者工作曲线（ReceiverOperating Curve，ROC）分析（P<0.05时，具有显著性差异，P<0.01时，具有极显著差异）。绘制包含误差线的散点图。
实施例 3
LCMS/MS方法测定血浆样本中的氨基酸和胆汁酸表达水平
样本收集：60例HC组、55例肺结核患者（TB组）、40例社区获得性肺炎（CAP组）和40例肺部肿瘤（LC组）。
按标准操作使用氨基酸代谢谱分析试剂盒（ClinMeta,Shanghai,CN）检测血浆氨基酸的表达水平。分离血清，取10μl待测样本于EP管中，加入40μl氨基酸样本稀释液，震荡混匀（2000rp，5min）。设置氮吹仪温度50℃吹干；加入复溶液100µl 至96孔板内，以600rpm震荡混匀5min，采用LC-20A液相色谱仪和API3200MD三重四级杆质谱仪进行检测，采用Analyst质谱工作站对数据和质谱图像进行收集。
色谱条件：使用ACE Excel3 C18（3.0mm×100mm）分析柱；柱温40℃；流动相A：超纯水与流动相添加剂的混合液；流动相B：甲醇与流动相添加剂的混合液；梯度洗脱, 流速550μL/min。
质谱条件：电喷雾离子源，正离子扫描，按试剂说明书进行参数设置：离子源参数为雾化气压力为50psi, 辅助加热器压力为50 psi，气帘气压力为30 psi，碰撞气压力为6psi；离子源电压为5000V；离子源温度为500℃。MRM扫描分析。
按标准操作使用胆汁酸代谢谱分析试剂盒（ClinMeta,Shanghai,CN）检测血浆胆汁酸的表达水平。分离血清，取血清样本100μl，加入含内标的提取液500μl，涡旋混匀(2500rpm，5min)；离心(13000rpm，10min)；取上清液400μl于96孔板中，60℃氮气吹干；加入复溶液100µl，将96孔板放置在微孔板恒温振荡器中混匀（700rmp 10min），转移96孔板中的复溶液到专用过滤板中，过滤板下放置新的96孔板，将过滤板及96孔板一起放置于多管架自动平衡离心机中进行过滤，离心（4000rpm，1min），收集滤液，采用LC-20A液相色谱仪和API3200MD三重四级杆质谱仪进行检测，采用Analyst质谱工作站对数据和质谱图像进行收集。
色谱条件：使用ACE Excel3 C18（3.0mm×100mm）分析柱；柱温40℃；流动相A：超纯水与流动相添加剂的混合液；流动相B：甲醇；梯度洗脱, 流速500μL/min。
质谱条件：电喷雾离子源，正离子扫描，按试剂说明书进行参数设置：离子源参数为雾化气压力为60psi, 辅助加热器压力为65 psi，气帘气压力为20 psi，碰撞气压力为8psi；离子源电压为-4500V；离子源温度为600℃。MRM扫描分析。
采用SPSS 22.0软件及MedCalc 15.0软件进行统计分析。正态性分布检验采用K-S检验。呈正态分布的数据以x±s表示，组间比较采用独立样本t检验；呈非正态分布的计量资料以M(Q1,Q3)表示，组间比较采用Mann-Whitney U检验。采用ROC曲线评估各指标的诊断性能。以P<0.05为差异有统计学意义。
实施例 4
活动性结核病快速诊断模型的构建：
采用SPSS 17.0对血浆蛋白标志物的表达进行整合分析，以80%的样本量作为训练集，20%的样本量作为测试集进行二元Logistics回归分析。利用GraphPad Prism 8软件进行诊断模型的ROC曲线的绘制。由APOA1, APOB, C3, Orn, DCA 和 TCDCA组成的活动性结核病快速诊断模型在鉴别结核与非结核的AUC达0.954，，敏感性为84.09%，特异性为95.31%，见图3所示。
通过对包括60例HC组、55例肺结核患者（TB组）、40例社区获得性肺炎（CAP组）和40例肺部肿瘤（LC组）的血浆来源的多组学标志物的分析，发现由APOA1, APOB, C3, Orn,DCA 和 TCDCA组成诊断模型有很好的诊断敏感性和不错的特异性，可为活动性结核病的鉴别诊断提供依据。
在本实施例中，基于血浆蛋白指标和氨基酸、胆汁酸的结核病风险预测模型的构建方法的构建系统，包括：
数据采集模块，至少用于数据采集，获取样本数据集；
数据处理模块，至少用于从样本数据集中提取可用于构建评估模型的有效样本；
模型构建模块，至少用于将所述有效样本的不完整数据集随机分割为训练集和验证集，并使用神经网络的方法拟合训练集，记录最优模型参数；
阈值计算模块，至少用于根据ROC曲线使用验证集计算模型分类阈值。
综上所述，本发明通过检测病人血浆蛋白、氨基酸和胆汁酸含量指标，然后利用建立的运算公式模型，计算各个标本值及诊断阈值，从而对病人进行肺结核的鉴别诊断。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
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Thr Gln Phe Asn Asn Asn Glu Tyr Ser Gln Asp Leu Asp Ala Tyr Asn
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2005 2010 2015
Leu Thr Leu Leu Asp Ser Pro Ile Lys Val Pro Leu Leu Leu Ser Glu
2020 2025 2030
Pro Ile Asn Ile Ile Asp Ala Leu Glu Met Arg Asp Ala Val Glu Lys
2035 2040 2045
Pro Gln Glu Phe Thr Ile Val Ala Phe Val Lys Tyr Asp Lys Asn Gln
2050 2055 2060
Asp Val His Ser Ile Asn Leu Pro Phe Phe Glu Thr Leu Gln Glu Tyr
2065 2070 2075 2080
Phe Glu Arg Asn Arg Gln Thr Ile Ile Val Val Leu Glu Asn Val Gln
2085 2090 2095
Arg Asn Leu Lys His Ile Asn Ile Asp Gln Phe Val Arg Lys Tyr Arg
2100 2105 2110
Ala Ala Leu Gly Lys Leu Pro Gln Gln Ala Asn Asp Tyr Leu Asn Ser
2115 2120 2125
Phe Asn Trp Glu Arg Gln Val Ser His Ala Lys Glu Lys Leu Thr Ala
2130 2135 2140
Leu Thr Lys Lys Tyr Arg Ile Thr Glu Asn Asp Ile Gln Ile Ala Leu
2145 2150 2155 2160
Asp Asp Ala Lys Ile Asn Phe Asn Glu Lys Leu Ser Gln Leu Gln Thr
2165 2170 2175
Tyr Met Ile Gln Phe Asp Gln Tyr Ile Lys Asp Ser Tyr Asp Leu His
2180 2185 2190
Asp Leu Lys Ile Ala Ile Ala Asn Ile Ile Asp Glu Ile Ile Glu Lys
2195 2200 2205
Leu Lys Ser Leu Asp Glu His Tyr His Ile Arg Val Asn Leu Val Lys
2210 2215 2220
Thr Ile His Asp Leu His Leu Phe Ile Glu Asn Ile Asp Phe Asn Lys
2225 2230 2235 2240
Ser Gly Ser Ser Thr Ala Ser Trp Ile Gln Asn Val Asp Thr Lys Tyr
2245 2250 2255
Gln Ile Arg Ile Gln Ile Gln Glu Lys Leu Gln Gln Leu Lys Arg His
2260 2265 2270
Ile Gln Asn Ile Asp Ile Gln His Leu Ala Gly Lys Leu Lys Gln His
2275 2280 2285
Ile Glu Ala Ile Asp Val Arg Val Leu Leu Asp Gln Leu Gly Thr Thr
2290 2295 2300
Ile Ser Phe Glu Arg Ile Asn Asp Ile Leu Glu His Val Lys His Phe
2305 2310 2315 2320
Val Ile Asn Leu Ile Gly Asp Phe Glu Val Ala Glu Lys Ile Asn Ala
2325 2330 2335
Phe Arg Ala Lys Val His Glu Leu Ile Glu Arg Tyr Glu Val Asp Gln
2340 2345 2350
Gln Ile Gln Val Leu Met Asp Lys Leu Val Glu Leu Ala His Gln Tyr
2355 2360 2365
Lys Leu Lys Glu Thr Ile Gln Lys Leu Ser Asn Val Leu Gln Gln Val
2370 2375 2380
Lys Ile Lys Asp Tyr Phe Glu Lys Leu Val Gly Phe Ile Asp Asp Ala
2385 2390 2395 2400
Val Lys Lys Leu Asn Glu Leu Ser Phe Lys Thr Phe Ile Glu Asp Val
2405 2410 2415
Asn Lys Phe Leu Asp Met Leu Ile Lys Lys Leu Lys Ser Phe Asp Tyr
2420 2425 2430
His Gln Phe Val Asp Glu Thr Asn Asp Lys Ile Arg Glu Val Thr Gln
2435 2440 2445
Arg Leu Asn Gly Glu Ile Gln Ala Leu Glu Leu Pro Gln Lys Ala Glu
2450 2455 2460
Ala Leu Lys Leu Phe Leu Glu Glu Thr Lys Ala Thr Val Ala Val Tyr
2465 2470 2475 2480
Leu Glu Ser Leu Gln Asp Thr Lys Ile Thr Leu Ile Ile Asn Trp Leu
2485 2490 2495
Gln Glu Ala Leu Ser Ser Ala Ser Leu Ala His Met Lys Ala Lys Phe
2500 2505 2510
Arg Glu Thr Leu Glu Asp Thr Arg Asp Arg Met Tyr Gln Met Asp Ile
2515 2520 2525
Gln Gln Glu Leu Gln Arg Tyr Leu Ser Leu Val Gly Gln Val Tyr Ser
2530 2535 2540
Thr Leu Val Thr Tyr Ile Ser Asp Trp Trp Thr Leu Ala Ala Lys Asn
2545 2550 2555 2560
Leu Thr Asp Phe Ala Glu Gln Tyr Ser Ile Gln Asp Trp Ala Lys Arg
2565 2570 2575
Met Lys Ala Leu Val Glu Gln Gly Phe Thr Val Pro Glu Ile Lys Thr
2580 2585 2590
Ile Leu Gly Thr Met Pro Ala Phe Glu Val Ser Leu Gln Ala Leu Gln
2595 2600 2605
Lys Ala Thr Phe Gln Thr Pro Asp Phe Ile Val Pro Leu Thr Asp Leu
2610 2615 2620
Arg Ile Pro Ser Val Gln Ile Asn Phe Lys Asp Leu Lys Asn Ile Lys
2625 2630 2635 2640
Ile Pro Ser Arg Phe Ser Thr Pro Glu Phe Thr Ile Leu Asn Thr Phe
2645 2650 2655
His Ile Pro Ser Phe Thr Ile Asp Phe Val Glu Met Lys Val Lys Ile
2660 2665 2670
Ile Arg Thr Ile Asp Gln Met Leu Asn Ser Glu Leu Gln Trp Pro Val
2675 2680 2685
Pro Asp Ile Tyr Leu Arg Asp Leu Lys Val Glu Asp Ile Pro Leu Ala
2690 2695 2700
Arg Ile Thr Leu Pro Asp Phe Arg Leu Pro Glu Ile Ala Ile Pro Glu
2705 2710 2715 2720
Phe Ile Ile Pro Thr Leu Asn Leu Asn Asp Phe Gln Val Pro Asp Leu
2725 2730 2735
His Ile Pro Glu Phe Gln Leu Pro His Ile Ser His Thr Ile Glu Val
2740 2745 2750
Pro Thr Phe Gly Lys Leu Tyr Ser Ile Leu Lys Ile Gln Ser Pro Leu
2755 2760 2765
Phe Thr Leu Asp Ala Asn Ala Asp Ile Gly Asn Gly Thr Thr Ser Ala
2770 2775 2780
Asn Glu Ala Gly Ile Ala Ala Ser Ile Thr Ala Lys Gly Glu Ser Lys
2785 2790 2795 2800
Leu Glu Val Leu Asn Phe Asp Phe Gln Ala Asn Ala Gln Leu Ser Asn
2805 2810 2815
Pro Lys Ile Asn Pro Leu Ala Leu Lys Glu Ser Val Lys Phe Ser Ser
2820 2825 2830
Lys Tyr Leu Arg Thr Glu His Gly Ser Glu Met Leu Phe Phe Gly Asn
2835 2840 2845
Ala Ile Glu Gly Lys Ser Asn Thr Val Ala Ser Leu His Thr Glu Lys
2850 2855 2860
Asn Thr Leu Glu Leu Ser Asn Gly Val Ile Val Lys Ile Asn Asn Gln
2865 2870 2875 2880
Leu Thr Leu Asp Ser Asn Thr Lys Tyr Phe His Lys Leu Asn Ile Pro
2885 2890 2895
Lys Leu Asp Phe Ser Ser Gln Ala Asp Leu Arg Asn Glu Ile Lys Thr
2900 2905 2910
Leu Leu Lys Ala Gly His Ile Ala Trp Thr Ser Ser Gly Lys Gly Ser
2915 2920 2925
Trp Lys Trp Ala Cys Pro Arg Phe Ser Asp Glu Gly Thr His Glu Ser
2930 2935 2940
Gln Ile Ser Phe Thr Ile Glu Gly Pro Leu Thr Ser Phe Gly Leu Ser
2945 2950 2955 2960
Asn Lys Ile Asn Ser Lys His Leu Arg Val Asn Gln Asn Leu Val Tyr
2965 2970 2975
Glu Ser Gly Ser Leu Asn Phe Ser Lys Leu Glu Ile Gln Ser Gln Val
2980 2985 2990
Asp Ser Gln His Val Gly His Ser Val Leu Thr Ala Lys Gly Met Ala
2995 3000 3005
Leu Phe Gly Glu Gly Lys Ala Glu Phe Thr Gly Arg His Asp Ala His
3010 3015 3020
Leu Asn Gly Lys Val Ile Gly Thr Leu Lys Asn Ser Leu Phe Phe Ser
3025 3030 3035 3040
Ala Gln Pro Phe Glu Ile Thr Ala Ser Thr Asn Asn Glu Gly Asn Leu
3045 3050 3055
Lys Val Arg Phe Pro Leu Arg Leu Thr Gly Lys Ile Asp Phe Leu Asn
3060 3065 3070
Asn Tyr Ala Leu Phe Leu Ser Pro Ser Ala Gln Gln Ala Ser Trp Gln
3075 3080 3085
Val Ser Ala Arg Phe Asn Gln Tyr Lys Tyr Asn Gln Asn Phe Ser Ala
3090 3095 3100
Gly Asn Asn Glu Asn Ile Met Glu Ala His Val Gly Ile Asn Gly Glu
3105 3110 3115 3120
Ala Asn Leu Asp Phe Leu Asn Ile Pro Leu Thr Ile Pro Glu Met Arg
3125 3130 3135
Leu Pro Tyr Thr Ile Ile Thr Thr Pro Pro Leu Lys Asp Phe Ser Leu
3140 3145 3150
Trp Glu Lys Thr Gly Leu Lys Glu Phe Leu Lys Thr Thr Lys Gln Ser
3155 3160 3165
Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys Lys Asn Lys His Arg His
3170 3175 3180
Ser Ile Thr Asn Pro Leu Ala Val Leu Cys Glu Phe Ile Ser Gln Ser
3185 3190 3195 3200
Ile Lys Ser Phe Asp Arg His Phe Glu Lys Asn Arg Asn Asn Ala Leu
3205 3210 3215
Asp Phe Val Thr Lys Ser Tyr Asn Glu Thr Lys Ile Lys Phe Asp Lys
3220 3225 3230
Tyr Lys Ala Glu Lys Ser His Asp Glu Leu Pro Arg Thr Phe Gln Ile
3235 3240 3245
Pro Gly Tyr Thr Val Pro Val Val Asn Val Glu Val Ser Pro Phe Thr
3250 3255 3260
Ile Glu Met Ser Ala Phe Gly Tyr Val Phe Pro Lys Ala Val Ser Met
3265 3270 3275 3280
Pro Ser Phe Ser Ile Leu Gly Ser Asp Val Arg Val Pro Ser Tyr Thr
3285 3290 3295
Leu Ile Leu Pro Ser Leu Glu Leu Pro Val Leu His Val Pro Arg Asn
3300 3305 3310
Leu Lys Leu Ser Leu Pro Asp Phe Lys Glu Leu Cys Thr Ile Ser His
3315 3320 3325
Ile Phe Ile Pro Ala Met Gly Asn Ile Thr Tyr Asp Phe Ser Phe Lys
3330 3335 3340
Ser Ser Val Ile Thr Leu Asn Thr Asn Ala Glu Leu Phe Asn Gln Ser
3345 3350 3355 3360
Asp Ile Val Ala His Leu Leu Ser Ser Ser Ser Ser Val Ile Asp Ala
3365 3370 3375
Leu Gln Tyr Lys Leu Glu Gly Thr Thr Arg Leu Thr Arg Lys Arg Gly
3380 3385 3390
Leu Lys Leu Ala Thr Ala Leu Ser Leu Ser Asn Lys Phe Val Glu Gly
3395 3400 3405
Ser His Asn Ser Thr Val Ser Leu Thr Thr Lys Asn Met Glu Val Ser
3410 3415 3420
Val Ala Thr Thr Thr Lys Ala Gln Ile Pro Ile Leu Arg Met Asn Phe
3425 3430 3435 3440
Lys Gln Glu Leu Asn Gly Asn Thr Lys Ser Lys Pro Thr Val Ser Ser
3445 3450 3455
Ser Met Glu Phe Lys Tyr Asp Phe Asn Ser Ser Met Leu Tyr Ser Thr
3460 3465 3470
Ala Lys Gly Ala Val Asp His Lys Leu Ser Leu Glu Ser Leu Thr Ser
3475 3480 3485
Tyr Phe Ser Ile Glu Ser Ser Thr Lys Gly Asp Val Lys Gly Ser Val
3490 3495 3500
Leu Ser Arg Glu Tyr Ser Gly Thr Ile Ala Ser Glu Ala Asn Thr Tyr
3505 3510 3515 3520
Leu Asn Ser Lys Ser Thr Arg Ser Ser Val Lys Leu Gln Gly Thr Ser
3525 3530 3535
Lys Ile Asp Asp Ile Trp Asn Leu Glu Val Lys Glu Asn Phe Ala Gly
3540 3545 3550
Glu Ala Thr Leu Gln Arg Ile Tyr Ser Leu Trp Glu His Ser Thr Lys
3555 3560 3565
Asn His Leu Gln Leu Glu Gly Leu Phe Phe Thr Asn Gly Glu His Thr
3570 3575 3580
Ser Lys Ala Thr Leu Glu Leu Ser Pro Trp Gln Met Ser Ala Leu Val
3585 3590 3595 3600
Gln Val His Ala Ser Gln Pro Ser Ser Phe His Asp Phe Pro Asp Leu
3605 3610 3615
Gly Gln Glu Val Ala Leu Asn Ala Asn Thr Lys Asn Gln Lys Ile Arg
3620 3625 3630
Trp Lys Asn Glu Val Arg Ile His Ser Gly Ser Phe Gln Ser Gln Val
3635 3640 3645
Glu Leu Ser Asn Asp Gln Glu Lys Ala His Leu Asp Ile Ala Gly Ser
3650 3655 3660
Leu Glu Gly His Leu Arg Phe Leu Lys Asn Ile Ile Leu Pro Val Tyr
3665 3670 3675 3680
Asp Lys Ser Leu Trp Asp Phe Leu Lys Leu Asp Val Thr Thr Ser Ile
3685 3690 3695
Gly Arg Arg Gln His Leu Arg Val Ser Thr Ala Phe Val Tyr Thr Lys
3700 3705 3710
Asn Pro Asn Gly Tyr Ser Phe Ser Ile Pro Val Lys Val Leu Ala Asp
3715 3720 3725
Lys Phe Ile Ile Pro Gly Leu Lys Leu Asn Asp Leu Asn Ser Val Leu
3730 3735 3740
Val Met Pro Thr Phe His Val Pro Phe Thr Asp Leu Gln Val Pro Ser
3745 3750 3755 3760
Cys Lys Leu Asp Phe Arg Glu Ile Gln Ile Tyr Lys Lys Leu Arg Thr
3765 3770 3775
Ser Ser Phe Ala Leu Asn Leu Pro Thr Leu Pro Glu Val Lys Phe Pro
3780 3785 3790
Glu Val Asp Val Leu Thr Lys Tyr Ser Gln Pro Glu Asp Ser Leu Ile
3795 3800 3805
Pro Phe Phe Glu Ile Thr Val Pro Glu Ser Gln Leu Thr Val Ser Gln
3810 3815 3820
Phe Thr Leu Pro Lys Ser Val Ser Asp Gly Ile Ala Ala Leu Asp Leu
3825 3830 3835 3840
Asn Ala Val Ala Asn Lys Ile Ala Asp Phe Glu Leu Pro Thr Ile Ile
3845 3850 3855
Val Pro Glu Gln Thr Ile Glu Ile Pro Ser Ile Lys Phe Ser Val Pro
3860 3865 3870
Ala Gly Ile Val Ile Pro Ser Phe Gln Ala Leu Thr Ala Arg Phe Glu
3875 3880 3885
Val Asp Ser Pro Val Tyr Asn Ala Thr Trp Ser Ala Ser Leu Lys Asn
3890 3895 3900
Lys Ala Asp Tyr Val Glu Thr Val Leu Asp Ser Thr Cys Ser Ser Thr
3905 3910 3915 3920
Val Gln Phe Leu Glu Tyr Glu Leu Asn Val Leu Gly Thr His Lys Ile
3925 3930 3935
Glu Asp Gly Thr Leu Ala Ser Lys Thr Lys Gly Thr Phe Ala His Arg
3940 3945 3950
Asp Phe Ser Ala Glu Tyr Glu Glu Asp Gly Lys Tyr Glu Gly Leu Gln
3955 3960 3965
Glu Trp Glu Gly Lys Ala His Leu Asn Ile Lys Ser Pro Ala Phe Thr
3970 3975 3980
Asp Leu His Leu Arg Tyr Gln Lys Asp Lys Lys Gly Ile Ser Thr Ser
3985 3990 3995 4000
Ala Ala Ser Pro Ala Val Gly Thr Val Gly Met Asp Met Asp Glu Asp
4005 4010 4015
Asp Asp Phe Ser Lys Trp Asn Phe Tyr Tyr Ser Pro Gln Ser Ser Pro
4020 4025 4030
Asp Lys Lys Leu Thr Ile Phe Lys Thr Glu Leu Arg Val Arg Glu Ser
4035 4040 4045
Asp Glu Glu Thr Gln Ile Lys Val Asn Trp Glu Glu Glu Ala Ala Ser
4050 4055 4060
Gly Leu Leu Thr Ser Leu Lys Asp Asn Val Pro Lys Ala Thr Gly Val
4065 4070 4075 4080
Leu Tyr Asp Tyr Val Asn Lys Tyr His Trp Glu His Thr Gly Leu Thr
4085 4090 4095
Leu Arg Glu Val Ser Ser Lys Leu Arg Arg Asn Leu Gln Asn Asn Ala
4100 4105 4110
Glu Trp Val Tyr Gln Gly Ala Ile Arg Gln Ile Asp Asp Ile Asp Val
4115 4120 4125
Arg Phe Gln Lys Ala Ala Ser Gly Thr Thr Gly Thr Tyr Gln Glu Trp
4130 4135 4140
Lys Asp Lys Ala Gln Asn Leu Tyr Gln Glu Leu Leu Thr Gln Glu Gly
4145 4150 4155 4160
Gln Ala Ser Phe Gln Gly Leu Lys Asp Asn Val Phe Asp Gly Leu Val
4165 4170 4175
Arg Val Thr Gln Glu Phe His Met Lys Val Lys His Leu Ile Asp Ser
4180 4185 4190
Leu Ile Asp Phe Leu Asn Phe Pro Arg Phe Gln Phe Pro Gly Lys Pro
4195 4200 4205
Gly Ile Tyr Thr Arg Glu Glu Leu Cys Thr Met Phe Ile Arg Glu Val
4210 4215 4220
Gly Thr Val Leu Ser Gln Val Tyr Ser Lys Val His Asn Gly Ser Glu
4225 4230 4235 4240
Ile Leu Phe Ser Tyr Phe Gln Asp Leu Val Ile Thr Leu Pro Phe Glu
4245 4250 4255
Leu Arg Lys His Lys Leu Ile Asp Val Ile Ser Met Tyr Arg Glu Leu
4260 4265 4270
Leu Lys Asp Leu Ser Lys Glu Ala Gln Glu Val Phe Lys Ala Ile Gln
4275 4280 4285
Ser Leu Lys Thr Thr Glu Val Leu Arg Asn Leu Gln Asp Leu Leu Gln
4290 4295 4300
Phe Ile Phe Gln Leu Ile Glu Asp Asn Ile Lys Gln Leu Lys Glu Met
4305 4310 4315 4320
Lys Phe Thr Tyr Leu Ile Asn Tyr Ile Gln Asp Glu Ile Asn Thr Ile
4325 4330 4335
Phe Ser Asp Tyr Ile Pro Tyr Val Phe Lys Leu Leu Lys Glu Asn Leu
4340 4345 4350
Cys Leu Asn Leu His Lys Phe Asn Glu Phe Ile Gln Asn Glu Leu Gln
4355 4360 4365
Glu Ala Ser Gln Glu Leu Gln Gln Ile His Gln Tyr Ile Met Ala Leu
4370 4375 4380
Arg Glu Glu Tyr Phe Asp Pro Ser Ile Val Gly Trp Thr Val Lys Tyr
4385 4390 4395 4400
Tyr Glu Leu Glu Glu Lys Ile Val Ser Leu Ile Lys Asn Leu Leu Val
4405 4410 4415
Ala Leu Lys Asp Phe His Ser Glu Tyr Ile Val Ser Ala Ser Asn Phe
4420 4425 4430
Thr Ser Gln Leu Ser Ser Gln Val Glu Gln Phe Leu His Arg Asn Ile
4435 4440 4445
Gln Glu Tyr Leu Ser Ile Leu Thr Asp Pro Asp Gly Lys Gly Lys Glu
4450 4455 4460
Lys Ile Ala Glu Leu Ser Ala Thr Ala Gln Glu Ile Ile Lys Ser Gln
4465 4470 4475 4480
Ala Ile Ala Thr Lys Lys Ile Ile Ser Asp Tyr His Gln Gln Phe Arg
4485 4490 4495
Tyr Lys Leu Gln Asp Phe Ser Asp Gln Leu Ser Asp Tyr Tyr Glu Lys
4500 4505 4510
Phe Ile Ala Glu Ser Lys Arg Leu Ile Asp Leu Ser Ile Gln Asn Tyr
4515 4520 4525
His Thr Phe Leu Ile Tyr Ile Thr Glu Leu Leu Lys Lys Leu Gln Ser
4530 4535 4540
Thr Thr Val Met Asn Pro Tyr Met Lys Leu Ala Pro Gly Glu Leu Thr
4545 4550 4555 4560
Ile Ile Leu
<210> 3
<211> 1663
<212> PRT
<213> 前原蛋白C3(Preproprotein C3)
<400> 3
Met Gly Pro Thr Ser Gly Pro Ser Leu Leu Leu Leu Leu Leu Thr His
1 5 10 15
Leu Pro Leu Ala Leu Gly Ser Pro Met Tyr Ser Ile Ile Thr Pro Asn
20 25 30
Ile Leu Arg Leu Glu Ser Glu Glu Thr Met Val Leu Glu Ala His Asp
35 40 45
Ala Gln Gly Asp Val Pro Val Thr Val Thr Val His Asp Phe Pro Gly
50 55 60
Lys Lys Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr
65 70 75 80
Asn His Met Gly Asn Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe
85 90 95
Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr Val Gln Ala Thr Phe
100 105 110
Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu Gln Ser Gly
115 120 125
Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr Thr Pro Gly Ser Thr
130 135 140
Val Leu Tyr Arg Ile Phe Thr Val Asn His Lys Leu Leu Pro Val Gly
145 150 155 160
Arg Thr Val Met Val Asn Ile Glu Asn Pro Glu Gly Ile Pro Val Lys
165 170 175
Gln Asp Ser Leu Ser Ser Gln Asn Gln Leu Gly Val Leu Pro Leu Ser
180 185 190
Trp Asp Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala
195 200 205
Tyr Tyr Glu Asn Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val
210 215 220
Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val Glu Pro Thr Glu
225 230 235 240
Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu Glu Val Thr Ile Thr
245 250 255
Ala Arg Phe Leu Tyr Gly Lys Lys Val Glu Gly Thr Ala Phe Val Ile
260 265 270
Phe Gly Ile Gln Asp Gly Glu Gln Arg Ile Ser Leu Pro Glu Ser Leu
275 280 285
Lys Arg Ile Pro Ile Glu Asp Gly Ser Gly Glu Val Val Leu Ser Arg
290 295 300
Lys Val Leu Leu Asp Gly Val Gln Asn Pro Arg Ala Glu Asp Leu Val
305 310 315 320
Gly Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser
325 330 335
Asp Met Val Gln Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro
340 345 350
Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr Phe Lys Pro Gly Met
355 360 365
Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala
370 375 380
Tyr Arg Val Pro Val Ala Val Gln Gly Glu Asp Thr Val Gln Ser Leu
385 390 395 400
Thr Gln Gly Asp Gly Val Ala Lys Leu Ser Ile Asn Thr His Pro Ser
405 410 415
Gln Lys Pro Leu Ser Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser
420 425 430
Glu Ala Glu Gln Ala Thr Arg Thr Met Gln Ala Leu Pro Tyr Ser Thr
435 440 445
Val Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu
450 455 460
Leu Arg Pro Gly Glu Thr Leu Asn Val Asn Phe Leu Leu Arg Met Asp
465 470 475 480
Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu Ile Met Asn
485 490 495
Lys Gly Arg Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln
500 505 510
Asp Leu Val Val Leu Pro Leu Ser Ile Thr Thr Asp Phe Ile Pro Ser
515 520 525
Phe Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg
530 535 540
Glu Val Val Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Val
545 550 555 560
Gly Ser Leu Val Val Lys Ser Gly Gln Ser Glu Asp Arg Gln Pro Val
565 570 575
Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly Ala Arg
580 585 590
Val Val Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys
595 600 605
Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu Lys Ala Asp
610 615 620
Ile Gly Cys Thr Pro Gly Ser Gly Lys Asp Tyr Ala Gly Val Phe Ser
625 630 635 640
Asp Ala Gly Leu Thr Phe Thr Ser Ser Ser Gly Gln Gln Thr Ala Gln
645 650 655
Arg Ala Glu Leu Gln Cys Pro Gln Pro Ala Ala Arg Arg Arg Arg Ser
660 665 670
Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys
675 680 685
Glu Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met Arg
690 695 700
Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala Cys
705 710 715 720
Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg Arg
725 730 735
Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg Ser Asn Leu Asp
740 745 750
Glu Asp Ile Ile Ala Glu Glu Asn Ile Val Ser Arg Ser Glu Phe Pro
755 760 765
Glu Ser Trp Leu Trp Asn Val Glu Asp Leu Lys Glu Pro Pro Lys Asn
770 775 780
Gly Ile Ser Thr Lys Leu Met Asn Ile Phe Leu Lys Asp Ser Ile Thr
785 790 795 800
Thr Trp Glu Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys
805 810 815
Val Ala Asp Pro Phe Glu Val Thr Val Met Gln Asp Phe Phe Ile Asp
820 825 830
Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg
835 840 845
Ala Val Leu Tyr Asn Tyr Arg Gln Asn Gln Glu Leu Lys Val Arg Val
850 855 860
Glu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala Thr Thr Lys Arg
865 870 875 880
Arg His Gln Gln Thr Val Thr Ile Pro Pro Lys Ser Ser Leu Ser Val
885 890 895
Pro Tyr Val Ile Val Pro Leu Lys Thr Gly Leu Gln Glu Val Glu Val
900 905 910
Lys Ala Ala Val Tyr His His Phe Ile Ser Asp Gly Val Arg Lys Ser
915 920 925
Leu Lys Val Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val
930 935 940
Arg Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu Gly Val Gln Lys Glu
945 950 955 960
Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Glu Ser
965 970 975
Glu Thr Arg Ile Leu Leu Gln Gly Thr Pro Val Ala Gln Met Thr Glu
980 985 990
Asp Ala Val Asp Ala Glu Arg Leu Lys His Leu Ile Val Thr Pro Ser
995 1000 1005
Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile Ala
1010 1015 1020
Val His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe Gly Leu Glu
1025 1030 1035 1040
Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr Thr Gln Gln
1045 1050 1055
Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala Phe Val Lys Arg
1060 1065 1070
Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val Phe Ser Leu
1075 1080 1085
Ala Val Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys Gly Ala Val
1090 1095 1100
Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val Phe Gln Glu
1105 1110 1115 1120
Asp Ala Pro Val Ile His Gln Glu Met Ile Gly Gly Leu Arg Asn Asn
1125 1130 1135
Asn Glu Lys Asp Met Ala Leu Thr Ala Phe Val Leu Ile Ser Leu Gln
1140 1145 1150
Glu Ala Lys Asp Ile Cys Glu Glu Gln Val Asn Ser Leu Pro Gly Ser
1155 1160 1165
Ile Thr Lys Ala Gly Asp Phe Leu Glu Ala Asn Tyr Met Asn Leu Gln
1170 1175 1180
Arg Ser Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala Gln Met Gly
1185 1190 1195 1200
Arg Leu Lys Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr Ala Lys Asp
1205 1210 1215
Lys Asn Arg Trp Glu Asp Pro Gly Lys Gln Leu Tyr Asn Val Glu Ala
1220 1225 1230
Thr Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp Phe Asp Phe
1235 1240 1245
Val Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly Gly
1250 1255 1260
Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala Leu Ala
1265 1270 1275 1280
Gln Tyr Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn Leu Asp Val
1285 1290 1295
Ser Leu Gln Leu Pro Ser Arg Ser Ser Lys Ile Thr His Arg Ile His
1300 1305 1310
Trp Glu Ser Ala Ser Leu Leu Arg Ser Glu Glu Thr Lys Glu Asn Glu
1315 1320 1325
Gly Phe Thr Val Thr Ala Glu Gly Lys Gly Gln Gly Thr Leu Ser Val
1330 1335 1340
Val Thr Met Tyr His Ala Lys Ala Lys Asp Gln Leu Thr Cys Asn Lys
1345 1350 1355 1360
Phe Asp Leu Lys Val Thr Ile Lys Pro Ala Pro Glu Thr Glu Lys Arg
1365 1370 1375
Pro Gln Asp Ala Lys Asn Thr Met Ile Leu Glu Ile Cys Thr Arg Tyr
1380 1385 1390
Arg Gly Asp Gln Asp Ala Thr Met Ser Ile Leu Asp Ile Ser Met Met
1395 1400 1405
Thr Gly Phe Ala Pro Asp Thr Asp Asp Leu Lys Gln Leu Ala Asn Gly
1410 1415 1420
Val Asp Arg Tyr Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser Asp
1425 1430 1435 1440
Arg Asn Thr Leu Ile Ile Tyr Leu Asp Lys Val Ser His Ser Glu Asp
1445 1450 1455
Asp Cys Leu Ala Phe Lys Val His Gln Tyr Phe Asn Val Glu Leu Ile
1460 1465 1470
Gln Pro Gly Ala Val Lys Val Tyr Ala Tyr Tyr Asn Leu Glu Glu Ser
1475 1480 1485
Cys Thr Arg Phe Tyr His Pro Glu Lys Glu Asp Gly Lys Leu Asn Lys
1490 1495 1500
Leu Cys Arg Asp Glu Leu Cys Arg Cys Ala Glu Glu Asn Cys Phe Ile
1505 1510 1515 1520
Gln Lys Ser Asp Asp Lys Val Thr Leu Glu Glu Arg Leu Asp Lys Ala
1525 1530 1535
Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Arg Leu Val Lys Val
1540 1545 1550
Gln Leu Ser Asn Asp Phe Asp Glu Tyr Ile Met Ala Ile Glu Gln Thr
1555 1560 1565
Ile Lys Ser Gly Ser Asp Glu Val Gln Val Gly Gln Gln Arg Thr Phe
1570 1575 1580
Ile Ser Pro Ile Lys Cys Arg Glu Ala Leu Lys Leu Glu Glu Lys Lys
1585 1590 1595 1600
His Tyr Leu Met Trp Gly Leu Ser Ser Asp Phe Trp Gly Glu Lys Pro
1605 1610 1615
Asn Leu Ser Tyr Ile Ile Gly Lys Asp Thr Trp Val Glu His Trp Pro
1620 1625 1630
Glu Glu Asp Glu Cys Gln Asp Glu Glu Asn Gln Lys Gln Cys Gln Asp
1635 1640 1645
Leu Gly Ala Phe Thr Glu Ser Met Val Val Phe Gly Cys Pro Asn
1650 1655 1660

Claims (8)

1.一种基于多组学整合联合标志物的活动性结核病快速诊断模型，其特征在于，采用检测算法构建APOA1、APOB、C3、Orn、DCA和TCDCA的活动性结核病鉴别诊断模型，所述检测算法包括以下步骤： S1、 从全血中分离血浆样本；S2、 检测血浆样本中血浆蛋白标志物APOA1、APOB、C3，氨基酸标志物Orn和胆汁酸标志物DCA、TCDCA的表达水平； S3、以80%的样本量作为训练集，20%的样本量作为测试集，对血浆蛋白和代谢分子标志物进行整合分析，绘制诊断模型的ROC曲线，并根据ROC曲线构建得到活动性结核病检测模型，其中所述受试者的血浆样品中APOA1、APOB的表达水平相对于正常人受试者的水平的下降及C3的表达水平相对于正常人受试者的水平的上升，同时Orn和TCDCA在受试者体中升高，而DCA下降指示所述受试者存在结核病。
2.一种活动性结核病检测诊断标志物，其特征在于，包括血液样本中的血浆蛋白生物标志物、氨基酸代谢标志物和胆汁酸代谢标志物，所述标志物包括APOA1、APOB、C3、Orn、DCA和TCDCA中的一种或几种的联合标志物。
3.一种基于多组学整合联合标志物的活动性结核病检测试剂盒，其特征在于，包括用于从全血中分离血浆样本的试剂，血浆样本的蛋白质组学、氨基酸代谢组学和胆汁酸代谢组学定量分析相关试剂，用于检测APOA1、APOB、C3、Orn、DCA和TCDCA表达水平的试剂和包括APOA1、APOB、C3、Orn、DCA和TCDCA中一种或多种的标准品。
4.一种基于多组学整合联合标志物的活动性结核病检测试剂盒的检测方法，其特征在于，试剂盒中的检测试剂用于测定血浆样品中的血浆生物标志物的水平，包括以下步骤：
S1、测定受试者血浆样品中APOA1、APOB和C3，以及Orn、DCA和TCDCA的表达水平；
S2、将测定的受试者血浆样品中的血浆生物标志物水平与正常受试者的血浆中标志物的水平进行对比；
S3、受试者的血浆样品中APOA1、APOB的表达水平相对于正常人受试者的水平的下降及C3的表达水平相对于正常人受试者的水平的上升，同时Orn和TCDCA在受试者体中升高，而DCA下降指示所述受试者存在结核病。
5.如权利要求4所述的一种基于多组学整合联合标志物的活动性结核病检测试剂盒的检测方法，其特征在于，通过TMT-LC/MS检测受试者的血浆样品中蛋白质的表达水平，其中所述受试者的血浆样品中APOA1、APOB的表达水平相对于正常人受试者的水平的下降及C3的表达水平相对于正常人受试者的水平的上升指示所述受试者存在结核病。
6.如权利要求4所述的一种基于多组学整合联合标志物的活动性结核病检测试剂盒的检测方法，其特征在于，使用免疫比浊方法检测APOA1、APOB、C3的表达水平。
7.如权利要求4所述的一种基于多组学整合联合标志物的活动性结核病检测试剂盒的检测方法，其特征在于，使用LCMS/MS方法检测Orn、DCA和TCDCA的表达水平。
8.如权利要求4所述的一种基于多组学整合联合标志物的活动性结核病检测试剂盒的检测方法，其特征在于，使用机器学习算法构建APOA1、APOB、C3、Orn、DCA和TCDCA的活动性结核病鉴别诊断模型。
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