CN117147842A - 尿液凝血因子ix及其多肽片段在系统性红斑狼疮中的应用 - Google Patents
尿液凝血因子ix及其多肽片段在系统性红斑狼疮中的应用 Download PDFInfo
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- CN117147842A CN117147842A CN202210241044.8A CN202210241044A CN117147842A CN 117147842 A CN117147842 A CN 117147842A CN 202210241044 A CN202210241044 A CN 202210241044A CN 117147842 A CN117147842 A CN 117147842A
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- Peptides Or Proteins (AREA)
Abstract
本发明提供一种尿液凝血因子IX（Coagulation factor IX）及其多肽片段的应用，具体为尿液凝血因子IX及其多肽片段在制备用于系统性红斑狼疮(Systemic lupus erythematosus，SLE)诊断、鉴别诊断、病情程度与活动性判断、治疗效果评价、监测、预后评估及机理研究等应用。SLE是由自身免疫介导的，以免疫炎症为突出表现的弥漫性结缔组织病，多数呈隐匿起病，常累及机体多个系统。本发明通过研究证实，尿液凝血因子IX及其多肽片段在SLE活动期患者中表达升高。可用于SLE患者的各种目的应用检测。本发明发挥尿液标本获取无创、可大规模重复取样、保存方便的优势，利用尿液标本检测尿液凝血因子IX及其多肽片段。
Description
技术领域
本发明涉及尿液凝血因子IX及其多肽片段的新用途，具体涉及尿液凝血因子IX及其多肽片段在SLE诊断、鉴别诊断、病情程度与活动性判断、治疗效果评价、监测、预后评估及机理研究等应用。
背景技术
系统性红斑狼疮（Systemic lupus erythematosus，SLE）是一种可累及全身多脏器的自身免疫性结缔组织疾病，好发于生育年龄女性。依据流行病学统计研究显示，我国SLE的患病率为70／10万人，妇女中则高达113／10万人。该病病因尚未十分明确，SLE常呈隐匿性起病，临床表现复杂多样，鼻梁和双颧颊部呈蝶形分布的红斑是SLE特征性的改变。SLE的皮肤损害还包括光敏感、脱发、手足掌面和甲周红斑、盘状红斑、雷诺现象等。除此之外，SLE病程发展还常涉及机体多个系统，尤其以重要脏器系统受累为著，如狼疮肾炎、神经精神狼疮、心包积液、血液及消化系统损害。可见，对于该病患者及时的诊断、有效的病情监测以及针对性的治疗尤为重要。
凝血因子IX（Coagulation factor IX，F9）是由肝脏实质细胞合成的单链糖蛋白，其在凝血过程中起重要作用，可酶解凝血因子X，使其转化为活化分子Xa。因子IX被转化为因子IXa的激活过程可分为内源性和外源性两种途径。正常情况下，体内F9以酶原形式存在，不具备酶活性。当其被激活后以转化为IXa，与钙离子结合，在磷脂膜表面与因子VIIIa形成复合物后激活因子X。本研究SLE活动期患者凝血因子IX在尿液中含量升高。
与常用的临床血液样本相比，尿液可以完全无创、连续、大量收集；没有稳态调节，可累积更多种类、更大幅度的变化，机体的很多病理生理变化可能体现在尿液中。一些激素和细胞因子等分子量相对较小的蛋白多肽入血后，会很快被排泄进入尿液，这些蛋白和多肽在尿液中被检测到的概率比在血中大很多；尿液收集之前，尿中可能的蛋白降解过程已经完成，所以尿蛋白可在较长时间内保持稳定。为减轻SLE患者多次采血的痛苦，本实验在前期方法学摸索的基础上，期望通过尿液蛋白或多肽研究，实现用无痛、方便、快捷、易重复的尿液检测辅助SLE患者的诊断及病情监测，也为进一步尿液多肽检测试剂盒的研究奠定基础。
发明内容
本发明的目的在于提供一种尿液凝血因子IX及其多肽片段在制备用于SLE诊断、鉴别诊断、病情程度与活动性判断、治疗效果评价、监测、预后评估及机理研究等的应用
优选地，所述尿液凝血因子IX的氨基酸序列包括SEQ ID NO.1所示（MQRVNMIMAESPGLITICLL GYLLSAECTV FLDHENANKI LNRPKRYNSG KLEEFVQGNL ERECMEEKCS FEEAREVFENTERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP FGFEGKNCEL DVTCNIKNGR CEQFCKNSADNKVVCSCTEG YRLAENQKSC EPAVPFPCGR VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQSFNDFTRVVGG EDAKPGQFPW QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHTEQKRNVIRII PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS
GYVSGWGRVF HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGDSGGPHVTEVE GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL T）、SEQ ID NO.2所示（MQRVNMIMAE SPGLITICLL GYLLSAECTV FLDHENANKI LNRPKRYNSG KLEEFVQGNL ERECMEEKCSFEEAREVFEN TERTTEFWKQ YVDVTCNIKN GRCEQFCKNS ADNKVVCSCT EGYRLAENQK SCEPAVPFPCGRVSVSQTSK LTRAETVFPD VDYVNSTEAE TILDNITQST QSFNDFTRVV GGEDAKPGQF PWQVVLNGKVDAFCGGSIVN EKWIVTAAHC VETGVKITVV AGEHNIEETE HTEQKRNVIR IIPHHNYNAA INKYNHDIALLELDEPLVLN SYVTPICIAD KEYTNIFLKF GSGYVSGWGR VFHKGRSALV LQYLRVPLVD RATCLRSTKF
TIYNNMFCAG FHEGGRDSCQ GDSGGPHVTE VEGTSFLTGI ISWGEECAMK GKYGIYTKVSRYVNWIKEKT KLT）；或由SEQ ID NO:1~2所示的氨基酸序列衍生的，且与SEQ ID NO:1~2所示的氨基酸序列具有相同功能的氨基酸序列。
优选地，所述制剂为SLE患者尿液凝血因子IX及其多肽片段检测试剂盒。
优选地，所述试剂盒包括抗原抗体反应的免疫方法及其试剂盒如能够特异性结合凝血因子IX及其多肽片段的适配体抗体或抗体片段中的一种或多种。
优选地，所述检测方法包括直接检测凝血因子IX及其多肽片段的质谱等方法及其相关试剂盒。
优选地，所述检测方法包括直接检测凝血因子IX及其多肽片段的相关核酸检测等方法及其相关试剂盒。
优选地，所述试剂盒还包括选自下组的成分：固相载体，稀释液，对照品，标准品，质控品，检测抗体，第二抗体、第二抗体稀释液，发光试剂，洗涤液、显色液、终止液中的任意一种或几种的组合。
优选地，所述标准品包括凝血因子IX标准品、人源化标签抗体标准品；较佳地，所述质控品包括：凝血因子IX质控品、人源化标签抗体质控品；较佳地，所述固相载体包括：微粒、微球、玻片、试纸条、塑料珠、液相芯片、微孔板或亲和膜等以及同等功能的其他载体。
优选地，所述固相载体的材质为聚氯乙烯、聚苯乙烯、聚丙酰胺、纤维素中的任意一种及具有类似功能的载体。
发明人首先收集了健康人、SLE活动期患者（aSLE）和稳定期患者（sSLE）的尿液标本，4000r/min离心5min后，吸取上清，采用Bradford法测定提取的蛋白浓度，进行SDS-PAGE酶解。尿液样本的Label-free质谱分析由OrbitrapFusion型质谱完成。将SLE组和正常对照组在质谱中得到的数据进行定量计算。以蛋白表达量差异在1.5倍以上且经统计检验P<0.05作为参考标准筛选差异性多肽。然后发明人对具有统计学意义的差异性多肽进行鉴定，利用数据库检索得到差异蛋白凝血因子IX。发明人利用Western Blot免疫印迹试验对SLE患者和健康人尿液中的凝血因子IX及其含量进行了验证。
本发明通过研究证实凝血因子IX及其多肽片段在SLE活动期患者的尿液中呈高表达，与临床诊断有较好的一致性。从而提出检测尿液凝血因子IX及其多肽片段可用于SLE的辅助诊断或病情监测。
本发明发挥尿液标本获取无创、可大规模重复取样、保存方便的优势，利用尿液标本检测尿液凝血因子IX及其多肽片段。
为让本发明的上述和其它目的、特征和优点能更明显易懂，下文特举较佳实施例，并配合附图，作详细说明如下。
附图说明
图1是尿液凝血因子IX及其多肽片段在SLE患者及健康对照组中含量图。
图2是尿液凝血因子IX及其多肽片段在SLE患者及健康对照组中Western Blot免疫印迹试验结果图。
具体实施方式
实施例1 尿液标本的收集与处理
选取SLE患者作为SLE组，选取同期健康体检者作为正常对照组。收集各组研究对象入院后的新鲜晨尿样本30ml，不能正常排尿者收集其早晨导尿管中尿液，置于干燥、洁净的容器内。将收集的尿液标本4000r/min离心5min后，吸取上清，每管2ml分装，-80℃冰箱保存。
实施例2 质谱分析和尿液多肽的筛选
对尿液样品蛋白提取，并对提取的蛋白浓度进行测定。尿液样本的质谱分析由OrbitrapFusion型质谱完成。将实验组和正常对照组在质谱中得到的数据进行定量计算。组间比较采用t-test进行差异分析，以蛋白表达量差异在1.5倍以上且经统计检验P<0.05作为参考标准筛选差异表达蛋白。
实施例3 差异多肽的鉴定及分析
使用的数据库为Uniprot_Homo数据库，产生的质谱原始文件采用MaxQuant软件处理，检索参数设置见表1。
实施例4 尿液凝血因子IX的验证
Label-free质谱分析结果显示凝血因子IX在SLE活动期患者的尿液中高表达，其在SLE患者及健康对照组尿液中的含量如图1所示，使用Western Blot免疫印迹试验对SLE患者和健康人尿液中的凝血因子IX进行检测，结果如图2所示。凝血因子IX在各组间表达具有显著性差异。
虽然本发明已以较佳实施例披露如上，然其并非用以限定本发明，任何所属技术领域的技术人员，在不脱离本发明的精神和范围内，当可作些许的更动与改进，因此本发明的保护范围当视权利要求所界定者为准。
序列表
<110> 张 曼
<120> 尿液凝血因子IX及其多肽片段在系统性红斑狼疮中的应用
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<140> 22PF9-CN
<141> 2022-02-17
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Claims (9)
1.尿液凝血因子IX及其多肽片段在制备用于SLE诊断、鉴别诊断、病情程度与活动性判断、治疗效果评价、监测、预后评估及机理研究等的应用。
2.根据权利要求1所述的应用，其特征在于，所述尿液凝血因子IX的氨基酸序列包括SEQ ID NO:1~2所示；或由SEQ ID NO:1~2所示的氨基酸序列衍生的，且与SEQ ID NO:1~2所示的氨基酸序列具有相同功能的氨基酸序列。
3.根据权利要求1所述的应用，其特征在于，所述制剂为SLE患者尿液凝血因子IX及其多肽片段检测试剂盒。
4.根据权利要求3所述的应用，其特征在于，所述试剂盒包括抗原抗体反应的免疫方法及其试剂盒如能够特异性结合凝血因子IX及其多肽片段的适配体抗体或抗体片段中的一种或多种。
5.根据权利要求3所述的应用，其特征在于，所述检测方法包括直接检测凝血因子IX及其多肽片段的质谱等方法及其相关试剂盒。
6.根据权利要求3所述的应用，其特征在于，所述检测方法包括直接检测凝血因子IX及其多肽片段或其相关核酸检测等方法及其相关试剂盒。
7.根据权利要求3所述的应用，其特征在于，所述试剂盒还包括选自下组的成分：固相载体，稀释液，对照品，标准品，质控品，检测抗体，第二抗体、第二抗体稀释液，发光试剂，洗涤液、显色液、终止液中的任意一种或几种的组合。
8.根据权利要求7所述的应用，其特征在于，所述标准品包括凝血因子IX标准品、人源化标签抗体标准品；较佳地，所述质控品包括：凝血因子IX控品、人源化标签抗体质控品；较佳地，所述固相载体包括：微粒、微球、玻片、试纸条、塑料珠、液相芯片、微孔板或亲和膜等以及同等功能的其他载体。
9.根据权利要求8所述的应用，其特征在于，所述固相载体的材质为聚氯乙烯、聚苯乙烯、聚丙酰胺、纤维素中的任意一种及具有类似功能的载体。
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