CN117147862A - 尿液α-胰蛋白酶抑制剂重链H3及其多肽片段在系统性红斑狼疮中的应用 - Google Patents

尿液α-胰蛋白酶抑制剂重链H3及其多肽片段在系统性红斑狼疮中的应用 Download PDF

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CN117147862A
CN117147862A CN202210241048.6A CN202210241048A CN117147862A CN 117147862 A CN117147862 A CN 117147862A CN 202210241048 A CN202210241048 A CN 202210241048A CN 117147862 A CN117147862 A CN 117147862A
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Abstract

本发明提供一种尿液α‑胰蛋白酶抑制剂重链H3（Inter‑alpha‑trypsin inhibitor heavy chain H3）及其多肽片段的应用，具体为尿液α‑胰蛋白酶抑制剂重链H3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，与尿液α‑胰蛋白酶抑制剂重链H3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α‑胰蛋白酶抑制剂重链H3及其多肽片段。

Description

尿液α-胰蛋白酶抑制剂重链H3及其多肽片段在系统性红斑狼 疮中的应用
技术领域
本发明涉及尿液α-胰蛋白酶抑制剂重链H3及其多肽片段的新用途，具体涉及尿液α-胰蛋白酶抑制剂重链H3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。
α-胰蛋白酶抑制剂（ITI）基因可表达葡萄糖胺聚糖复合物蛋白，在血浆中以高分子聚合物形式出现。传统意义上认为ITI为一种单肽链，随着分子生物学技术的广泛应用，ITI目前已被证实为一种血浆蛋白酶抑制剂分子家族，包含一条轻链bikumin（由AMBP编码）和5条同源重链（由ITIH1、ITIH2、ITIH3、ITIH4和ITIH5编码），通过软骨素硫酸桥与透明质酸共价连接。研究证实ITI的生物学功能与炎症、肿瘤和免疫反应关系密切。本研究SLE活动期患者α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3的氨基酸序列包括SEQ ID NO.1所示（MAFAWWPCLI LALLSSLAAS GFPRSPFRLL GKRSLPEGVA NGIEVYSTKI NSKVTSRFAH NVVTMRAVNRADTAKEVSFD VELPKTAFIT NFTLTIDGVT YPGNVKEKEV AKKQYEKAVS QGKTAGLVKA SGRKLEKFTVSVNVAAGSKV TFELTYEELL KRHKGKYEMY LKVQPKQLVK HFEIEVDIFE PQGISMLDAE ASFITNDLLGSALTKSFSGK KGHVSFKPSL DQQRSCPTCT DSLLNGDFTI TYDVNRESPG NVQIVNGYFV HFFAPQGLPVVPKNVAFVID ISGSMAGRKL EQTKEALLRI LEDMQEEDYL NFILFSGDVS TWKEHLVQAT PENLQEARTF
VKSMEDKGMT NINDGLLRGI SMLNKAREEH RIPERSTSIV IMLTDGDANV GESRPEKIQENVRNAIGGKF PLYNLGFGNN LNYNFLENMA LENHGFARRI YEDSDADLQL QGFYEEVANP LLTGVEMEYPENAILDLTQN TYQHFYDGSE IVVAGRLVDE DMNSFKADVK GHGATNDLTF TEEVDMKEME KALQERDYIFGNYIERLWAY LTIEQLLEKR KNAHGEEKEN LTARALDLSL KYHFVTPLTS MVVTKPEDNE DERAIADKPGEGGDSHTGEP RHVLPDQLPA SSKPLLLCGR
GSPLHHPNSG ERRCPLLQHR）；或由SEQ ID NO:1所示的氨基酸序列衍生的，且与SEQID NO:1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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3标准品、人源化标签抗体标准品；较佳地，所述质控品包括：α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3。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α-胰蛋白酶抑制剂重链H3及其含量进行了验证。
本发明通过研究证实α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3的验证
Label-free质谱分析结果显示α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3进行检测，结果如图2所示。α-胰蛋白酶抑制剂重链H3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。
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<110> 张 曼
<120> 尿液α-胰蛋白酶抑制剂重链H3及其多肽片段在系统性红斑狼疮中的应用
<130> 22PITIH3-CN
<140> 22PITIH3-CN
<141> 2022-02-17
<160> 1
<170> SIPOSequenceListing 1.0
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<213> Human Urine
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Lys Ile Asn Ser Lys Val Thr Ser Arg Phe Ala His Asn Val Val Thr
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Val Glu Leu Pro Lys Thr Ala Phe Ile Thr Asn Phe Thr Leu Thr Ile
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Asp Gly Val Thr Tyr Pro Gly Asn Val Lys Glu Lys Glu Val Ala Lys
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Lys Gln Tyr Glu Lys Ala Val Ser Gln Gly Lys Thr Ala Gly Leu Val
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Lys Ala Ser Gly Arg Lys Leu Glu Lys Phe Thr Val Ser Val Asn Val
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Ala Ala Gly Ser Lys Val Thr Phe Glu Leu Thr Tyr Glu Glu Leu Leu
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Gln Leu Val Lys His Phe Glu Ile Glu Val Asp Ile Phe Glu Pro Gln
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Leu Gly Ser Ala Leu Thr Lys Ser Phe Ser Gly Lys Lys Gly His Val
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Asp Ser Leu Leu Asn Gly Asp Phe Thr Ile Thr Tyr Asp Val Asn Arg
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Glu Ser Pro Gly Asn Val Gln Ile Val Asn Gly Tyr Phe Val His Phe
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Phe Ala Pro Gln Gly Leu Pro Val Val Pro Lys Asn Val Ala Phe Val
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Ile Asp Ile Ser Gly Ser Met Ala Gly Arg Lys Leu Glu Gln Thr Lys
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Glu Ala Leu Leu Arg Ile Leu Glu Asp Met Gln Glu Glu Asp Tyr Leu
305 310 315 320
Asn Phe Ile Leu Phe Ser Gly Asp Val Ser Thr Trp Lys Glu His Leu
325 330 335
Val Gln Ala Thr Pro Glu Asn Leu Gln Glu Ala Arg Thr Phe Val Lys
340 345 350
Ser Met Glu Asp Lys Gly Met Thr Asn Ile Asn Asp Gly Leu Leu Arg
355 360 365
Gly Ile Ser Met Leu Asn Lys Ala Arg Glu Glu His Arg Ile Pro Glu
370 375 380
Arg Ser Thr Ser Ile Val Ile Met Leu Thr Asp Gly Asp Ala Asn Val
385 390 395 400
Gly Glu Ser Arg Pro Glu Lys Ile Gln Glu Asn Val Arg Asn Ala Ile
405 410 415
Gly Gly Lys Phe Pro Leu Tyr Asn Leu Gly Phe Gly Asn Asn Leu Asn
420 425 430
Tyr Asn Phe Leu Glu Asn Met Ala Leu Glu Asn His Gly Phe Ala Arg
435 440 445
Arg Ile Tyr Glu Asp Ser Asp Ala Asp Leu Gln Leu Gln Gly Phe Tyr
450 455 460
Glu Glu Val Ala Asn Pro Leu Leu Thr Gly Val Glu Met Glu Tyr Pro
465 470 475 480
Glu Asn Ala Ile Leu Asp Leu Thr Gln Asn Thr Tyr Gln His Phe Tyr
485 490 495
Asp Gly Ser Glu Ile Val Val Ala Gly Arg Leu Val Asp Glu Asp Met
500 505 510
Asn Ser Phe Lys Ala Asp Val Lys Gly His Gly Ala Thr Asn Asp Leu
515 520 525
Thr Phe Thr Glu Glu Val Asp Met Lys Glu Met Glu Lys Ala Leu Gln
530 535 540
Glu Arg Asp Tyr Ile Phe Gly Asn Tyr Ile Glu Arg Leu Trp Ala Tyr
545 550 555 560
Leu Thr Ile Glu Gln Leu Leu Glu Lys Arg Lys Asn Ala His Gly Glu
565 570 575
Glu Lys Glu Asn Leu Thr Ala Arg Ala Leu Asp Leu Ser Leu Lys Tyr
580 585 590
His Phe Val Thr Pro Leu Thr Ser Met Val Val Thr Lys Pro Glu Asp
595 600 605
Asn Glu Asp Glu Arg Ala Ile Ala Asp Lys Pro Gly Glu Gly Gly Asp
610 615 620
Ser His Thr Gly Glu Pro Arg His Val Leu Pro Asp Gln Leu Pro Ala
625 630 635 640
Ser Ser Lys Pro Leu Leu Leu Cys Gly Arg Gly Ser Pro Leu His His
645 650 655
Pro Asn Ser Gly Glu Arg Arg Cys Pro Leu Leu Gln His Arg
660 665 670

Claims (9)

1.尿液α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3的氨基酸序列包括SEQ ID NO:1所示；或由SEQ ID NO:1所示的氨基酸序列衍生的，且与SEQ IDNO:1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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3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α-胰蛋白酶抑制剂重链H3标准品、人源化标签抗体标准品；较佳地，所述质控品包括：α-胰蛋白酶抑制剂重链H3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。
CN202210241048.6A 2022-03-13 2022-03-13 尿液α-胰蛋白酶抑制剂重链H3及其多肽片段在系统性红斑狼疮中的应用 Pending CN117147862A (zh)

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