CN117147861A - 尿液α-胰蛋白酶抑制剂重链H2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H2及其多肽片段在系统性红斑狼疮中的应用 Download PDFInfo
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- CN117147861A CN117147861A CN202210241047.1A CN202210241047A CN117147861A CN 117147861 A CN117147861 A CN 117147861A CN 202210241047 A CN202210241047 A CN 202210241047A CN 117147861 A CN117147861 A CN 117147861A
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Abstract
本发明提供一种尿液α‑胰蛋白酶抑制剂重链H2(Inter‑alpha‑trypsin inhibitor heavy chain H2)及其多肽片段的应用,具体为尿液α‑胰蛋白酶抑制剂重链H2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α‑胰蛋白酶抑制剂重链H2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H2及其多肽片段。
Description
技术领域
本发明涉及尿液α-胰蛋白酶抑制剂重链H2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H2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ITIH2、ITIH2、ITIH2、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H2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H2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H2的氨基酸序列包括SEQ ID NO.1所示(MKRLTCFFIC FFLSEVSGFE IPINGLSEFV DYEDLVELAP GKFQLVAENR RYQEEVDQVT LYSYKVQSTITSRMATTMIQ SKVVNNSPQP QNVVFDVQIP KGAFISNFSM TVDGKTFRSS IKEKTVGRAL YAQARAKGKTAGLVRSSALD MENFRTEVNV LPGAKVQFEL HYQEVKWRKL GSYEHRIYLQ PGRLAKHLEV DVWVIEPQGLRFLHVPDTFE GHFDGVPVIS KGQQKAHVSF KPTVAQQRIC PNCRETAVDG ELVVLYDVKR EEKAGELEVFNGYFVHFFAP DNLDPIPKNI LFVIDVSGSM WGVKMKQTVE AMKTILDDLR AEDHFSVIDF NQNIRTWRND
LISATKTQVA DAKRYIEKIQ PSGGTNINEA LLRAIFILNE ANNLGLLDPN SVSLIILVSDGDPTVGELKL SKIQKNVKEN IQDNISLFSL GMGFDVDYDF LKRLSNENHG IAQRIYGNQD TSSQLKKFYNQVSTPLLRNV QFNYPHTSVT DVTQNNFHNY FGGSEIVVAG KFDPAKLDQI ESVITATSAN TQLVLETLAQMDDLQDFLSK DKHADPDFTR KLWAYLTINQ LLAERSLAPT AAAKRRITRS ILQMSLDHHI VTPLTSLVIENEAGDERMLA DAPPQDPSCC SGALYYGSKV
VPDSTPSWAN PSPTPVISML AQGSQVLEST PPPHVMRVEN DPHFIIYLPK SQKNICFNIDSEPGKILNLV SDPESGIVVN GQLVGAKKPN NGKLSTYFGK LGFYFQSEDI KIEISTETIT LSHGSSTFSLSWSDTAQVTN QRVQISVKKE KVVTITLDKE MSFSVLLHRV WKKHPVNVDF LGIYIPPTNK FSPKAHGLIGQFMQEPKIHI FNERPGKDPE KPEASMEVKG QKLIITRGLQ KDYRTDLVFG TDVTCWFVHN SGKGFIDGHYKDYFVPQLYS FLKRP);或由SEQ ID NO:1所示的氨基酸序列衍生的,且与SEQ ID 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H2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H2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H2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H2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H2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H2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H2。发明人利用Western Blot免疫印迹试验和平行反应监测技术(PRM)对SLE患者和健康人尿液中的α-胰蛋白酶抑制剂重链H2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H2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H2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H2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H2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H2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。
图3是尿液α-胰蛋白酶抑制剂重链H2及其多肽片段在SLE患者及健康对照组PRM定量图。
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H2的验证
Label-free质谱分析结果显示α-胰蛋白酶抑制剂重链H2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H2进行检测,结果如图2所示,使用平行反应监测技术(PRM)对SLE患者及健康人尿液中α-胰蛋白酶抑制剂重链H2进行检测,结果如图3所示。验证结果一致,α-胰蛋白酶抑制剂重链H2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α-胰蛋白酶抑制剂重链H2及其多肽片段在系统性红斑狼疮中的应用
<130> 22PITIH2-CN
<140> 22PITIH2-CN
<141> 2022-02-17
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 935
<212> PRT
<213> Human Urine
<400> 1
Met Lys Arg Leu Thr Cys Phe Phe Ile Cys Phe Phe Leu Ser Glu Val
1 5 10 15
Ser Gly Phe Glu Ile Pro Ile Asn Gly Leu Ser Glu Phe Val Asp Tyr
20 25 30
Glu Asp Leu Val Glu Leu Ala Pro Gly Lys Phe Gln Leu Val Ala Glu
35 40 45
Asn Arg Arg Tyr Gln Glu Glu Val Asp Gln Val Thr Leu Tyr Ser Tyr
50 55 60
Lys Val Gln Ser Thr Ile Thr Ser Arg Met Ala Thr Thr Met Ile Gln
65 70 75 80
Ser Lys Val Val Asn Asn Ser Pro Gln Pro Gln Asn Val Val Phe Asp
85 90 95
Val Gln Ile Pro Lys Gly Ala Phe Ile Ser Asn Phe Ser Met Thr Val
100 105 110
Asp Gly Lys Thr Phe Arg Ser Ser Ile Lys Glu Lys Thr Val Gly Arg
115 120 125
Ala Leu Tyr Ala Gln Ala Arg Ala Lys Gly Lys Thr Ala Gly Leu Val
130 135 140
Arg Ser Ser Ala Leu Asp Met Glu Asn Phe Arg Thr Glu Val Asn Val
145 150 155 160
Leu Pro Gly Ala Lys Val Gln Phe Glu Leu His Tyr Gln Glu Val Lys
165 170 175
Trp Arg Lys Leu Gly Ser Tyr Glu His Arg Ile Tyr Leu Gln Pro Gly
180 185 190
Arg Leu Ala Lys His Leu Glu Val Asp Val Trp Val Ile Glu Pro Gln
195 200 205
Gly Leu Arg Phe Leu His Val Pro Asp Thr Phe Glu Gly His Phe Asp
210 215 220
Gly Val Pro Val Ile Ser Lys Gly Gln Gln Lys Ala His Val Ser Phe
225 230 235 240
Lys Pro Thr Val Ala Gln Gln Arg Ile Cys Pro Asn Cys Arg Glu Thr
245 250 255
Ala Val Asp Gly Glu Leu Val Val Leu Tyr Asp Val Lys Arg Glu Glu
260 265 270
Lys Ala Gly Glu Leu Glu Val Phe Asn Gly Tyr Phe Val His Phe Phe
275 280 285
Ala Pro Asp Asn Leu Asp Pro Ile Pro Lys Asn Ile Leu Phe Val Ile
290 295 300
Asp Val Ser Gly Ser Met Trp Gly Val Lys Met Lys Gln Thr Val Glu
305 310 315 320
Ala Met Lys Thr Ile Leu Asp Asp Leu Arg Ala Glu Asp His Phe Ser
325 330 335
Val Ile Asp Phe Asn Gln Asn Ile Arg Thr Trp Arg Asn Asp Leu Ile
340 345 350
Ser Ala Thr Lys Thr Gln Val Ala Asp Ala Lys Arg Tyr Ile Glu Lys
355 360 365
Ile Gln Pro Ser Gly Gly Thr Asn Ile Asn Glu Ala Leu Leu Arg Ala
370 375 380
Ile Phe Ile Leu Asn Glu Ala Asn Asn Leu Gly Leu Leu Asp Pro Asn
385 390 395 400
Ser Val Ser Leu Ile Ile Leu Val Ser Asp Gly Asp Pro Thr Val Gly
405 410 415
Glu Leu Lys Leu Ser Lys Ile Gln Lys Asn Val Lys Glu Asn Ile Gln
420 425 430
Asp Asn Ile Ser Leu Phe Ser Leu Gly Met Gly Phe Asp Val Asp Tyr
435 440 445
Asp Phe Leu Lys Arg Leu Ser Asn Glu Asn His Gly Ile Ala Gln Arg
450 455 460
Ile Tyr Gly Asn Gln Asp Thr Ser Ser Gln Leu Lys Lys Phe Tyr Asn
465 470 475 480
Gln Val Ser Thr Pro Leu Leu Arg Asn Val Gln Phe Asn Tyr Pro His
485 490 495
Thr Ser Val Thr Asp Val Thr Gln Asn Asn Phe His Asn Tyr Phe Gly
500 505 510
Gly Ser Glu Ile Val Val Ala Gly Lys Phe Asp Pro Ala Lys Leu Asp
515 520 525
Gln Ile Glu Ser Val Ile Thr Ala Thr Ser Ala Asn Thr Gln Leu Val
530 535 540
Leu Glu Thr Leu Ala Gln Met Asp Asp Leu Gln Asp Phe Leu Ser Lys
545 550 555 560
Asp Lys His Ala Asp Pro Asp Phe Thr Arg Lys Leu Trp Ala Tyr Leu
565 570 575
Thr Ile Asn Gln Leu Leu Ala Glu Arg Ser Leu Ala Pro Thr Ala Ala
580 585 590
Ala Lys Arg Arg Ile Thr Arg Ser Ile Leu Gln Met Ser Leu Asp His
595 600 605
His Ile Val Thr Pro Leu Thr Ser Leu Val Ile Glu Asn Glu Ala Gly
610 615 620
Asp Glu Arg Met Leu Ala Asp Ala Pro Pro Gln Asp Pro Ser Cys Cys
625 630 635 640
Ser Gly Ala Leu Tyr Tyr Gly Ser Lys Val Val Pro Asp Ser Thr Pro
645 650 655
Ser Trp Ala Asn Pro Ser Pro Thr Pro Val Ile Ser Met Leu Ala Gln
660 665 670
Gly Ser Gln Val Leu Glu Ser Thr Pro Pro Pro His Val Met Arg Val
675 680 685
Glu Asn Asp Pro His Phe Ile Ile Tyr Leu Pro Lys Ser Gln Lys Asn
690 695 700
Ile Cys Phe Asn Ile Asp Ser Glu Pro Gly Lys Ile Leu Asn Leu Val
705 710 715 720
Ser Asp Pro Glu Ser Gly Ile Val Val Asn Gly Gln Leu Val Gly Ala
725 730 735
Lys Lys Pro Asn Asn Gly Lys Leu Ser Thr Tyr Phe Gly Lys Leu Gly
740 745 750
Phe Tyr Phe Gln Ser Glu Asp Ile Lys Ile Glu Ile Ser Thr Glu Thr
755 760 765
Ile Thr Leu Ser His Gly Ser Ser Thr Phe Ser Leu Ser Trp Ser Asp
770 775 780
Thr Ala Gln Val Thr Asn Gln Arg Val Gln Ile Ser Val Lys Lys Glu
785 790 795 800
Lys Val Val Thr Ile Thr Leu Asp Lys Glu Met Ser Phe Ser Val Leu
805 810 815
Leu His Arg Val Trp Lys Lys His Pro Val Asn Val Asp Phe Leu Gly
820 825 830
Ile Tyr Ile Pro Pro Thr Asn Lys Phe Ser Pro Lys Ala His Gly Leu
835 840 845
Ile Gly Gln Phe Met Gln Glu Pro Lys Ile His Ile Phe Asn Glu Arg
850 855 860
Pro Gly Lys Asp Pro Glu Lys Pro Glu Ala Ser Met Glu Val Lys Gly
865 870 875 880
Gln Lys Leu Ile Ile Thr Arg Gly Leu Gln Lys Asp Tyr Arg Thr Asp
885 890 895
Leu Val Phe Gly Thr Asp Val Thr Cys Trp Phe Val His Asn Ser Gly
900 905 910
Lys Gly Phe Ile Asp Gly His Tyr Lys Asp Tyr Phe Val Pro Gln Leu
915 920 925
Tyr Ser Phe Leu Lys Arg Pro
930 935
Claims (9)
1.尿液α-胰蛋白酶抑制剂重链H2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H2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H2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H2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H2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H2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H2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H2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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