CN112114126B - 一种***性红斑狼疮的诊断标志物及其应用 - Google Patents

一种***性红斑狼疮的诊断标志物及其应用 Download PDF

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CN112114126B
CN112114126B CN202010813838.8A CN202010813838A CN112114126B CN 112114126 B CN112114126 B CN 112114126B CN 202010813838 A CN202010813838 A CN 202010813838A CN 112114126 B CN112114126 B CN 112114126B
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CN112114126A (zh
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邢艳
何仁栋
龙思琪
郭斌
祝静
王强
蒋瑶
晏波
雷燕
秦倩
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North Sichuan Medical College
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]

Abstract

本发明公开了一种***性红斑狼疮的诊断标志物及其应用，涉及生物医学检测技术领域，本发明公开了信号素4A(Sema4A)膜结合型和可溶型对***性红斑狼疮患者具有较高的诊断效能，可用于评估病情活动度和脏器损伤，同时可很好地将***性红斑狼疮与类风湿性关节炎进行鉴别，因而Sema4A能够作为***性红斑狼疮的诊断标志物。且Sema4A对***性红斑狼疮的敏感性较高，与自身抗体联合检测有助于提高疾病早期阶段的检出率，这在一定程度上弥补当前临床实验室诊断敏感性低的缺陷。

Description

一种***性红斑狼疮的诊断标志物及其应用
技术领域
本发明属于生物医学检测技术领域，尤其涉及一种***性红斑狼疮的诊断标志物及其应用。
背景技术
***性红斑狼疮(systemic lupus erythematosus,SLE)是以产生针对细胞核成分的自身抗体以及多***、多器官受累出现多种临床表现为特征的自身免疫性疾病。SLE临床表现复杂多样，早期症状不典型，导致疾病早期诊断困难。
SLE的诊断需要综合患者的症状、体格检查以及实验室检查。但SLE早期临床表现不典型，高效准确的实验室检测指标成为疾病早期的重要诊断依据。目前SLE的实验室常用诊断指标包括抗核抗体(Antinuclear antibody,ANA)以及可提取核抗原抗体(Antibodiesto extractable nuclear antigens,ENA)，如致病性抗体——抗dsDNA抗体和特征性抗体——抗Sm抗体。这些抗体对SLE均具有较高的特异性，但其灵敏度较低，使得早期SLE患者漏诊率较高。
此外，部分SLE患者以指关节肿痛为首发症状，而RA患者临床表现以关节症状为主。同时二者实验室检测均可出现ANA阳性、RF阳性、ESR和CRP增高，导致二者难以区分。因此有必要继续寻找新的诊断标志物以满足疾病早期诊断、病情评估和疗效监测的临床需求。
发明内容
本发明的目的在于：针对上述现有技术的用于SLE诊断的指标敏感性均很低，易造成漏检的问题，提供一种敏感性高的***性红斑狼疮的诊断标志物及其应用。
本发明采用的技术方案如下：
一种***性红斑狼疮的诊断标志物，诊断标志物的氨基酸序列如SEQ ID NO：1～SEQ ID NO：4所示中的至少一种；具体序列如下：
SEQ ID NO：1
MetAlaLeuProAlaLeuGlyLeuAspProTrpSerLeuLeuGlyLeuPheLeuPheGlnLeuLeuGlnLeuLeuLeuProThrThrThrAlaGlyGlyGlyGlyGlnGlyProMetProArgValArgTyrTyrAlaGlyAspGluArgArgAlaLeuSerPhePheHisGlnLysGlyLeuGlnAspPheAspThrLeuLeuLeuSerGlyAspGlyAsnThrLeuTyrValGlyAlaArgGluAlaIleLeuAlaLeuAspIleGlnAspProGlyValProArgLeuLysAsnMetIleProTrpProAlaSerAspArgLysLysSerGluCysAlaPheLysLysLysSerAsnGluThrGlnCysPheAsnPheIleArgValLeuValSerTyrAsnValThrHisLeuTyrThrCysGlyThrPheAlaPheSerProAlaCysThrPheIleGluLeuGlnAspSerTyrLeuLeuProIleSerGluAspLysValMetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
SEQ ID NO：2
MetIleProTrpProAlaSerAspArgLysLysSerGluCysAlaPheLysLysLysSerAsnGluGluLeuGlnAspSerTyrLeuLeuProIleSerGluAspLysValMetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
SEQ ID NO：3
MetIleProTrpProAlaSerAspArgLysLysSerGluCysAlaPheLysLysLysSerAsnGluThrGlnCysPheAsnPheIleArgValLeuValSerTyrAsnValThrHisLeuTyrThrCysGlyThrPheAlaPheSerProAlaCysThrPheIleGluLeuGlnAspSerTyrLeuLeuProIleSerGluAspLysValMetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
SEQ ID NO：4
MetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
进一步地，标志物的氨基酸序列为上述SEQ ID NO：1～SEQ ID NO：4所示的序列经取代、缺失和/或替换一个或多个碱基后，能表达相同功能的。
上述的诊断标志物在制备用于检测***性红斑狼疮的制剂中的应用。
一种制剂，包括能与上述的诊断标志物发生特异性结合的抗体。
一种用于检测***性红斑狼疮的试剂盒，包括能与上述的诊断标志物发生特异性结合的抗体。
信号素(semaphorin)是一大类膜结合型或分泌型的糖蛋白分子，在多种生理过程中具有重要调节作用，包括神经轴突导向、血管生长、骨的吸收与重塑、免疫调节和心脏发育等。信号素可分为7类，第1、2类信号素仅在无脊椎动物中表达，第3、4、6和7类信号素只在脊椎动物中表达，而第5类信号素在两者中均表达。研究表明，信号素广泛参与免疫功能调节，可以诱导DC的成熟及迁移，调节T、B细胞的活化、增殖及分化，促使炎症细胞因子的产生等。第4类信号素有A、B、C、D4个亚型，本发明通过研究发现Sema4A(包括膜结合型和可溶型)在SLE的临床诊断中具有重要价值。
本发明中，当以膜结合型Sema4A为诊断标志物时，采用流式细胞术(FCM)分析外周血细胞上膜结合型Sema4A的表达，具体步骤如下：
(1)采集外周血标本；
(2)将外周血标本分别加入4个5ml离心管中，分别命名为1号离心管、2号离心管、3号离心管和4号离心管；
(3)1号离心管中加入抗人CD100-Alexa 647抗体、抗人CD4-PE抗体和抗人CD19-FITC抗体后室温下避光孵育15min；3号离心管设置同型对照；2号离心管中加入抗人Sema4A-PerCP-eFluor 710抗体、抗人CD11c-APC抗体、抗人CD4-PE抗体和抗人CD19-FITC抗体后室温下避光孵育15min，4号离心管设置同型对照；
(4)分别向每个离心管中加入红细胞裂解液，避光放置10min待红细胞裂解完全；
(5)每管加入PBS混匀后，离心，弃上清，重复两次；
(6)上机检测。
本发明中，当以可溶型Sema4A为诊断标志物时，采用酶联免疫吸附试验(ELISA)检测血浆中可溶型Sema4A的浓度，具体步骤如下：
(1)试剂配制：①标准品配制：从ELISA试剂盒中取出Sema4A抗体标准品，离心，用1ml样本稀释液溶解，充分混匀后得到标准品S7；然后取7个1.5mlEp管(分别命名为S0-S6)依序排列，分别加入250pl样本稀释液，吸取250mlS7标准品置于第一个Ep管中(S6)，从S6中吸取250ml到第二个Ep管中(S5)，以此类推进行标准品的倍比稀释，S0为样本稀释液；②洗涤工作液配制：用去离子水将浓洗涤液按l：25倍进行稀释，以备用；③生物素标记抗体工作液配制：生物素标记抗体液按1：100倍用生物素标记抗体稀释液进行稀释，以备用；④辣根过氧化物酶标记亲和素工作液配制：辣根过氧化物酶标记亲和素按1：100倍用辣根过氧化物酶标记亲和素稀释液进行稀释，以备用；
(2)按照ELISA说明书进行以下操作：
①将各种试剂置于室温平衡至少30min，按前述方法配制试剂，备用；
②分别设置标准品孔及待测样品孔，每孔分别加标准品或待测样品100μl，轻轻晃动混匀，覆上板贴后于37℃温育2h；
③弃去液体，甩干；
④每孔加入生物素标记抗体工作液，覆上新的板贴，37℃温育1h；
⑤弃去孔内液体，甩干，洗涤，再甩干；
⑥每孔加入辣根过氧化物酶标记亲和素工作液，覆上新的板贴，37℃温育1h；
⑦弃去孔内液体，甩干，洗涤，再甩干；
⑧每孔加底物溶液，37℃避光显色30min；
⑨每孔加终止溶液，终止反应；在五分钟内用酶标仪在450nm波长下测量各孔的光密度(OD值)。
综上所述，由于采用了上述技术方案，本发明的有益效果是：
1、本发明中，以膜结合型和可溶型Sema4A作为***性红斑狼疮的诊断标志物，具有较高的诊断效能，同时可用于评估病情活动度，并具有一定的评估脏器损伤的价值；
2、本发明的***性红斑狼疮诊断标志物Sema4A对SLE的敏感性较高，与自身抗体联合检测有助于提高疾病早期阶段的检出率，解决了现有技术中用于SLE诊断的指标敏感性均很低，易造成漏检的问题，因而本发明的诊断标志物的应用对于早期临床表现不典型而诊断困难的***性红斑狼疮的早期诊断有着极为重要的意义；
3、本发明的膜结合型和可溶型Sema4A可用于***性红斑狼疮与类风湿性关节炎的鉴别诊断，由于***性红斑狼疮患者膜结合型和可溶型Sema4A显著高于健康人群，而类风湿关节炎患者膜结合型和可溶型Sema4A显著低于健康人群，因此能够鉴别临床表现相似的此两种疾病；
4、本发明的应用包括采用Sema4A的检测试剂，用于定量检测血浆中可溶型Sema4A浓度或使用Sema4A抗体用于流式细胞术检测外周血细胞表面膜结合型Sema4A水平，根据不同场景采用该两类检测方法单独使用或共同使用，均能获得较好的诊断效果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
因此，以下对提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，术语“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
流式细胞术(FCM)分析外周血细胞上膜结合型Sema4A的表达：
(1)采集外周血标本5ml置于EDTA抗凝管中；
(2)将外周血标本按200μl每管加入至4个5ml离心管中，分别命名为1号离心管、2号离心管、3号离心管和4号离心管；
(3)1号离心管中加入抗人CD100-Alexa 647抗体、抗人CD4-PE抗体和抗人CD19-FITC抗体后室温下避光孵育15min；3号离心管设置同型对照；2号离心管中加入抗人Sema4A-PerCP-eFluor 710抗体、抗人CD11c-APC抗体、抗人CD4-PE抗体和抗人CD19-FITC抗体后室温下避光孵育15min，4号离心管设置同型对照；
(4)分别向每个离心管中加入700μl红细胞裂解液，避光放置10min待红细胞裂解完全；
(5)每管加入2～3mlPBS混匀后，以1000转/min离心5min，弃上清，重复两次；
(6)分别向每管加入200μlPBS混匀后上机检测。
采用上述方法分别对年龄和性别相匹配的***性红斑狼疮患者、类风湿关节炎(RA)患者和健康者进行采样检测，结果如下表1所示：
表1 SLE、RA和健康者mDCs上Sema4A表达表
Figure BDA0002631976780000081
由上表可知，SLE患者mDCs上膜结合型Sema4A的浓度较RA患者(p＝0.000)和健康者(p＝0.000)明显升高。该结果表明膜结合型Sema4A能够用于***性红斑狼疮的临床诊断，也能够与类风湿关节炎进行鉴别诊断。
实施例2
酶联免疫吸附试验(ELISA)检测血浆中可溶型Sema4A的浓度：
(1)将试剂盒中所有试剂置于室温下冻融30min，将-80℃冰箱中保存的血浆样品取出放置至室温后离心待用；
(2)试剂配制：①标准品配制：从ELISA试剂盒中取出一支Sema4A抗体标准品，于10000转/每分钟离心30秒钟，用1ml样本稀释液溶解，并用枪头对准EP管底部反复吹打以助溶解，充分混匀后得到标准品S7；然后取7个1.5mlEp管(分别命名为S0-S6)依序排列，分别加入250pl样本稀释液，吸取250mlS7标准品置于第一个Ep管中(S6)，并轻轻吹打混匀，从S6中吸取250ml到第二个Ep管中(S5)，同样轻轻吹打混匀，以此类推进行标准品的倍比稀释，S0为样本稀释液；②洗涤工作液配制：用去离子水将浓洗涤液按l：25倍进行稀释，以备用；③生物素标记抗体工作液配制：生物素标记抗体液按1：100倍用生物素标记抗体稀释液进行稀释，以备用；④辣根过氧化物酶标记亲和素工作液配制：辣根过氧化物酶标记亲和素按1：100倍用辣根过氧化物酶标记亲和素稀释液进行稀释，以备用；
(3)按照ELISA说明书进行以下操作：
①将各种试剂置于室温(18-25℃)平衡至少30min，按前述方法配制试剂，备用；
②分别设置标准品孔及待测样品孔，每孔分别加标准品或待测样品100μl，轻轻晃动混匀，覆上板贴后于37℃温育2h；
③弃去液体，甩干；
④每孔加入生物素标记抗体工作液100μl，覆上新的板贴，37℃温育1h；
⑤弃去孔内液体，甩干，使用自动洗板机洗涤5次，每次浸泡30s，200μl/每孔，甩干；
⑥每孔加入辣根过氧化物酶标记亲和素工作液100μl，覆上新的板贴，37℃温育1h；
⑦弃去孔内液体，甩干，使用自动洗板机洗涤6次，每次浸泡30s，200μl/每孔，甩干；
⑧每孔加底物溶液90μl，37℃避光显色30min；
⑨每孔加终止溶液50μl，终止反应；在5min内用酶标仪在450nm波长下测量各孔的光密度(OD值)。
采用上述方法分别对***性红斑狼疮患者、类风湿关节炎(RA)患者和健康者进行采样检测，结果如下表2所示：
表2 SLE、RA和健康者血浆中可溶型Sema4A浓度表
Figure BDA0002631976780000091
由上表可知，SLE患者血浆中可溶型Sema4A的表达较RA患者(p＝0.000)和健康者(p＝0.000)明显升高。该结果表明分泌型Sema4A能用于***性红斑狼疮的临床诊断，也能与类风湿关节炎进行鉴别诊断。
实施例3
在实施例1和实施例2的基础上，根据Pearson相关系数，进一步分析SLE患者Sema4A与临床指标的相关性，结果发现：SLE患者可溶型Sema4A的浓度与抗dsDNA抗体水平呈正相关(p＝0.000，r＝0.742)；②与C3、C4和Hb的水平均呈负相关(p＝0.001,r＝-0.583；p＝0.013,r＝-0.462；p＝0.000,r＝-0.734)；③尿蛋白阳性的SLE患者血浆中可溶型Sema4A的浓度较阴性组明显升高(p＝0.029)。
由此可见，Sema4A与目前用于诊断SLE的成熟指标抗dsDNA抗体具有强相关性，因此能够作为标志物用于评估SLE病情活动度(与C3、C4负相关)，以及具有一定评估血液***损伤(与Hb水平负相关)和肾脏损伤的价值(尿蛋白阳性的SLE患者血浆中可溶型Sema4A的浓度较尿蛋白阴性组明显升高)。
实施例4
在实施例1和实施例2的基础上，通过SPSS分析软件，建立ROC曲线评价可溶性Sema4A对SLE的诊断效能，计算得曲线下面积(AUC)为0.799，对应敏感性为0.833，特异性为0.696，Youden指数为0.529；ROC曲线评估mDC细胞上膜结合型Sema4A对SLE的诊断效能，计算得AUC为0.830，对应敏感性为0.864，特异性为0.733，Youden指数为0.597。
实施例5
在实施例1和实施例2的基础上，将Sema4A与现有的标志物的诊断效能相比较，如下表3所示：
表3诊断效能比较表
Figure BDA0002631976780000101
本发明标志物Sema4A计算ROC曲线下的面积AUC(Area Under Curve)分别为：可溶型0.799，膜结合型0.830.与现有的其他标志物比较，Sema4A的AUC值较高，仅次于ANA。表明Sema4A具有较好的诊断效能。
实施例6
在实施例1和实施例2的基础上，将Sema4A与现有的标志物的敏感性及特异性相比较，如下表4所示:
表4敏感性及特异性比较表
Figure BDA0002631976780000111
抗dsDNA抗体和抗Sm抗体为目前用于SLE诊断特异性最高的指标，但两者敏感性均很低，易造成漏检。而本发明的Sema4A敏感性＞80％(ROC曲线分析结果)，远高于抗dsDNA抗体和抗Sm抗体，能够弥补因敏感性不足而造成的对SLE诊断和治疗的延误。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
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565 570 575
Ala Gln Gln Ser Tyr Trp Pro His Phe Val Thr Val Thr Val Leu Phe
580 585 590
Ala Leu Val Leu Ser Gly Ala Leu Ile Ile Leu Val Ala Ser Pro Leu
595 600 605
Arg Ala Leu Arg Ala Arg Gly Lys Val Gln Gly Cys Glu Thr Leu Arg
610 615 620
Pro Gly Glu Lys Ala Pro Leu Ser Arg Glu Gln His Leu Gln Ser Pro
625 630 635 640
Lys Glu Cys Arg Thr Ser Ala Ser Asp Val Asp Ala Asp Asn Asn Cys
645 650 655
Leu Gly Thr Glu Val Ala
660
<210> 4
<211> 592
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Glu Gly Lys Gly Gln Ser Pro Phe Asp Pro Ala His Lys His Thr
1 5 10 15
Ala Val Leu Val Asp Gly Met Leu Tyr Ser Gly Thr Met Asn Asn Phe
20 25 30
Leu Gly Ser Glu Pro Ile Leu Met Arg Thr Leu Gly Ser Gln Pro Val
35 40 45
Leu Lys Thr Asp Asn Phe Leu Arg Trp Leu His His Asp Ala Ser Phe
50 55 60
Val Ala Ala Ile Pro Ser Thr Gln Val Val Tyr Phe Phe Phe Glu Glu
65 70 75 80
Thr Ala Ser Glu Phe Asp Phe Phe Glu Arg Leu His Thr Ser Arg Val
85 90 95
Ala Arg Val Cys Lys Asn Asp Val Gly Gly Glu Lys Leu Leu Gln Lys
100 105 110
Lys Trp Thr Thr Phe Leu Lys Ala Gln Leu Leu Cys Thr Gln Pro Gly
115 120 125
Gln Leu Pro Phe Asn Val Ile Arg His Ala Val Leu Leu Pro Ala Asp
130 135 140
Ser Pro Thr Ala Pro His Ile Tyr Ala Val Phe Thr Ser Gln Trp Gln
145 150 155 160
Val Gly Gly Thr Arg Ser Ser Ala Val Cys Ala Phe Ser Leu Leu Asp
165 170 175
Ile Glu Arg Val Phe Lys Gly Lys Tyr Lys Glu Leu Asn Lys Glu Thr
180 185 190
Ser Arg Trp Thr Thr Tyr Arg Gly Pro Glu Thr Asn Pro Arg Pro Gly
195 200 205
Ser Cys Ser Val Gly Pro Ser Ser Asp Lys Ala Leu Thr Phe Met Lys
210 215 220
Asp His Phe Leu Met Asp Glu Gln Val Val Gly Thr Pro Leu Leu Val
225 230 235 240
Lys Ser Gly Val Glu Tyr Thr Arg Leu Ala Val Glu Thr Ala Gln Gly
245 250 255
Leu Asp Gly His Ser His Leu Val Met Tyr Leu Gly Thr Thr Thr Gly
260 265 270
Ser Leu His Lys Ala Val Val Ser Gly Asp Ser Ser Ala His Leu Val
275 280 285
Glu Glu Ile Gln Leu Phe Pro Asp Pro Glu Pro Val Arg Asn Leu Gln
290 295 300
Leu Ala Pro Thr Gln Gly Ala Val Phe Val Gly Phe Ser Gly Gly Val
305 310 315 320
Trp Arg Val Pro Arg Ala Asn Cys Ser Val Tyr Glu Ser Cys Val Asp
325 330 335
Cys Val Leu Ala Arg Asp Pro His Cys Ala Trp Asp Pro Glu Ser Arg
340 345 350
Thr Cys Cys Leu Leu Ser Ala Pro Asn Leu Asn Ser Trp Lys Gln Asp
355 360 365
Met Glu Arg Gly Asn Pro Glu Trp Ala Cys Ala Ser Gly Pro Met Ser
370 375 380
Arg Ser Leu Arg Pro Gln Ser Arg Pro Gln Ile Ile Lys Glu Val Leu
385 390 395 400
Ala Val Pro Asn Ser Ile Leu Glu Leu Pro Cys Pro His Leu Ser Ala
405 410 415
Leu Ala Ser Tyr Tyr Trp Ser His Gly Pro Ala Ala Val Pro Glu Ala
420 425 430
Ser Ser Thr Val Tyr Asn Gly Ser Leu Leu Leu Ile Val Gln Asp Gly
435 440 445
Val Gly Gly Leu Tyr Gln Cys Trp Ala Thr Glu Asn Gly Phe Ser Tyr
450 455 460
Pro Val Ile Ser Tyr Trp Val Asp Ser Gln Asp Gln Thr Leu Ala Leu
465 470 475 480
Asp Pro Glu Leu Ala Gly Ile Pro Arg Glu His Val Lys Val Pro Leu
485 490 495
Thr Arg Val Ser Gly Gly Ala Ala Leu Ala Ala Gln Gln Ser Tyr Trp
500 505 510
Pro His Phe Val Thr Val Thr Val Leu Phe Ala Leu Val Leu Ser Gly
515 520 525
Ala Leu Ile Ile Leu Val Ala Ser Pro Leu Arg Ala Leu Arg Ala Arg
530 535 540
Gly Lys Val Gln Gly Cys Glu Thr Leu Arg Pro Gly Glu Lys Ala Pro
545 550 555 560
Leu Ser Arg Glu Gln His Leu Gln Ser Pro Lys Glu Cys Arg Thr Ser
565 570 575
Ala Ser Asp Val Asp Ala Asp Asn Asn Cys Leu Gly Thr Glu Val Ala
580 585 590

Claims (3)

1.一种***性红斑狼疮的诊断标志物在制备用于鉴别区分***性红斑狼疮和类风湿性关节炎的制剂中的应用，其特征在于，所述诊断标志物的氨基酸序列如SEQ ID NO：1~SEQID NO：4所示中的至少一种；
其中，***性红斑狼疮患者膜结合型和可溶型Sema4A显著高于健康人群，类风湿关节炎患者膜结合型和可溶型Sema4A显著低于健康人群；
所述诊断标志物来自于外周血细胞。
2.根据权利要求1所述的应用，其特征在于，所述制剂包括能与权利要求1中所述的诊断标志物发生特异性结合的抗体。
3.一种***性红斑狼疮的诊断标志物在制备用于鉴别区分***性红斑狼疮和类风湿性关节炎的试剂盒中的用途，其特征在于，所述试剂盒包括能与权利要求1中所述的诊断标志物发生特异性结合的抗体。
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104105706A (zh) * 2011-10-06 2014-10-15 德克萨斯州立大学董事会 用于治疗疾病的抗人sema4A抗体

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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IN2012DN02485A (zh) * 2009-09-03 2015-08-28 Hoffmann La Roche
CN102688283B (zh) * 2012-06-15 2014-07-02 复旦大学附属中山医院 一种***的中药胶囊及其制备方法

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* Cited by examiner, † Cited by third party
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CN104105706A (zh) * 2011-10-06 2014-10-15 德克萨斯州立大学董事会 用于治疗疾病的抗人sema4A抗体

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