CN106543277B - 一种与移植排斥相关的内皮细胞抗原及其应用、试剂盒 - Google Patents
一种与移植排斥相关的内皮细胞抗原及其应用、试剂盒 Download PDFInfo
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Abstract
本发明公开了一种与移植排斥相关的人体内皮细胞移植抗原及其应用和临床检测试剂盒。该内皮细胞抗原为角蛋白1。检测角蛋白1抗体的试剂盒用于评估临床移植排斥的风险和状态。本发明为探究临床器官移植排斥发生的原因与如何预测排斥反应的发生提供新的研究目标，为建立新的临床器官移植风险评估系统提供新思路。
Description
技术领域
本发明属于器官移植免疫排斥评估预测技术领域，具体涉及一种与移植排斥相关的人体内皮细胞新移植抗原及其应用。
背景技术
器官移植排斥反应仍然是移植成功的重要屏障。肾移植是迄今挽救终末期肾脏功能衰竭患者生命最理想的治疗手段。而器官移植后出现的超急性、急性和慢性排斥反应使移植物不能在受者体内发挥正常的生理功能，器官被排斥，功能丢失，严重影响移植受者的生活质量和生命安全。移植物主要组织相容性抗原包括ABO血型抗原和人类白细胞抗原(HLA)。然而，在不存在供体HLA抗体的情况下，急性排斥反应也可以发生。预示尚有Non-HLA抗原参与移植排斥反应。
目前临床上公认的移植抗原包括ABO血型抗原，HLA与主要组织相容性复合体I类相关链A(MICA)。Non-HLA，如MICA/B，波形蛋白，血管紧张素II 1型受体，微管蛋白，肌球蛋白，和胶原蛋白均可成为相应抗体攻击的靶抗原，使移植物遭受免疫损伤。临床上观察到有病例出现在红细胞血型相合和交叉配型阴性的情况下，接受肾移植后仍然发生移植排斥反应的现象。这提示还有其他的因素在移植排斥反应中起作用。移植物的血管内皮是接触受者血液循环的前沿阵地，也是遭受宿主免疫攻击的首要部位。有研究提出，抗内皮细胞抗体在临床器官移植方面具有重要作用。临床上HLA交叉配型反应阴性的肾移植病例出现不明原因的排斥反应很可能与该内皮细胞抗原有关。尽管越来越多的证据表明抗内皮细胞抗体参与了宿主对同种异体移植物的响应，但抗内皮细胞抗体确切针对的是何种内皮细胞表面的抗原分子尚不清楚。
角蛋白(Keratin，KRT)是存在于上皮细胞并具有一定物理化学性质的蛋白质。根据角蛋白呈电中性时的pH值(等电点)将其分为I型(酸性)和II型(碱性)。在人类中，I型角蛋白包括KRT9-KRT23、KRT25-KRT28和KRT31-KRT38；II型角蛋白包括KRT1-KRT8、KRT71-KRT80和KRT81-KRT86。角蛋白1(genebank ID：3848)是上皮细胞中重要的蛋白质，与角蛋白10组成的异二聚体是组成中间纤维的重要组分，在维持细胞核组织的形态中发挥重要作用。角蛋白1主要表达在基底上皮细胞，在血管内皮细胞，如毛细血管内皮细胞、静脉、动脉的内皮细胞也能检测到角蛋白1的表达。角蛋白1不仅在维持细胞核组织的形态中起着至关重要的作用，而且还参与了受体识别、信号传导，通过血管内皮细胞的氧化应激触发凝集素补体途径等生物学过程。本发现的线索来自临床肾移植病人存在有抗人体血管内皮细胞表面特异性抗体，这类病人常出现移植肾功能缺失。可以应用从这类病人血清中纯化出来的抗体来鉴定其作用的抗原。
发明内容
本发明的目的是提供一种与移植排斥相关的人体血管内皮细胞新的移植抗原及其应用，为探究临床器官移植排斥发生的原因与如何预测排斥反应的发生提供新的研究目标，为建立新的移植排斥评估和诊断体系提供检测试剂。
本发明提供了一种与临床器官移植排斥相关的人体内皮细胞抗原为角蛋白1(Keratin 1，KRT1)。
本发明还将检测角蛋白1抗体的试剂用于制备评估和诊断移植排斥风险和状况的制剂。
本发明还提供一种评估和诊断移植排斥状况的试剂盒，检测人体血清中角蛋白1抗体的试剂盒。
本发明的抗原的筛选、鉴定与验证，包括步骤如下：
(1)临床样本的筛选：
肾移植病人血清首先用随机的人脐带静脉内皮细胞(HUVEC)流式细胞检测AECAs，再用HLA抗体检测试剂盒和MICA抗体检测试剂盒进行HLA抗体和MICA抗体的检测，符合以下条件的血清作为下一步的研究标本：(1)血清肌酐水平>400umol/L，(2)随机的HUVEC流式交叉试验反应阳性，(3)抗HLA或MICA抗体阳性，或HUVEC阳性。(4)C4d的染色阳性。针对有HLA抗体存在的样本，用人体血小板试剂吸收血清中的HLA I类抗体，吸收后的血清再做抗体测试证实HLA I类抗体已经去除，同时避免使用含有MICA抗体的血清标本。挑选出的不含HLA抗体和MICA抗体的样本分别和来自不同个体的体外分离培养的HUVEC进行细胞流式反应，筛选出可能含有血管内皮细胞特异性抗体的血清样本。
(2)免疫沉淀法捕获抗内皮细胞抗体相应的抗原：
筛选出的血清样本与体外分离培养的HUVEC进行孵育，富集特异性抗体后收集细胞沉淀。进行细胞裂解处理，在裂解液中加入蛋白A免疫微珠，待蛋白A免疫微珠与裂解液中抗原-抗体复合物充分反应后，加入酸性洗脱缓冲液将蛋白A免疫微珠表面的抗原-抗体复合物洗脱；
(3)应用质谱技术鉴定捕获的抗原编码基因：
步骤(2)洗脱下来的抗原抗体复合物进行SDS聚丙烯酰胺凝胶电泳，用银染的方法显色，被特异性抗体捕获到的相应的免疫沉淀物与胶一起取出，应用蛋白质质谱技术对该抗原的编码基因进行鉴定；
(4)对鉴定的新移植抗原的验证：
以含有血管内皮细胞特异性抗体的血清作为抗体对经过基因工程表达的角蛋白1重组蛋白进行免疫印迹反应，还原抗体与特定抗原的结合反应。
将所述的筛选和验证方法用于为研究临床与移植排斥相关预测及检测试剂盒。
本发明的技术方案是基于抗原抗体相互作用的基本原理，对特异性抗原以免疫沉淀的方法来捕获，经SDS-聚丙烯酰胺凝胶电泳展示，并进行蛋白质质谱鉴定。
本发明的优势在于首次发现大量肾移植排斥的病人血清与人体血管内皮细胞表面的角蛋白1有抗原抗体反应，与体液排斥反应相关。为深入研究临床器官移植排斥的抗原因素提供新靶标；为完善器官移植临床配型试验和方案提供一种新的移植抗原作为检测试剂。
附图说明
图1免疫沉淀过程流程图；
图2洗脱的抗原-抗体复合物经SDS-PAGE后的银染结果；与健康正常人血清相比，移植受者血清中捕获到人体血管内皮细胞特异性抗原；
图3对鉴定的移植抗原的验证；以人群中频率最高的三种角蛋白1的重组蛋白作为抗原，以含有内皮细胞抗原的肾移植病人血清和健康正常人血清作为抗体进行免疫印迹实验，结果显示肾移植病人血清确实存在抗内皮细胞抗原角蛋白1的抗体。
具体实施方式：
以下是本发明的具体实例，进一步描述本发明，但是本发明不仅限于此。
实施例
试剂来源：
M199培养基、血管内皮生长因子、胶原蛋白酶、胎牛血清、蛋白A微球珠为商品化产品，购自Invitrogen公司；混合健康人血小板试剂购自Sigma公司；HLA抗体检测试剂盒购自One Lambda公司；MICA抗体检测试剂；蛋白质质谱鉴定由华大基因完成。
临床样本的筛选
肾移植病人血清首先用随机的人脐带静脉内皮细胞(HUVEC)流式细胞检测AECAs，再用HLA抗体检测试剂盒和MICA抗体检测试剂盒进行HLA抗体和MICA抗体的检测，符合以下条件的血清作为下一步的研究标本：(1)血清肌酐水平>400umol/L，(2)随机的HUVEC流式交叉试验反应阳性，(3)抗HLA或MICA抗体阳性，或HUVEC阳性。(4)C4d的染色阳性。针对有HLA抗体存在的样本，用人体血小板试剂吸收血清中的HLA I类抗体，吸收后的血清再做抗体测试证实HLA I类抗体已经去除，同时避免使用含有MICA抗体的血清标本。挑选出的不含HLA抗体和MICA抗体的样本分别和来自不同个体的体外分离培养的HUVEC进行细胞流式反应，筛选出可能含有血管内皮细胞特异性抗体的血清样本。
免疫沉淀法捕获抗内皮细胞抗体相应的抗原
筛选出的血清样本与体外分离培养的HUVEC进行孵育，富集特异性抗体后收集细胞沉淀。进行细胞裂解处理，在裂解液中加入蛋白A免疫微珠，待蛋白A免疫微珠与裂解液中抗原-抗体复合物充分反应后，加入酸性洗脱缓冲液将蛋白A免疫微珠表面的抗原-抗体复合物洗脱；
应用质谱技术鉴定捕获的新移植抗原编码基因
洗脱下来的抗原抗体复合物进行SDS聚丙烯酰胺凝胶电泳，用银染的方法显色，被特异性抗体捕获到的相应的抗原与胶一起取出，应用蛋白质质谱技术对该抗原的编码基因进行鉴定。运用Mascot蛋白质鉴定软件鉴定出SDS-PAGE标本中蛋白质。
蛋白质质谱鉴定结果显示角蛋白1的蛋白分数最高(11578)，在检测到的2116个肽段中的315个肽段(14.9％)属于角蛋白1。该315个肽段共包括38种(见表1)，覆盖了角蛋白1氨基酸序列的64％。
表1.质谱鉴定结果中角蛋白1的相关肽段信息
对鉴定的新移植抗原的验证
将人群中频率最高的三种角蛋白1(野生型、突变型、缺失型)进行原核表达获得重组蛋白(序列见序列表)，以该重组蛋白作为抗原，以角蛋白1的兔多克隆抗体、含有血管内皮细胞特异性抗体的血清和正常健康人血清作为抗体对重组表达的角蛋白1进行免疫印迹来验证，结果显示，商业化的抗角蛋白1的多克隆兔血清与三种重组蛋白发生特异性结合，说明重组蛋白具有功能活性。应用已知的三种角蛋白作为抗原，还原了病人血清中抗体与鉴定的角蛋白1的免疫反应性。结果见附图。
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<211> 12
<212> PRT
<213> 编号为21的角蛋白1肽段
<400> 21
Ser Lys Ala Glu Ala Glu Ser Leu Tyr Gln Ser Lys
1 5 10
<210> 22
<211> 10
<212> PRT
<213> 编号为22的角蛋白1肽段
<400> 22
Ala Glu Ala Glu Ser Leu Tyr Gln Ser Lys
1 5 10
<210> 23
<211> 20
<212> PRT
<213> 编号为23的角蛋白1肽段
<400> 23
Ala Glu Ala Glu Ser Leu Tyr Gln Ser Lys Tyr Glu Glu Leu Gln Ile
1 5 10 15
Thr Ala Gly Arg
20
<210> 24
<211> 10
<212> PRT
<213> 编号为24的角蛋白1肽段
<400> 24
Tyr Glu Glu Leu Gln Ile Thr Ala Gly Arg
1 5 10
<210> 25
<211> 8
<212> PRT
<213> 编号为25的角蛋白1肽段
<400> 25
Ile Glu Ile Ser Glu Leu Asn Arg
1 5
<210> 26
<211> 16
<212> PRT
<213> 编号为26的角蛋白1肽段
<400> 26
Lys Gln Ile Ser Asn Leu Gln Gln Ser Ile Ser Asp Ala Glu Gln Arg
1 5 10 15
<210> 27
<211> 15
<212> PRT
<213> 编号为27的角蛋白1肽段
<400> 27
Gln Ile Ser Asn Leu Gln Gln Ser Ile Ser Asp Ala Glu Gln Arg
1 5 10 15
<210> 28
<211> 21
<212> PRT
<213> 编号为28的角蛋白1肽段
<400> 28
Gln Ile Ser Asn Leu Gln Gln Ser Ile Ser Asp Ala Glu Gln Arg Gly
1 5 10 15
Glu Asn Ala Leu Lys
20
<210> 29
<211> 14
<212> PRT
<213> 编号为29的角蛋白1肽段
<400> 29
Asn Lys Leu Asn Asp Leu Glu Asp Ala Leu Gln Gln Ala Lys
1 5 10
<210> 30
<211> 12
<212> PRT
<213> 编号为30的角蛋白1肽段
<400> 30
Leu Asn Asp Leu Glu Asp Ala Leu Gln Gln Ala Lys
1 5 10
<210> 31
<211> 17
<212> PRT
<213> 编号为31的角蛋白1肽段
<400> 31
Leu Asn Asp Leu Glu Asp Ala Leu Gln Gln Ala Lys Glu Asp Leu Ala
1 5 10 15
Arg
<210> 32
<211> 12
<212> PRT
<213> 编号为32的角蛋白1肽段
<400> 32
Leu Leu Arg Asp Tyr Gln Glu Leu Met Asn Thr Lys
1 5 10
<210> 33
<211> 9
<212> PRT
<213> 编号为33的角蛋白1肽段
<400> 33
Asp Tyr Gln Glu Leu Met Asn Thr Lys
1 5
<210> 34
<211> 11
<212> PRT
<213> 编号为34的角蛋白1肽段
<400> 34
Leu Ala Leu Asp Leu Glu Ile Ala Thr Tyr Arg
1 5 10
<210> 35
<211> 9
<212> PRT
<213> 编号为35的角蛋白1肽段
<400> 35
Thr Leu Leu Glu Gly Glu Glu Ser Arg
1 5
<210> 36
<211> 26
<212> PRT
<213> 编号为36的角蛋白1肽段
<400> 36
Met Ser Gly Glu Cys Ala Pro Asn Val Ser Val Ser Val Ser Thr Ser
1 5 10 15
His Thr Thr Ile Ser Gly Gly Gly Ser Arg
20 25
<210> 37
<211> 31
<212> PRT
<213> 编号为37的角蛋白1肽段
<400> 37
Gly Gly Gly Gly Gly Gly Tyr Gly Ser Gly Gly Ser Ser Tyr Gly Ser
1 5 10 15
Gly Gly Gly Ser Tyr Gly Ser Gly Gly Gly Gly Gly Gly Gly Arg
20 25 30
<210> 38
<211> 39
<212> PRT
<213> 编号为38的角蛋白1肽段
<400> 38
Gly Ser Tyr Gly Ser Gly Gly Ser Ser Tyr Gly Ser Gly Gly Gly Ser
1 5 10 15
Tyr Gly Ser Gly Gly Gly Gly Gly Gly His Gly Ser Tyr Gly Ser Gly
20 25 30
Ser Ser Ser Gly Gly Tyr Arg
35
<210> 39
<211> 651
<212> PRT
<213> 野生型角蛋白1氨基酸序列
<400> 39
Met Ser Arg Gln Phe Ser Ser Arg Ser Gly Tyr Arg Ser Gly Gly Gly
1 5 10 15
Phe Ser Ser Gly Ser Ala Gly Ile Ile Asn Tyr Gln Arg Arg Thr Thr
20 25 30
Ser Ser Ser Thr Arg Arg Ser Gly Gly Gly Gly Gly Arg Phe Ser Ser
35 40 45
Cys Gly Gly Gly Gly Gly Ser Phe Gly Ala Gly Gly Gly Phe Gly Ser
50 55 60
Arg Ser Leu Val Asn Leu Gly Gly Ser Lys Ser Ile Ser Ile Ser Val
65 70 75 80
Ala Arg Gly Gly Gly Arg Gly Ser Gly Phe Gly Gly Gly Tyr Gly Gly
85 90 95
Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly
100 105 110
Gly Ile Gly Gly Gly Gly Phe Gly Gly Phe Gly Ser Gly Gly Gly Gly
115 120 125
Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Tyr Gly Gly Gly Tyr Gly
130 135 140
Pro Val Cys Pro Pro Gly Gly Ile Gln Glu Val Thr Ile Asn Gln Ser
145 150 155 160
Leu Leu Gln Pro Leu Asn Val Glu Ile Asp Pro Glu Ile Gln Lys Val
165 170 175
Lys Ser Arg Glu Arg Glu Gln Ile Lys Ser Leu Asn Asn Gln Phe Ala
180 185 190
Ser Phe Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn Gln Val Leu
195 200 205
Gln Thr Lys Trp Glu Leu Leu Gln Gln Val Asp Thr Ser Thr Arg Thr
210 215 220
His Asn Leu Glu Pro Tyr Phe Glu Ser Phe Ile Asn Asn Leu Arg Arg
225 230 235 240
Arg Val Asp Gln Leu Lys Ser Asp Gln Ser Arg Leu Asp Ser Glu Leu
245 250 255
Lys Asn Met Gln Asp Met Val Glu Asp Tyr Arg Asn Lys Tyr Glu Asp
260 265 270
Glu Ile Asn Lys Arg Thr Asn Ala Glu Asn Glu Phe Val Thr Ile Lys
275 280 285
Lys Asp Val Asp Gly Ala Tyr Met Thr Lys Val Asp Leu Gln Ala Lys
290 295 300
Leu Asp Asn Leu Gln Gln Glu Ile Asp Phe Leu Thr Ala Leu Tyr Gln
305 310 315 320
Ala Glu Leu Ser Gln Met Gln Thr Gln Ile Ser Glu Thr Asn Val Ile
325 330 335
Leu Ser Met Asp Asn Asn Arg Ser Leu Asp Leu Asp Ser Ile Ile Ala
340 345 350
Glu Val Lys Ala Gln Tyr Glu Asp Ile Ala Gln Lys Ser Lys Ala Glu
355 360 365
Ala Glu Ser Leu Tyr Gln Ser Lys Tyr Glu Glu Leu Gln Ile Thr Ala
370 375 380
Gly Arg His Gly Asp Ser Val Arg Asn Ser Lys Ile Glu Ile Ser Glu
385 390 395 400
Leu Asn Arg Val Ile Gln Arg Leu Arg Ser Glu Ile Asp Asn Val Lys
405 410 415
Lys Gln Ile Ser Asn Leu Gln Gln Ser Ile Ser Asp Ala Glu Gln Arg
420 425 430
Gly Glu Asn Ala Leu Lys Asp Ala Lys Asn Lys Leu Asn Asp Leu Glu
435 440 445
Asp Ala Leu Gln Gln Ala Lys Glu Asp Leu Ala Arg Leu Leu Arg Asp
450 455 460
Tyr Gln Glu Leu Met Asn Thr Lys Leu Ala Leu Asp Leu Glu Ile Ala
465 470 475 480
Thr Tyr Arg Thr Leu Leu Glu Gly Glu Glu Ser Arg Met Ser Gly Glu
485 490 495
Cys Ala Pro Asn Val Ser Val Ser Val Ser Thr Ser His Thr Thr Ile
500 505 510
Ser Gly Gly Gly Ser Arg Gly Gly Gly Gly Gly Gly Tyr Gly Ser Gly
515 520 525
Gly Ser Ser Tyr Gly Ser Gly Gly Gly Ser Tyr Gly Ser Gly Gly Gly
530 535 540
Gly Gly Gly Gly Arg Gly Ser Tyr Gly Ser Gly Gly Ser Ser Tyr Gly
545 550 555 560
Ser Gly Gly Gly Ser Tyr Gly Ser Gly Gly Gly Gly Gly Gly His Gly
565 570 575
Ser Tyr Gly Ser Gly Ser Ser Ser Gly Gly Tyr Arg Gly Gly Ser Gly
580 585 590
Gly Gly Gly Gly Gly Ser Ser Gly Gly Arg Gly Ser Gly Gly Gly Ser
595 600 605
Ser Gly Gly Ser Ile Gly Gly Arg Gly Ser Ser Ser Gly Gly Val Lys
610 615 620
Ser Ser Gly Gly Ser Ser Ser Val Lys Phe Val Ser Thr Thr Tyr Ser
625 630 635 640
Gly Val Thr Arg Lys Leu Ala Ala Ala Leu Glu
645 650
<210> 40
<211> 651
<212> PRT
<213> 突变型角蛋白1氨基酸序列
<400> 40
Met Ser Arg Gln Phe Ser Ser Arg Ser Gly Tyr Arg Ser Gly Gly Gly
1 5 10 15
Phe Ser Ser Gly Ser Ala Gly Ile Ile Asn Tyr Gln Arg Arg Thr Thr
20 25 30
Ser Ser Ser Thr Arg Arg Ser Gly Gly Gly Gly Gly Arg Phe Ser Ser
35 40 45
Cys Gly Gly Gly Gly Gly Ser Phe Gly Ala Gly Gly Gly Phe Gly Ser
50 55 60
Arg Ser Leu Val Asn Leu Gly Gly Ser Lys Ser Ile Ser Ile Ser Val
65 70 75 80
Ala Arg Gly Gly Gly Arg Gly Ser Gly Phe Gly Gly Gly Tyr Gly Gly
85 90 95
Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly
100 105 110
Gly Ile Gly Gly Gly Gly Phe Gly Gly Phe Gly Ser Gly Gly Gly Gly
115 120 125
Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Tyr Gly Gly Gly Tyr Gly
130 135 140
Pro Val Cys Pro Pro Gly Gly Ile Gln Glu Val Thr Ile Asn Gln Ser
145 150 155 160
Leu Leu Gln Pro Leu Asn Val Glu Ile Asp Pro Glu Ile Gln Lys Val
165 170 175
Lys Ser Arg Glu Arg Glu Gln Ile Lys Ser Leu Asn Asn Gln Phe Ala
180 185 190
Ser Phe Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn Gln Val Leu
195 200 205
Gln Thr Lys Trp Glu Leu Leu Gln Gln Val Asp Thr Ser Thr Arg Thr
210 215 220
His Asn Leu Glu Pro Tyr Phe Glu Ser Phe Ile Asn Asn Leu Arg Arg
225 230 235 240
Arg Val Asp Gln Leu Lys Ser Asp Gln Ser Arg Leu Asp Ser Glu Leu
245 250 255
Lys Asn Met Gln Asp Met Val Glu Asp Tyr Arg Asn Lys Tyr Glu Asp
260 265 270
Glu Ile Asn Lys Arg Thr Asn Ala Glu Asn Glu Phe Val Thr Ile Lys
275 280 285
Lys Asp Val Asp Gly Ala Tyr Met Thr Lys Val Asp Leu Gln Ala Lys
290 295 300
Leu Asp Asn Leu Gln Gln Glu Ile Asp Phe Leu Thr Ala Leu Tyr Gln
305 310 315 320
Ala Glu Leu Ser Gln Met Gln Thr Gln Ile Ser Glu Thr Asn Val Ile
325 330 335
Leu Ser Met Asp Asn Asn Arg Ser Leu Asp Leu Asp Ser Ile Ile Ala
340 345 350
Glu Val Lys Ala Gln Tyr Glu Asp Ile Ala Gln Lys Ser Lys Ala Glu
355 360 365
Ala Glu Ser Leu Tyr Gln Ser Lys Tyr Glu Glu Leu Gln Ile Thr Ala
370 375 380
Gly Arg His Gly Asp Ser Val Arg Asn Ser Lys Ile Glu Ile Ser Glu
385 390 395 400
Leu Asn Arg Val Ile Gln Arg Leu Arg Ser Glu Ile Asp Asn Val Lys
405 410 415
Lys Gln Ile Ser Asn Leu Gln Gln Ser Ile Ser Asp Ala Glu Gln Arg
420 425 430
Gly Glu Asn Ala Leu Lys Asp Ala Lys Asn Lys Leu Asn Asp Leu Glu
435 440 445
Asp Ala Leu Gln Gln Ala Lys Glu Asp Leu Ala Arg Leu Leu Arg Asp
450 455 460
Tyr Gln Glu Leu Met Asn Thr Lys Leu Ala Leu Asp Leu Glu Ile Ala
465 470 475 480
Thr Tyr Arg Thr Leu Leu Glu Gly Glu Glu Ser Arg Met Ser Gly Glu
485 490 495
Cys Ala Pro Asn Val Ser Val Ser Val Ser Thr Ser His Thr Thr Ile
500 505 510
Ser Gly Gly Gly Ser Arg Gly Gly Gly Gly Gly Gly Tyr Gly Ser Gly
515 520 525
Gly Ser Ser Tyr Gly Ser Gly Gly Gly Ser Tyr Gly Ser Gly Gly Gly
530 535 540
Gly Gly Gly Gly Arg Gly Ser Tyr Gly Ser Gly Gly Ser Ser Tyr Gly
545 550 555 560
Ser Gly Gly Gly Ser Tyr Gly Ser Gly Gly Gly Gly Gly Gly His Gly
565 570 575
Ser Tyr Gly Ser Gly Ser Ser Ser Gly Gly Tyr Arg Gly Gly Ser Gly
580 585 590
Gly Gly Gly Gly Gly Ser Ser Gly Gly Arg Gly Ser Gly Gly Gly Ser
595 600 605
Ser Gly Gly Ser Ile Gly Gly Arg Gly Ser Ser Ser Gly Gly Val Lys
610 615 620
Ser Ser Gly Gly Ser Ser Ser Val Arg Phe Val Ser Thr Thr Tyr Ser
625 630 635 640
Gly Val Thr Arg Lys Leu Ala Ala Ala Leu Glu
645 650
<210> 41
<211> 644
<212> PRT
<213> 缺失型角蛋白1氨基酸序列
<400> 41
Met Ser Arg Gln Phe Ser Ser Arg Ser Gly Tyr Arg Ser Gly Gly Gly
1 5 10 15
Phe Ser Ser Gly Ser Ala Gly Ile Ile Asn Tyr Gln Arg Arg Thr Thr
20 25 30
Ser Ser Ser Thr Arg Arg Ser Gly Gly Gly Gly Gly Arg Phe Ser Ser
35 40 45
Cys Gly Gly Gly Gly Gly Ser Phe Gly Ala Gly Gly Gly Phe Gly Ser
50 55 60
Arg Ser Leu Val Asn Leu Gly Gly Ser Lys Ser Ile Ser Ile Ser Val
65 70 75 80
Ala Arg Gly Gly Gly Arg Gly Ser Gly Phe Gly Gly Gly Tyr Gly Gly
85 90 95
Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly
100 105 110
Gly Ile Gly Gly Gly Gly Phe Gly Gly Phe Gly Ser Gly Gly Gly Gly
115 120 125
Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Tyr Gly Gly Gly Tyr Gly
130 135 140
Pro Val Cys Pro Pro Gly Gly Ile Gln Glu Val Thr Ile Asn Gln Ser
145 150 155 160
Leu Leu Gln Pro Leu Asn Val Glu Ile Asp Pro Glu Ile Gln Lys Val
165 170 175
Lys Ser Arg Glu Arg Glu Gln Ile Lys Ser Leu Asn Asn Gln Phe Ala
180 185 190
Ser Phe Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn Gln Val Leu
195 200 205
Gln Thr Lys Trp Glu Leu Leu Gln Gln Val Asp Thr Ser Thr Arg Thr
210 215 220
His Asn Leu Glu Pro Tyr Phe Glu Ser Phe Ile Asn Asn Leu Arg Arg
225 230 235 240
Arg Val Asp Gln Leu Lys Ser Asp Gln Ser Arg Leu Asp Ser Glu Leu
245 250 255
Lys Asn Met Gln Asp Met Val Glu Asp Tyr Arg Asn Lys Tyr Glu Asp
260 265 270
Glu Ile Asn Lys Arg Thr Asn Ala Glu Asn Glu Phe Val Thr Ile Lys
275 280 285
Lys Asp Val Asp Gly Ala Tyr Met Thr Lys Val Asp Leu Gln Ala Lys
290 295 300
Leu Asp Asn Leu Gln Gln Glu Ile Asp Phe Leu Thr Ala Leu Tyr Gln
305 310 315 320
Ala Glu Leu Ser Gln Met Gln Thr Gln Ile Ser Glu Thr Asn Val Ile
325 330 335
Leu Ser Met Asp Asn Asn Arg Ser Leu Asp Leu Asp Ser Ile Ile Ala
340 345 350
Glu Val Lys Ala Gln Tyr Glu Asp Ile Ala Gln Lys Ser Lys Ala Glu
355 360 365
Ala Glu Ser Leu Tyr Gln Ser Lys Tyr Glu Glu Leu Gln Ile Thr Ala
370 375 380
Gly Arg His Gly Asp Ser Val Arg Asn Ser Lys Ile Glu Ile Ser Glu
385 390 395 400
Leu Asn Arg Val Ile Gln Arg Leu Arg Ser Glu Ile Asp Asn Val Lys
405 410 415
Lys Gln Ile Ser Asn Leu Gln Gln Ser Ile Ser Asp Ala Glu Gln Arg
420 425 430
Gly Glu Asn Ala Leu Lys Asp Ala Lys Asn Lys Leu Asn Asp Leu Glu
435 440 445
Asp Ala Leu Gln Gln Ala Lys Glu Asp Leu Ala Arg Leu Leu Arg Asp
450 455 460
Tyr Gln Glu Leu Met Asn Thr Lys Leu Ala Leu Asp Leu Glu Ile Ala
465 470 475 480
Thr Tyr Arg Thr Leu Leu Glu Gly Glu Glu Ser Arg Met Ser Gly Glu
485 490 495
Cys Ala Pro Asn Val Ser Val Ser Val Ser Thr Ser His Thr Thr Ile
500 505 510
Ser Gly Gly Gly Ser Arg Gly Gly Gly Gly Gly Gly Tyr Gly Ser Gly
515 520 525
Gly Ser Ser Tyr Gly Ser Gly Gly Gly Ser Tyr Gly Ser Gly Gly Gly
530 535 540
Gly Gly Gly Gly Arg Gly Ser Tyr Gly Ser Gly Gly Gly Ser Tyr Gly
545 550 555 560
Ser Gly Gly Gly Gly Gly Gly His Gly Ser Tyr Gly Ser Gly Ser Ser
565 570 575
Ser Gly Gly Tyr Arg Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser
580 585 590
Gly Gly Arg Gly Ser Gly Gly Gly Ser Ser Gly Gly Ser Ile Gly Gly
595 600 605
Arg Gly Ser Ser Ser Gly Gly Val Lys Ser Ser Gly Gly Ser Ser Ser
610 615 620
Val Lys Phe Val Ser Thr Thr Tyr Ser Gly Val Thr Arg Lys Leu Ala
625 630 635 640
Ala Ala Leu Glu
Claims (1)
1.检测角蛋白1抗体的试剂在制备用于评估和诊断移植排斥风险和状况的制剂中的应用。
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|---|---|---|---|---|
| CN1602426A (zh) * | 2001-12-11 | 2005-03-30 | 技术科学基金会 | 从患有类风湿性关节炎的患者中检测自身抗体的方法、肽和检测试剂盒 |
| CN104619864A (zh) * | 2012-07-13 | 2015-05-13 | 科学发展实验室 | 用于鉴定儿童皮肤的分子标记物的方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1602426A (zh) * | 2001-12-11 | 2005-03-30 | 技术科学基金会 | 从患有类风湿性关节炎的患者中检测自身抗体的方法、肽和检测试剂盒 |
| CN104619864A (zh) * | 2012-07-13 | 2015-05-13 | 科学发展实验室 | 用于鉴定儿童皮肤的分子标记物的方法 |
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| Analysis of Sera of Recipients with Allograft Rejection Indicates That Keratin 1 Is the Target of Anti-Endothelial Antibodies;Xuli Guo et al.,;《Journal of Immunology Research》;20170228;1-11 * |
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| 肾移植受者血清抗血管内皮细胞特异性抗体的提取和纯化;刘丹等;《器官移植》;20150531;第6卷(第3期);890-892 * |
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