CN114729019A - 重组钙卫蛋白 - Google Patents
重组钙卫蛋白 Download PDFInfo
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- CN114729019A CN114729019A CN202180006638.4A CN202180006638A CN114729019A CN 114729019 A CN114729019 A CN 114729019A CN 202180006638 A CN202180006638 A CN 202180006638A CN 114729019 A CN114729019 A CN 114729019A
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- polypeptide
- leu
- glu
- lys
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Abstract
本发明涉及一种多肽,该多肽包含与SEQ D NO:1具有至少80%序列同一性的氨基酸序列的第一链;包含与SEO D NO:2具有至少80%序列同一性的氨基酸序列的第二链:和连接第一链和第二链的接头。此外,本发明涉及使用这些多肽作为诊断方法中的校准物和标准物的方法。
Description
技术领域
本发明涉及多肽,其包括包含与SEQ ID NO:1具有至少80%序列同一性的氨基酸序列的第一链;包含与SEQ ID NO:2具有至少80%序列同一性的氨基酸序列的第二链;以及连接第一和第二链的接头。此外,本发明涉及使用这些多肽作为诊断方法中的校准物和标准物的方法。
背景技术
钙卫蛋白(CP)是一种细胞质蛋白,表达于各种骨髓细胞类型,如中性粒细胞、单核细胞和巨噬细胞。在中性粒细胞中,钙卫蛋白是组成型表达的,可能构成总细胞质蛋白的约40%,而在上皮细胞和角质形成细胞中,可以诱导钙卫蛋白表达。
钙卫蛋白由两条多肽链Mrp8(同义词:S100A8,钙粒蛋白A)和Mrp14(同义词:S100A9,钙粒蛋白B)组成,形成稳定的二聚体。在大约150μM Ca2+的存在下,两个Mrp8/Mrp14异二聚体可以形成一个异四聚体,因其能够作为其他二价阳离子,如Zn2+的螯合复合物,可在营养免疫过程中饿死微生物,在营养免疫中具有重要作用(Zygiel EM,Nolan EM.宿主防御蛋白钙卫蛋白对过渡金属的螯合作用(Transition Metal Sequestration by theHost-Defense Protein Calprotectin)(2018).Annu.Rev.Biochem.87:621-43)。
由于其在炎症部位的释放,钙卫蛋白被认为是一种警报素,经常被用作监测炎症过程的生物标志物。例如,粪便钙卫蛋白目前是诊断和监测炎症性肠病(IBD)的金标准,例如克罗恩病(CD)和溃疡性结肠炎(UC)(Konikoff MR,Denson LA.粪便钙卫蛋白作为炎症性肠病肠道炎症生物标志物的作用(Role of Fecal Calprotectin as a Biomarker ofIntestinal Inflammation in Inflammatory Bowel Disease)(2006).Inflamm.BowelDis.12(6):524-34)。
此外,血清CP被验证为监测各种(慢性)炎症性疾病的生物标志物,例如类风湿性关节炎(Austermann J et al.风湿病中的S100蛋白(S100 proteins in rheumaticdiseases)(2018).Nat.Rev.Rheumatol.14:528~541;Ometto F et al.钙卫蛋白在风湿病中的作用(Calprotectin in rheumatic diseases)(2017).Exp.Biol.Med.242:859~873)。
为了开发可靠的免疫测定来测量钙卫蛋白,使用高度纯化的钙卫蛋白抗原非常重要,该抗原必须尽可能与天然钙卫蛋白相似。这避免了由校准品、对照品和样品之间的抗体反应性差异引起的测定问题。目前,钙卫蛋白主要作为粪便生物标志物用于在有机IBD和无机肠易激综合征之间进行区分,但没有普遍接受和/或验证的参考材料可用于校准血液或粪便分析测定。
方法比较表明,来自不同制造商的粪便钙卫蛋白测定之间存在明显的校准差异(De Sloovere MW et al.两种粪便钙卫蛋白自动免疫分析检测炎症性肠病的分析和诊断性能(Analytical and Diagnostic Performance of Two Automated FecalCalprotectin Immunoassays for Detection of Inflammatory Bowel Disease)(2017).Clin.Chem.Lab.Med.55(9):1435~1446),这严重加剧了医生对测试结果的解释。
获得纯钙卫蛋白的最先进方法包括在不溶性包涵体中通过精细的重折叠过程中的Mrp8和Mrp14的细菌表达(Hadley and Nolan,分子生物学方法(Methods in MolecularBiology),2019);或从人血中分离的粒细胞中纯化钙卫蛋白(Nilsen T,Haugen SH.从粒细胞中提取、分离和浓缩钙卫蛋白抗原(S100A8/S100A9)(Extraction,isolation,andconcentration of calprotectin antigen(S100A8/S100A9)from granulocytes)(2018).Health Sci.Rep.1:e35)。
然而,这两种方法都有几个缺点。从包涵体获得的Mrp8和Mrp14的重折叠可能会导致同源二聚体或其他重折叠伪影(Vogl et al.S100A8和S100A9在不存在和存在二价阳离子情况下的生物物理特性(Biophysical Characterization of S100A8 and S100A9 inthe Absence and Presence of Bivalent Cations)(2006).Biochim.Biophys.Acta 1763(11):1298-12306)。另一方面,从献血者中获取纯化钙卫蛋白的收率低,劳动强度大且成本高,并且不允许对钙卫蛋白进行定点诱变。
本领域需要与天然钙卫蛋白高度相似并且可以用作免疫测定和其他分析测定的可靠校准物和对照的人工钙卫蛋白。
发明内容
因此,本发明要解决的问题是提供保持天然存在的钙卫蛋白的重要特征的重组钙卫蛋白。
通过表达可溶性钙卫蛋白的方法解决了该问题,该方法包括从包含第一链的载体表达钙卫蛋白,该第一链包含与SEQ ID NO:11具有至少80%序列同源性的核苷酸序列,第二链包含具有与SEQ ID NO:12具有至少80%序列同源性的核苷酸序列,以及连接第一链和第二链的接头。
该问题还通过一种多肽来解决,该多肽包括:
a)包含与SEQ ID NO:1具有至少80%序列同一性的氨基酸序列的第一链;
b)包含与SEQ ID NO:2具有至少80%序列同一性的氨基酸序列的第二链;和
c)连接第一链和第二链的接头,
其中该接头是包含蛋白酶识别序列的肽接头。
本发明还涉及在分析测定中使用本发明的多肽测量样品中的S100A8、S100A9、S100A8/A9或其寡聚体的方法,包括以下步骤:
a)使用分析测定法测量不同含量的所述的多肽;
b)使用步骤a)中获得的分析结果建立校准曲线;
c)测量样品;
d)将样品的分析结果与步骤b)和的校准曲线进行比较;
e)量化样品中S100A8、S100A9、S100A8/A9二聚体或寡聚体的浓度。
本发明还涉及将本发明的多肽用于诊断受试者的急性或慢性炎症性疾病的方法中,该方法包括:
a)提供受试者的生物样本;
b)通过使用本发明的多肽寡聚体作为校准参考物质来量化步骤a)的生物样本中S100A8、S100A9、S100A8/A9或其寡聚体;和
c)将步骤b)中确定的S100A8、S100A9、S100A8/A9或其寡聚体的量与来自已知患有急性或慢性炎症性疾病的受试者的参考数据进行比较。
本发明还涉及一种试剂盒,包括:
a)本发明的多肽和/或其寡聚体;
b)测试容器;
c)缓冲溶液;
d)第一结合试剂。
附图说明
图1:本发明的融合蛋白在缓冲液A(20mM HEPES pH7.5、100mM NaCl、2.5%甘油、1mM DTT)中在HiPrep Sephacryl S200 16/60(通用电气医疗(GE Healthcare))上的尺寸排阻色谱。纯化的融合蛋白是单分散的,并作为单体(26.8kDa)从柱中洗脱。
图2:融合蛋白的3C消化。15ug纯化的rCAL(Mrp14-Mrp8融合)单体在室温下用150ng(++)或50ng(+)纯化的3C蛋白酶消化3小时。然后用LDS缓冲液(×4)制备样品用于SDS-PAGE,并在95℃下变性12分钟。SDS-PAGE显示融合蛋白被3C蛋白酶有效切割成S100A8和S100A9链。
图3:向缓冲液A添加2mM CaCl2显着改变了实施例1的重组融合多肽的洗脱体积,这表明二聚化。这相当于内源性钙卫蛋白中S100A8/S100A9的钙依赖性异四聚体。
图4:单克隆抗体27E10与钙卫蛋白的结合需要S100A8和S100A9的异二聚化(Hessian PA,Fisher L.MRP-8(S100A8)和MRP-14(S100A9)的异二聚体复合物(Theheterodimeric complex of MRP-8(S100A8)and MRP-14(S100A9))。抗体识别、表位定义及其对结构的影响(Antibody recognition,epitope definition and the implicationsfor structure)(2001).Eur.J.Biochem.268:353~363)。该融合蛋白在BLI(生物层干涉测量)测量中显示出对27E10 mAB的高亲和力,表明本发明的融合多肽的3D结构模拟了内源性钙卫蛋白异四聚体的异二聚体表面结构。
图5:采用一系列稀释的重组融合多肽(SEQ-ID NO:3)与单克隆捕获和检测抗体的夹心ELISA测量。捕获抗体与HRP连接,在加入TMB后导致在OD450处的浓度依赖性读数。加标融合多肽浓度(从0.5μg/ml到10μg/ml)与OD 450信号的线性相关表明融合多肽(SEQ-IDNO:3)能够以浓度依赖性方式被单克隆钙卫蛋白抗体识别。
图6:使用一系列稀释重组融合多肽(SEQ-ID NO:3)与多克隆抗体的浊度测量。PETIA(粒子增强比浊免疫测定法)也显示出与加标融合多肽浓度(从0到21.7ug/ml)相关的吸收信号。因此,针对S100A8/S100A9的多克隆抗体也容易识别本发明的融合多肽(SEQ-IDNO:3),证实其结构模拟内源性钙卫蛋白。
图7:从23个人血清样本中获得的结果与图6中描述的比浊免疫测定法进行比较,其中在一种配方中,免疫测定法用纯化的内源性钙卫蛋白(S100A8/A9异四聚体)校准,而在第二种配方中,用重组融合多肽校准(本发明的SEQ-ID NO:3)。然后将两个结果集关联并表示为Passing-Bablok图。
图8:用实施例5(图5)中描述的使用单克隆抗体的ELISA比较从本发明的重组融合多肽(SEQ-ID NO:3)的4种不同稀释液和23个人血清样品获得的结果(图5)与使用如实施例5(图6)中所述的多克隆抗体的比浊免疫测定法。对于该实验,两种测定均使用内源性钙卫蛋白进行校准。然后将两个结果集关联并表示为Passing-Bablok图。
图9:SEQ-ID NO:3融合蛋白的定点诱变及其后果。先前描述的突变(EP3248015A1)被引入融合蛋白中,该突变,即S100A9多肽链中的E78A,会损害S100A8/S100A9的异四聚化。这个E78A突变体保持单体,即使在CaCl2存在下。S200尺寸排阻色谱在缓冲液A+2mMCaCl2中将非突变融合蛋白转移到洗脱体积,表明与不添加钙的缓冲液A相比,二聚化(约63ml)不会改变含有E78A突变的融合蛋白的洗脱曲线(约56ml)。
具体实施方式
本发明涉及一种表达可溶性钙卫蛋白的方法,该方法包括从包含第一链的载体表达钙卫蛋白,该第一链包含与SEQ ID NO:11具有至少80%序列同源性的核苷酸序列,第二链包含与SEQ ID NO:12具有至少80%序列同源性的核苷酸序列和连接第一链和第二链的接头。
在优选的实施方案中,位于载体上连接第一链和第二链的接头具有18~180个核苷酸、18~120个核苷酸、18~90个核苷酸或18~60个核苷酸的长度,最优选为21~30个核苷酸长度。具有该长度的接头确保两条链足够接近以允许正确折叠。
在本文中,术语“S100A8”是指具有SEQ ID NO:1的多肽。由SEQ ID NO:11编码的人S100A8在本领域中也称为Mrp8或钙粒蛋白A。
在本文中,术语“S100A9”是指具有SEQ ID NO:2的多肽。由SEQ ID NO:12编码的人S100A9在本领域中也称为Mrp14或钙粒蛋白B。
一旦这两个分子相互接触,S100A8和S100A9的异二聚体就会立即自发形成。在金属离子,特别是二价金属离子如Ca2+的存在下,两个S100A8/S100A9异二聚体可以形成异四聚体。在本文中,术语“S100A8/S100A9的寡聚体”是指异二聚体、异四聚体和寡聚体,包括包含一种或多种S100A8和S100A9单体的多聚体。特别地,术语“S100A8/S100A9的寡聚体”是指内源性“钙卫蛋白”,即,异四聚体,包含两个单元,每个单元为S100A8和S100A9。
在本发明之前,重组钙卫蛋白的获取是通过将每个亚基分别表达到包涵体中,然后将亚基纯化、再折叠和二聚化以形成天然存在的异二聚体和异四聚体。这个过程不仅劳动强度大,而且容易出错,因为该过程通常会形成几乎没有分析价值的同源二聚体。
发明人惊奇地发现,通过连接两个亚基并使其表达为一种融合蛋白,在表达时会直接获得正确折叠的异二聚体,从而极大地促进了随后的纯化。因此,在一个优选的实施方案中,本发明的方法不包括钙卫蛋白的细胞外重折叠步骤。换句话说,本发明的方法确保钙卫蛋白在表达时已经正确折叠。
通过本发明的方法获得的异二聚体随后在二价金属离子的存在下组装成异四聚体,类似于天然钙卫蛋白异二聚体。通过本发明的方法获得的可溶性钙卫蛋白在结构上与天然钙卫蛋白基本相同,因此能够结合对S100A8/S100A9异二聚体具有特异性的单克隆抗体,特别是单克隆抗体mAb27E10。
具有克隆ID mAb27E10的单克隆抗体可以从商业上获得,例如从Abcam,UK(ab17050)或Santa Cruz Biotechnology,TX,USA(sc-33714)。mAb27E10已在Hessian和Fisher,Eur.J.Biochem.268,353~363(2001)中进一步描述。
本发明进一步涉及一种多肽,其包含:
a)第一链,其包含与SEQ ID NO:1具有至少80%序列同一性的氨基酸序列;
b)第二链,其包含与SEQ ID NO:2具有至少80%序列同一性的氨基酸序列;
c)连接第一链和第二链的接头,
其中,接头是包含蛋白酶识别序列的肽接头。
本发明的多肽包含两条链,其包含分别与SEQ ID NOs:1和2具有至少80%序列同一性的氨基酸序列。在本发明的上下文中,术语“具有至少80%序列同一性的序列”包括具有至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、至少99%、至少99.5%、最优选100%与相应序列的序列同一性。在所有情况下,同一性是指在相应氨基酸序列的总长度上的同一性。
SEQ ID NO:1对应于人S100A8并且SEQ ID NO:2对应于人S100A9。因此,本发明的多肽包含与构成人钙卫蛋白的两个单体相似或相同的氨基酸序列。在本文中,设想第一链是本领域已知的S100A8亚型之一,特别是具有93个氨基酸的亚型(SEQ ID NO:1)。同样,第一链可以对应于具有101,116或117个氨基酸S100A8亚型。
本发明的多肽的第一链优选地具有至少80,85或90个氨基酸长度。在一个特别优选的实施方案中,第一链正好是具有93、101、116或117个氨基酸长度。
本发明的多肽的第二链优选地具有至少90、100或110个氨基酸长度。在一个特别优选的实施方案中,第一链正好是具有114个氨基酸长度。
在本发明的多肽中,两条链通过肽接头连接。这种接头具有很好的适应性并且容易整合到本发明的多肽中。
优选地,肽接头的长度在1到20个氨基酸之间。在一个特别优选的实施方案中,肽接头的长度在5到10个氨基酸之间。具有该长度的肽接头特别适合以允许两条链之间相互作用的方式连接本发明的多肽的两条链,这种相互作用的方式就如同内源性钙卫蛋白中S100A8与S100A9的二聚体相互作用。
肽接头包含蛋白酶识别序列,即可以被蛋白酶识别和切割的氨基酸序列。这具有本发明的多肽的第一链和第二链可以通过添加各自的蛋白酶而彼此分离的优点。因此,本实施方式的多肽一旦被表达组装就可以被切割成与Mrp8和Mrp14对应的两条链,从而使Mrp8和Mrp14没有永久连接的生理状态得以恢复。
蛋白酶识别序列的许多例子是本领域已知的。例如,蛋白质识别序列对于鼻病毒3C蛋白酶(Prescission蛋白酶)可以是LEVLFQGP,对于TEV蛋白酶可以是ENLYFQG,对于凝血酶可以是LVPRGS。对于Xa因子来说时IEDGR,或者对于肠肽酶可以是DDDDK。
接头可以将第一链的N端或C端与第二链连接。在一个优选的实施方案中,第一链在N端与接头和第二条链相连。这种安排是有利的,因为它促进了多肽在大肠杆菌中的可溶性表达。
在本发明的一个方面,本发明的多肽能够在金属离子存在下彼此寡聚化,特别是二聚化。优选地,金属离子是二价金属离子,最优选地,Ca2+。当寡聚化时,本发明的多肽能够形成与天然存在的钙卫蛋白异四聚体高度相似的结构。这对于检测和校准内源性钙卫蛋白的免疫分析是有用的。
在本发明的另一方面,本发明的多肽能够形成一种结构,该结构能够被识别S100A8单体、S100A9单体、S100A8/S100A9二聚体和/或其寡聚体,包括内源性钙卫蛋白的结合试剂识别。
结合试剂可以是识别S100A8单体、S100A9单体、S100A8/S100A9二聚体和/或它们的寡聚体的任何实体,特别是钙卫蛋白。特别地,结合试剂可以是抗体或其片段、sybody、纳米抗体、单体、CAMELID HC抗体、affibody、TandAb、双环肽、DARPin、avimer、锚蛋白重复序列、BiTE、DART、anticalin或核苷酸序列。
本发明的多肽可以另外包含本领域已知的标签、标记、识别序列或信号,以允许对本发明的多肽的纯化、鉴定、定位和识别。在一个实施方案中,本发明的多肽包含允许使用色谱法有效纯化多肽的His标签(His-Tag)。可用于本发明的多肽的其他标签或标记是技术人员已知的FLAG标签(FLAG-Tag)、HA标签(HA-Tag)、Strep标签(Strep-Tag)或绿色荧光蛋白(GFP)。
在一个优选的实施方案中,本发明的多肽与SEQ ID NO:7、8、9、10、18或19具有至少80%的序列同一性。在一个优选的实施方案中,本发明的多肽与SEQ ID NO:7、8、9、10、18或19具有85%、90%或95%的序列同一性。在一个特别优选的实施方案中,本发明的多肽与SEQ ID NO:7、8、9、10、18或19具有100%的序列同一性。SEQ ID NO:7至10包含一个通过短氨基酸序列连接到第一链或第二链的His-Tag,该短氨基酸序列包含用于鼻病毒SC蛋白酶的蛋白酶序列,从而,通过添加鼻病毒3C蛋白酶,可以从包含两条链和接头的多肽部分切割His-Tag。因此,可以有效地纯化具有SEQ ID NO:7至10的多肽,并且随后去除His-Tag。此外,在SEQ ID NO:7至10中位于两条链之间的接头包含TEV蛋白酶的识别序列,使得两条链可以通过加入TEV蛋白酶而解离。
在其他实施方案中,如SEQ ID NO:3和4所示,本发明的多肽在His-Tag和两条链之间包含相同的蛋白酶识别序列,因此允许在一步中去除His-Tag和两条链之间的切割。
在进一步的实施方案中,与天然存在的S100A8和S100A9相比,本发明的多肽链携带一个或多个特定突变。突变可能影响链的寡聚化和/或特异性结合剂的识别。
例如,本发明的多肽可以携带突变,如在EP3248015 A1中描述,其阻止S100A8和S100A9的异二聚体之间的寡聚化。这些多肽可以有利地用作校准物或标准品,因为它们提供了不受外部条件影响的明确结构。例如,在存在钙离子和/或存在于测定缓冲液或生物样品中的其他物质的情况下,它们不会异四聚化。
本发明还涉及编码本发明的多肽的多核苷酸序列。在一个实施方案中,多核苷酸序列是在高度严格条件下与SEQ ID NO:5的多核苷酸序列杂交而成的。在另一个实施方案中,多核苷酸序列是在高度严格条件下与多核苷酸序列SEQ ID NO:6杂交的。SEQ ID NO:5编码SEQ ID NO:3的氨基酸序列,而SEQ ID NO:6编码SEQ ID NO:4的氨基酸序列。
本文使用的术语“杂交”或“使杂交”这里包括核酸分子链通过碱基配对与互补链连接的任何过程(J.Coombs(1994)Dictionary of Biotechnology,Stockton Press,NewYork)。杂交和杂交强度(即核酸分子之间的结合强度)受诸如核酸分子之间的互补程度、所涉及条件的严格性、形成的杂交体的Tm和核酸分子内的G:C比率影响。
在本文中,术语“Tm”用指“熔化温度”。熔化温度是双链核酸分子群半解离成单链时的温度。用于计算核酸分子Tm的方程式是本领域公知的。如标准参考文献所示,当核酸分子在1M NaCl的水溶液中时,Tm值的简单估值可以通过以下等式计算:Tm=81.5+0.41(%G+C)(参见,Anderson和Young,在核酸杂交中的定量筛选杂交(Quantitative FilterHybridization,in Nucleic Acid Hybridization)(1985))。其他参考资料包括更复杂的计算,在计算Tm时考虑了结构和序列特征。严格条件是本领域技术人员已知的,并且可以在分子生物学现代方法(Current Protocols in Molecular Biology),John Wiley&Sons,N.Y.(1989),6.3.1~6.3.6中找到。
特别地,术语“严格条件”是指100个或更多的连续核苷酸,150个或更多的连续核苷酸,200个连续核苷酸,250个或更多的连续核苷酸是一个片段或与互补核酸分子(DNA、DNA、RNA、ssDNA或ssRNA)在7%十二烷基硫酸钠(SDS)、0.5M NaPO4、1mM EDTA中在50℃的条件下进行杂交,并在50℃或65℃时,在2×SSC、0.1%SDS中洗涤,最好为65℃,带有特定的核酸分子(DNA;RNA,ssDNA或ss RNA)。优选地,杂交条件等同于50℃时,在7%十二烷基硫酸钠(SDS)、0.5M NaPO4、1mM EDTA中杂交,在50℃或65℃时,在1×SSC、0.1%SDS中洗涤,优选为65℃,更优选的杂交条件等同于50℃时,在7%十二烷基硫酸钠(SDS)、0.5M NaPO4、1mMEDTA中杂交,在50℃或65°时,在0.1×SSC、0.1%SDS中洗涤,优选65℃。优选地,互补核苷酸与一个片段或整个核酸杂交。或者,优选的杂交条件包括在65℃,1×SSC中或在42℃,1×SSC中与50%甲酰胺中杂交,然后65℃,在0.3×SSC中洗涤或50℃,在4×SSC中或40℃,在6×SSC与50%甲酰胺杂交,然后50℃,在2×SSC中洗涤。进一步优选的杂交条件是0.1%SDS、0.1×SSD和65℃。
本发明的多核苷酸序列可以另外携带启动子、核酸酶识别位点、标记基因、增强子和其他用于转化和表达多核苷酸序列的遗传元素。
本发明的多肽可以形成寡聚体。因此,在一个实施方案中,本发明涉及包含两种或更多种本发明中多肽的多肽寡聚体。在一个优选的实施方案中,多肽寡聚体正好包含两个本发明中的多肽。这种多肽寡聚体对应于包含两个S100A8和两个S100A9单体的内源性钙卫蛋白异四聚体。在一个特别优选的实施方案中,多肽寡聚体在二价金属离子,优选Ca2+的存在下形成。
本发明的多肽或其寡聚体可用于动物的免疫。“免疫”在本文中是指将物质施用于受试者或动物,目的是在受试者或动物中诱导产生免疫反应,免疫反应可能涉及抗体的产生。
动物可以是可用于临床研究或工业应用的任何动物,例如小鼠、大鼠、兔子、山羊、狗、母鸡、鲨鱼、骆驼、猪或猴子。当应用本发明中的多肽对动物进行免疫时,可以以本领域已知的任何方式配制多肽,例如通过添加其他免疫原性化合物。
在免疫过程中,本发明中的多肽可以替代内源性钙卫蛋白以获得针对钙卫蛋白的抗体。
本发明的多肽或其寡聚体还可用作表位,用于体外选择或用于识别S100A8单体、S100A9单体、S100A8/S100A9二聚体和/或其寡聚体,特别是内源性钙卫蛋白的结合试剂的亲和纯化。由于本发明的多肽可以容易地大量获得,因此在体外选择和纯化方法中使用它们允许以可重复的方式经济有效地选择和纯化合适的结合试剂。
在另一个实施方案中,本发明的多肽可以用作药物。在一个实施方案中,药物可用于治疗或预防需要调节免疫反应或减少/抑制炎症过程的疾病。
在另一个优选的实施方案中,本发明的多肽可以用作校准参考物质。校准参考物质在本文中被理解为指一种物质,在用于任何类型的分析测定的可追溯性和/或可比性中用作(国际公认的)主要参考物质(最高级别),或在建立新的分析方法时用作标准。因此,校准参考物质可用于测试新型结合剂或测定系统的灵敏度和特异性。同样,校准参考物质可用于校准分析测定。因此,本发明的多肽可用于建立新测定法,特别是在免疫测定法中,以及用于测试结合剂和校准测定方法。在这些实施方案中,本发明的多肽可以代替标准异二聚体或异四聚体钙卫蛋白作为校准参考物质,因为本发明的多肽显示出天然钙卫蛋白的所有基本特征,例如表位呈递和结合特性,但其比钙卫蛋白更容易获得以用作校准参考物质。
本发明还涉及在分析测定中使用本发明的多肽测量样品中S100A8、S100A9、S100A8/A9或其寡聚体,特别是钙卫蛋白的方法,包括以下步骤:
a)使用分析测定法测量不同数量的所述的多肽;
b)使用步骤a)中获得的分析结果建立校准曲线;
c)测量样品;
d)将样品的分析结果与步骤b)的校准曲线进行比较;
e)量化样品中S100A8、S100A9、S100A8/A9或其低聚物的浓度。
本领域技术人员知道如何校准分析测定。简而言之,本发明中不同数量的多肽使用分析测定法测量以获得分析结果,然后用于建立校准曲线。换言之,本发明的多肽或其寡聚体可被用作分析测定的校准参考物质。由于它们的单分散性和同质性,使用本发明的多肽的校准是快速且高度可靠的。
在本发明的方法中,使用相同的分析测试来测量样品,并将这些测量的结果与校准曲线进行比较。通过比较,可以定量测定样品中的S100A8、S100A9、S100A8/A9或其低聚物的浓度。
根据本发明的多肽或其寡聚体还可以用于诊断受试者的急性或慢性炎性疾病的方法,该方法包括:
a)提供受试者的生物样本;
b)通过使用本发明的多肽寡聚体作为校准参考物质来量化步骤a)的生物样本中S100A8、S100A9、S100A8/A9或其寡聚体,特别是钙卫蛋白的量;
c)将步骤b)中确定的S100A8、S100A9、S100A8/A9或其寡聚体的量与来自已知患有急性或慢性炎症性疾病的受试者的参考数据进行比较。
本领域技术人员知道各种类型的急性或慢性炎症性疾病,例如过敏、哮喘、自身免疫性疾病、乳糜泻、肾小球肾炎、肝炎、炎症性肠病、溃疡性结肠炎、克罗恩病、灌注前损伤、移植排斥、感染性结肠炎、坏死性小肠结肠炎、(肠)囊性纤维化、类风湿性关节炎、幼年特发性关节炎、强直性脊椎炎、关节肿胀、牛皮癣、银屑病关节炎、白塞病、牙龈炎、扁桃体炎、阑尾炎、狼疮、不明原因的发热、败血症、心脏病、心肌梗塞、多发性硬化症和癌症如结肠直肠癌。在一个优选的实施方案中,炎性疾病选自炎性肠病,特别是溃疡性结肠炎或克罗恩病,和炎性关节炎,特别是类风湿性关节炎、幼年特发性关节炎或强直性脊柱炎。
在本发明的一方面,与参考数据相比,S100A8、S100A9、S100A8/A9或其寡聚体,特别是钙卫蛋白的量显著增加,表明受试者患有急性或慢性炎性疾病。
使用本发明的多肽或其寡聚物,可以容易且可靠地诊断炎性疾病并监测它们的治疗。
在本发明的方法中,样品可以是本领域中使用的任何生物样品,特别是血液、血清、血浆、滑液、唾液、尿液、眼泪、汗液、龈沟液、粪便、胃肠道灌洗液、支气管灌洗液,细胞培养上清或组织提取剂。在一个优选的实施方案中,样品是粪便样品或血液样品。
分析测定可以是本领域已知的任何分析测定,例如免疫测定、生化测定、生物物理测定或物理测定。在一个优选的实施方案中,所述的分析测定为免疫测定,该测定是基于需要通过高亲和力结合试剂识别分析物的方法,例如酶联免疫吸收测定(ELISA)、侧向流动免疫测定(LFIA)或粒子增强免疫比浊测定(PETIA)。
在本发明的方法中,样品的分析结果可以基于光学读数、吸收、UV/VIS光谱、光散射、比浊法、光散射、反射光、荧光、发光、化学发光、表面等离子共振、安培法、磁力测量法、伏安法、电位法、电导法、库仑法、极谱法、重量法或悬臂梁。在一个优选的实施方案中,分析结果基于通过吸收、UV/VIS光谱、光散射、比浊法、反射法、荧光、发光或化学发光测量的免疫测定法。在最优选的实施方案中,分析结果基于通过吸收光谱、光散射或化学发光测量的免疫测定。
本发明还涉及一种试剂盒,包括:
a)本发明的多肽和/或其寡聚体;
b)测试容器;
c)缓冲溶液;
d)第一结合试剂,优选固定在固体支持物上,特异于S100A8、S100A9、S100A8/A9或其寡聚物,特别是钙卫蛋白;
在一个实施方案中,第一结合试剂用物质标记或与允许定量测定所述多肽和S100A8、S100A9、S100A8/A9或其寡聚体的材料结合。
测试容器用于在其中进行分析测定。例如,测试容器可以是测试盒、膜、管、滴定板或容器。
本领域技术人员知道如何选择合适的缓冲溶液。例如,缓冲溶液可以是磷酸盐、马来酸盐、氯乙酸盐、甲酸盐、苯甲酸盐、吡啶、哌嗪、丙酸盐、3-N-吗啉代丙磺酸(MOPS)、1,3-双三羟甲基)甲基氨基丙烷(Bis-TRIS)、三(羟甲基)氨基甲烷(TRIS),三(羟甲基)氨基甲烷-马来酸(TRIS-马来酸盐),2-(三-(羟甲基)甲基氨基)乙磺酸(TES),1,4-哌嗪双乙磺酸(PIPES),4-吗啉代乙磺酸(MES),N-2-羟乙基哌嗪-N'-2-乙磺酸(HEPES),N,N-双(2-羟乙基)-2-氨基乙磺酸(BES),N-(2-乙酰氨基)亚氨基二乙酸(ADA)、N-(2-乙酰氨基)-2-氨基乙磺酸(ACES)和本领域技术人员已知的其它缓冲溶液。该缓冲溶剂还可以包括盐、抗菌剂、洗涤剂、螯合剂、离液剂、和/或者防沫剂。
根据本发明的试剂盒还包含第一粘合剂和任选的第二粘合剂,其对S100A8、S100A9、S100A8/S100A9或其寡聚物具有特异性。在一个优选的实施方案中,第一/第二粘合剂对包含S100A8和S100A9的异四聚体,即钙卫蛋白具有特异性。在另一个优选的实施方案中,第一/第二粘合剂对包含S100A8和S100A9的异二聚体具有特异性。然而,本发明还设想第一/第二粘合剂对S100A8或S100A9单体具有特异性。
由于本发明的多肽的序列,对S100A8、S100A9、S100A8/S100A9或其寡聚物具有特异性的粘合剂也识别根据发明的多肽。
在一个优选的实施方案中,第一粘合剂固定在固体支持物上。这允许该试剂盒用于ELISA、LFIA或PETIA。
根据本发明,第一/第二粘合试剂可以用物质标记或与允许定量测定所述多肽和S100A8、S100A9、S100A8/A9或其寡聚体的材料结合。本领域技术人员知道哪些物质或标记可用于分析物的定量测定。例如,粘合剂可以用乳胶、荧光、(顺)磁性、有色纤维素、铁、金或二氧化硅(纳米)颗粒标记;荧光团或荧光染料;量子点;酶如辣根过氧化物酶(HRP)、碱性磷酸盐(AP)葡萄糖氧化酶、葡萄糖-6-磷酸脱氢酶、苹果酸脱氢酶、NADH脱氢酶、乙酰胆碱酯酶;化学发光反应物,例如萤光素酶或鲁米诺及其衍生物;荧光蛋白如绿色荧光蛋白(GFP)、红色荧光蛋白或黄色荧光蛋白;和放射性同位素。
任选地,该试剂盒可以包括纯化生物样品的装置。
因此,根据本发明的试剂盒允许定量测定样品中的钙卫蛋白。因为本发明的多肽可以特异性地形成异二聚体或异四聚体,它们在试剂盒中的使用避免了假阳性并因此能够对样品中的钙卫蛋白进行可靠的定量。
实施例
实施例1:本发明多肽的制备。
将SEQ ID NO:5克隆到pET21载体(Novagen)中用于IPTG诱导型T7转录。用相应的质粒转化表达菌株大肠杆菌BL21。根据标准方案通过IPTG诱导蛋白质表达以获得具有SEQ-ID NO:3的可溶性多肽。更具体地,各表达菌株在具有100μg/ml羧苄青霉素的LB培养基中,在37℃、200rpm条件下生长至OD600=0.5。随后,将细胞培养物冷却至18℃并在OD600=0.8时加入0.2mM IPTG开始诱导蛋白表达。在7,000xg下离心收获大肠杆菌表达菌株并在缓冲液A(20mM HEPES pH 7.5、100mM NaCl、2.5%甘油、1mM DTT)中洗涤。表达过程在18℃,200rpm下进行14~16小时。
在裂解缓冲液(缓冲液A+20mM咪唑)中重新悬浮诱导的表达菌株,随后通过超声处理(Branson Sonifier)破坏细胞,然后以20,000xg离心将可溶性蛋白质从细胞碎片中分离出来。将上清液应用于NiNTA树脂,纯化的融合蛋白用咪唑梯度洗脱。使用Q Sepharose柱,用NaCl梯度将融合蛋白与核酸污染物分离。最终纯化步骤使用S200尺寸排阻柱(图1)并最终在缓冲液A中产生纯的和单分散的融合蛋白(图2,泳道3)。
实施例2:融合多肽蛋白水解切割成其单体。
由于在这个版本中融合蛋白(SEQ ID NO:3)中3C蛋白酶(PreScissionTM)识别序列连接His-Tag与S100A9以及S100A9与S100A8,单一融合多肽可被蛋白水解切割成三个多肽,其对应于游离His-Tag、S100A8和S100A9以及来自3C识别序列的相应残留氨基酸(图2,泳道4)。
实施例3:在Ca2+离子存在下重组融合多肽的二聚化。
重组融合多肽与内源性钙卫蛋白具有共同特征,内源性钙卫蛋白是S100A8和S100A9的异二聚体。含有2mM CaCl2的缓冲液A中的尺寸排阻色谱法使融合蛋白的洗脱体积发生显着变化,这相当于两个S100A8和S100A9异二聚体的钙依赖性异四聚体(图3)。重组多肽单体(相当于S100A8/S100A9二聚体)在大约62ml时洗脱,而重组多肽二聚体(相当于S100A8/A9四聚体)在大约58ml时洗脱得更快。
实施例4:本发明的重组融合多肽的免疫原性特性。
所描述的融合蛋白(SEQ ID NO:3)显示出与内源性钙卫蛋白相似的免疫原性特性。单克隆抗体27E10仅结合S100A8和S100A9的二聚体,但不结合单个单体(Hessian PA,Fisher L.MRP-8(S100A8)和MRP-14(S100A9)的异二聚体复合物。抗体识别、表位定义和对结构的影响(2001).Eur.J.Biochem.268:353~363)。单克隆抗体27E10固定在Biacore芯片传感器上并识别融合蛋白(SEQ ID NO:3),其通过使用表面等离子共振与融合蛋白(SEQ IDNO:3)相互作用时的浓度依赖性反应单位表示(图4)。
实施例5:基于本发明的重组融合多肽的免疫测定。
在CaCl2存在下用不同浓度的融合蛋白(SEQ ID NO:3)进行单克隆捕获抗体的ELISA。HRP偶联检测抗体用于诱导基于TMB(3,3',5,5-四甲基联苯胺)的颜色变化。添加酸性终止溶液后,测量450nm处的OD并显示出与先前通过A280吸收测量评估的纯化融合蛋白(SEQ-ID NO:3)的加标浓度相比呈线性相关性(图5)。类似地,用多克隆抗体进行的免疫比浊测试也与不同浓度的融合蛋白(SEQ ID NO:3)相关(图6),这证实了融合蛋白(SEQ IDNO:3)表现出与内源性钙卫蛋白基本相同的特征(S100A8/S100A9),因此可以与从人类内源性来源如粪便样本或血液来源的粒细胞中纯化的S100A8/S100A9互换使用。
实施例6:由本发明的内源性钙卫蛋白或重组融合多肽组成的校准物的互换性。
使用本发明的重组融合多肽(SEQ ID NO:3)作为校准物对23个人的血清样品进行比浊测定,并与使用纯化的内源性钙卫蛋白(S100A8/A9异四聚体)作为校准物的相同免疫测定获得的结果进行比较。将结果相互绘制并显示出完美的相关性(斜率=1.009;R2=0.999),与使用的校准材料无关(图7)。
实施例7:本发明的融合多肽对多克隆和单克隆抗体显示出相同的免疫学特性。
重组融合多肽的四种不同稀释液以及23份血清人样品用实施例5中描述的使用单克隆抗体的ELISA法与实施例5中描述的使用多克隆抗体的比浊免疫测定法进行分析。对于该实验,两种测定均使用内源性钙卫蛋白作为校准材料。两个结果集相互关联并表示为Passing-Bablok图(图8)。对于本发明的融合多肽(SEQ ID NO:3)和人血清样品,在多克隆抗体与单克隆抗体产生的结果之间几乎没有观察到定量差异。
实施例8:通过引入点突变来防止本发明的融合多肽(SEQ-ID NO:3)的二聚或寡聚。
定向诱变用于在融合蛋白(SEQ ID NO:3)的链S100A9(参见EP3248015A1)中引入突变E78A。在内源性钙卫蛋白中,这种突变消除了异二聚化(Leukert N et al.Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biologicalactivity(2006).J.Mol.Biol.359:961~972)。类似地,将这种突变引入融合蛋白(SEQ-IDNO:3)可防止CaCl2依赖性二聚化。如实施例1和3(图9)中所述,在含有2mM CaCl2的缓冲液A中进行突变和非突变融合蛋白(SEQ ID NO:3)的S200尺寸排阻色谱。
序列表
<110> 伯尔曼实验室股份有限公司(Buehlmann Laboratories AG)
<120> 重组钙卫蛋白
<130> 38366-BHL-P-WO
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<211> 93
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<220>
<223> 人类S100A8/Mrp8蛋白序列
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Ala Ala His Lys Lys Ser His Glu Glu Ser His Lys Glu
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<210> 2
<211> 114
<212> PRT
<213> 智人(Homo sapiens)
<220>
<223> 人类S100A9/Mrp14蛋白序列
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Met Thr Cys Lys Met Ser Gln Leu Glu Arg Asn Ile Glu Thr Ile Ile
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Thr Pro
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Met His His His His His His Leu Glu Val Leu Phe Gln Gly Pro Met
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Gln Gly Glu Phe Lys Glu Leu Val Arg Lys Asp Leu Gln Asn Phe Leu
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Lys Lys Glu Asn Lys Asn Glu Lys Val Ile Glu His Ile Met Glu Asp
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Asp Glu Gly Pro Gly His His His Lys Pro Gly Leu Gly Glu Gly Thr
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Pro Leu Glu Val Leu Phe Gln Gly Pro Met Leu Thr Glu Leu Glu Lys
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Ala Leu Asn Ser Ile Ile Asp Val Tyr His Lys Tyr Ser Leu Ile Lys
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Gly Asn Phe His Ala Val Tyr Arg Asp Asp Leu Lys Lys Leu Leu Glu
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Lys Glu Leu Asp Ile Asn Thr Asp Gly Ala Val Asn Phe Gln Glu Phe
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Leu Ile Leu Val Ile Lys Met Gly Val Ala Ala His Lys Lys Ser His
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Glu Glu Ser His Lys Glu
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<210> 4
<211> 229
<212> PRT
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<223> MRP8-3C蛋白酶 Seq.[接头]-MRP14-3C蛋白酶 Seq.-HisTag
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His Lys Tyr Ser Leu Ile Lys Gly Asn Phe His Ala Val Tyr Arg Asp
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Asp Leu Lys Lys Leu Leu Glu Thr Glu Cys Pro Gln Tyr Ile Arg Lys
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Ala Val Asn Phe Gln Glu Phe Leu Ile Leu Val Ile Lys Met Gly Val
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<400> 5
atgcaccatc atcaccatca cttggaagtt ctgtttcaag gcccaatgac gtgcaaaatg 60
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atgctgacgg aactggagaa agcgcttaac agcattattg acgtttacca caaatacagt 60
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gaatgtcccc agtatatccg caagaaaggt gccgatgtgt ggttcaaaga actggacatt 180
aacactgacg gtgcagtgaa tttccaggaa tttctcattc tggtgatcaa aatgggcgtt 240
gctgctcaca agaaatcgca tgaagagtct cacaaagaat tggaagttct gtttcaaggc 300
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attatgctga tggcacgtct gacatgggcc tcacatgaga aaatgcatga aggcgatgag 600
ggtcctggac atcatcacaa accggggtta ggcgaaggaa ctccgttgga agttctgttt 660
caaggcccac accatcatca ccatcactaa 690
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<212> PRT
<213> 人工序列(Artificial)
<220>
<223> HisTag-3C蛋白酶 Seq.-MRP14-TEV蛋白酶 seq.[接头]-MRP8
<400> 7
Met His His His His His His Leu Glu Val Leu Phe Gln Gly Pro Met
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Thr Cys Lys Met Ser Gln Leu Glu Arg Asn Ile Glu Thr Ile Ile Asn
20 25 30
Thr Phe His Gln Tyr Ser Val Lys Leu Gly His Pro Asp Thr Leu Asn
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Gln Gly Glu Phe Lys Glu Leu Val Arg Lys Asp Leu Gln Asn Phe Leu
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Lys Lys Glu Asn Lys Asn Glu Lys Val Ile Glu His Ile Met Glu Asp
65 70 75 80
Leu Asp Thr Asn Ala Asp Lys Gln Leu Ser Phe Glu Glu Phe Ile Met
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Leu Met Ala Arg Leu Thr Trp Ala Ser His Glu Lys Met His Glu Gly
100 105 110
Asp Glu Gly Pro Gly His His His Lys Pro Gly Leu Gly Glu Gly Thr
115 120 125
Pro Glu Asn Leu Tyr Phe Gln Ser Met Leu Thr Glu Leu Glu Lys Ala
130 135 140
Leu Asn Ser Ile Ile Asp Val Tyr His Lys Tyr Ser Leu Ile Lys Gly
145 150 155 160
Asn Phe His Ala Val Tyr Arg Asp Asp Leu Lys Lys Leu Leu Glu Thr
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Glu Cys Pro Gln Tyr Ile Arg Lys Lys Gly Ala Asp Val Trp Phe Lys
180 185 190
Glu Leu Asp Ile Asn Thr Asp Gly Ala Val Asn Phe Gln Glu Phe Leu
195 200 205
Ile Leu Val Ile Lys Met Gly Val Ala Ala His Lys Lys Ser His Glu
210 215 220
Glu Ser His Lys Glu
225
<210> 8
<211> 229
<212> PRT
<213> 人工序列(Artificial)
<220>
<223> HisTag-3C蛋白酶 Seq.-MRP8-TEV蛋白酶 seq.[接头]-MRP14
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<210> 10
<211> 229
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<220>
<223> HisTag-TEV蛋白酶 seq.-MRP8-3C蛋白酶 Seq.[接头]-MRP14
<400> 10
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100 105 110
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115 120 125
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<210> 11
<211> 282
<212> DNA
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<220>
<221> misc_feature
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<400> 11
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gagtgtcctc agtatatcag gaaaaagggt gcagacgtct ggttcaaaga gttggatatc 180
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gcagcccaca aaaaaagcca tgaagaaagc cacaaagagt ag 282
<210> 12
<211> 282
<212> DNA
<213> 智人(Homo sapiens)
<220>
<221> misc_feature
<223> 人类S100A9/Mrp14 DNA序列
<400> 12
atgttgaccg agctggagaa agccttgaac tctatcatcg acgtctacca caagtactcc 60
ctgataaagg ggaatttcca tgccgtctac agggatgacc tgaagaaatt gctagagacc 120
gagtgtcctc agtatatcag gaaaaagggt gcagacgtct ggttcaaaga gttggatatc 180
aacactgatg gtgcagttaa cttccaggag ttcctcattc tggtgataaa gatgggcgtg 240
gcagcccaca aaaaaagcca tgaagaaagc cacaaagagt ag 282
<210> 13
<211> 8
<212> PRT
<213> 人工序列(Artificial)
<220>
<223> 鼻病毒3C蛋白酶的识别序列(Prescission蛋白酶)
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Leu Glu Val Leu Phe Gln Gly Pro
1 5
<210> 14
<211> 7
<212> PRT
<213> 人工序列(Artificial)
<220>
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<400> 14
Glu Asn Leu Tyr Phe Gln Gly
1 5
<210> 15
<211> 6
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<213> 人工序列(Artificial)
<220>
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<400> 15
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1 5
<210> 16
<211> 4
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<220>
<223> 因子Xa的识别序列;X为E或者D
<220>
<222> (2)..(2)
<223> D或者E
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 16
Ile Xaa Gly Arg
1
<210> 17
<211> 5
<212> PRT
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<400> 17
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<210> 18
<211> 254
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<220>
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<210> 19
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Claims (26)
1.一种获得可溶性钙卫蛋白的方法,其特征在于,所述的方法包括从载体表达钙卫蛋白,所述的载体包含第一链,所述的第一链包含与SEQ ID NO:11具有至少80%序列同源性的核苷酸序列,所述的第二链包含与SEQ ID NO:12具有至少80%序列同源性的核苷酸序列以及连接第一链和第二链的接头。
2.根据权利要求1所述的方法,其特征在于,所述的可溶性钙卫蛋白能够结合单克隆抗体mAb27E10。
3.根据权利要求1或2所述的方法,其特征在于,所述的方法不包括所述的钙卫蛋白的细胞外重折叠步骤。
4.根据权利要求1至3中任一项所述的方法,其特征在于,所述的接头的长度为18至180个核苷酸,优选为21至30个核苷酸。
5.根据权利要求1至4中任一项所述的方法,其特征在于,所述的接头包含编码蛋白酶识别序列,特别是鼻病毒3C蛋白酶识别序列、TEV蛋白酶识别序列、凝血酶识别序列、因子Xa识别序列或肠肽酶识别序列。
6.一种多肽,其包含:
a)包含与SEQ ID NO:1具有至少80%序列同一性的氨基酸序列的第一链;
b)包含与SEQ ID NO:2具有至少80%序列同一性的氨基酸序列的第二链;和
c)连接第一链和第二链的接头,
其特征在于,所述的接头是包含蛋白酶识别序列的肽接头。
7.根据权利要求4所述的多肽,其特征在于,所述的蛋白酶识别序列是鼻病毒3C蛋白酶识别序列、TEV蛋白酶识别序列、凝血酶识别序列、因子Xa识别序列或肠肽酶识别序列。
8.根据权利要求4或5所述的多肽,其特征在于,所述的多肽能够在金属离子存在下寡聚化。
9.根据权利要求6所述的多肽,其特征在于,所述的多肽能够在二价金属离子,优选Ca2+离子的存在下形成二聚体。
10.根据权利要求4至7中任一项所述的多肽,其特征在于,所述的多肽能够形成一种结构,所述的结构能够被识别S100A8单体、S100A9单体、S100A8/S100A9二聚体和/或它们的寡聚体的结合试剂识别。
11.根据权利要求4所述的多肽,其特征在于,所述的多肽包含与SEQ ID NO:3的多肽具有至少80%序列同一性的氨基酸序列。
12.根据权利要求4所述的多肽,其特征在于,所述的多肽包含与SEQ ID NO:4、7至10、18或19的多肽具有至少80%序列同一性的氨基酸序列。
13.一种多肽寡聚体,其特征在于,包含两种或更多种根据权利要求6至12中任一项所述的多肽。
14.根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体,其特征在于,用于动物免疫的用途。
15.根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体,其特征在于,作为用于体外选择识别S100A8单体、S100A9单体、S100A8/S100A9二聚体和/或其低聚物的结合试剂的表位的用途。
16.根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体,其特征在于,用于在亲和纯化识别S100A8单体、S100A9单体S100A8/S100A9二聚体和/或其寡聚体的结合试剂中的用途。
17.根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体,其特征在于,用于作为药物。
18.根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体,其特征在于,作为校准参考物质的用途。
19.使用根据前述权利要求中任一项所述的多肽在分析测定测量样品中的S100A8、S100A9、S100A8/A9二聚体或寡聚体的方法,其特征在于,所述的方法包括以下步骤:
a)使用分析测定法测量不同含量的所述的多肽;
b)使用步骤a)中获得的分析结果建立校准曲线;
c)使用分析测定法测量样品;
d)将样品的分析结果与步骤b)和的校准曲线进行比较;
e)量化样品中S100A8、S100A9、S100A8/A9二聚体或寡聚体的浓度。
20.根据权利要求19所述的方法,其特征在于,所述的样品是血液、血清、血浆、滑液、唾液、尿液、泪液、汗液、龈沟液、粪便、胃肠灌洗液、支气管灌洗液、细胞培养上清液或组织提取物。
21.根据权利要求19或20所述的方法,其特征在于,所述的分析测定法是免疫测定、生化测定、生物物理测定或物理测定。
22.根据权利要求19至21中任一项所述的方法,其特征在于,所述的样品的分析结果基于光学读数、吸收、UV/VIS光谱、比浊法、浊度测定法、光散射、反射法、荧光、发光、化学发光、表面等离子体共振、电流测定法、磁力测定法、伏安法、电位测定法、电导法、库仑法、极谱法、重量法或悬臂梁。
23.根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体,用于诊断受试者的急性或慢性炎性疾病的方法,其特征在于,包括:
a)提供受试者的生物样本;
b)通过使用根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体作为校准参考物质来量化步骤a)的生物样本中S100A8、S100A9、S100A8/A9或其寡聚体;
c)将步骤b)中确定的S100A8、S100A9、S100A8/A9或其寡聚体的量与来自已知患有急性或慢性炎症性疾病的受试者的参考数据进行比较。
24.一种试剂盒,其特征在于,包括:
a)根据权利要求6至12中任一项所述的多肽或通过根据权利要求1至5中任一项所述的方法获得的多肽或根据权利要求13所述的多肽寡聚体;
b)测试容器;
c)缓冲溶液;和
d)第一结合试剂,其特异性针对S100A8、S100A9、S100A8/A9二聚体或寡聚体。
25.根据权利要求24所述的试剂盒,其特征在于,所述的第一结合试剂用物质标记或结合到允许定量测定所述的多肽及S100A8、S100A9、S100A8/A9二聚体或寡聚体的材料上。
26.根据权利要求25所述的试剂盒,还包含对S100A8、S100A9、S100A8/A9二聚体或寡聚体具有特异性的第二结合试剂,其特征在于,所述的第一结合试剂固定在固体支持物上,所述的第二结合试剂用物质标记或结合到允许定量测定所述的多肽及S100A8、S100A9、S100A8/A9二聚体或寡聚体的材料上。
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CN117607461A (zh) * | 2023-12-06 | 2024-02-27 | 陕西省动物研究所 | 一种s100a8蛋白的检测方法及其应用 |
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US20230074277A1 (en) | 2023-03-09 |
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