CN103974978A - 用于检测snap/clip标记物的单克隆抗体 - Google Patents

用于检测snap/clip标记物的单克隆抗体 Download PDF

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CN103974978A
CN103974978A CN201280057908.5A CN201280057908A CN103974978A CN 103974978 A CN103974978 A CN 103974978A CN 201280057908 A CN201280057908 A CN 201280057908A CN 103974978 A CN103974978 A CN 103974978A
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S·巴思
K·科尔伯格
C·帕特曼
S·施密斯
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Abstract

一种与SNAP基序和CLIP标记物特异性结合的单克隆抗体，包含具有氨基酸序列SEQ ID No.3,4,5和8,9,10的CDR。

Description

用于检测SNAP/CLIP标记物的单克隆抗体
本发明涉及用于检测SNAP/CLIP标记物的单克隆抗体,编码该抗体的核酸和该抗体用于检测含有SNAP/CLIP标记物的蛋白的用途。
SNAP和CLIP标记技术是一种相对年轻的技术。它是给靶蛋白，特别是融合蛋白提供所需配体的精细方法。
WO20009/114748A1披露了SNAP-25组合物，制造α-SNAP-25抗体的方法，α-SNAP-25抗体，检测BoNT/A活性的方法和检测中和α-BoNT/A抗体的方法，所述α-SNAP-25抗体与某种表位结合，而所述表位包含SNAP-25切割产物的BoNT/A切割位点易切断键的P1残基处的羧基末端。
M.Yamamoto,L.Hassinger,J.E.Crandall在Journal of Neurocytology19,619-627(1990)报道了阶段特异性神经突相关蛋白在发育中大鼠大脑和小脑皮质中的超微结构定位。SNAP/TAG-1是135kDa的糖蛋白，在发育的啮齿动物CNS中生长轴突子集的表面表达。采用免疫电子显微术和识别SNAP/TAG-1(4D7)的单克隆抗体以及过氧化物酶偶联的二抗，分析了大鼠的胚胎期第17天大脑皮层和出生后第4天和第8天小脑皮层中这种抗原的超微结构定位。在胚胎皮层，免疫反应性与位于中间区(intermediate zone)，基底板(subplate)和皮质板(cortical plate)的有限组的神经轴突的质膜，神经元胞体(neuronal somata)以及它们的主导过程相关。免疫反应性轴突结合在一起成为10-20个的一组，并与非免疫反应性轴突隔开。有些生长锥呈免疫反应性，然而不是所有4D7-免疫反应性轴突的生长锥显示染色。在出生后的小脑中，免疫反应活性与位于外部颗粒细胞层(external granule cell layer)的最内部分的颗粒细胞的胞体和轴突相关。在大脑和小脑皮层，免疫反应活性以间插方式出现在相邻细胞膜的对应点，规律性的周期为100-200nm。该文献讨论了SNAP/TAG-1用作发育CNS中特定亚组轴突的粘附分子的可能性。
Richard J Ward,John D Pediani,和Graeme Milligan在British JournalPharmacology(2011),162,1439-1452中报道了通过N-端SNAP和CLIP-标记技术评估配体诱导的食欲肽OX1和大麻素CB1受体的内在化。通过抗体识别表位标签和荧光团与O6-烷基鸟嘌呤-DNA烷基转移酶变体的共价结合检测每一种受体结构的细胞表面形态。可采用各方法监测对激动剂而非拮抗剂作起反应的受体内在化，但当使用SNAP标记物的新型、时间分辨荧光探针时，灵敏度最高是其它方法的6-10倍以上。然而，就CLIP标记物而言，灵敏度没有提高，可能由于非特异性结合水平较高。
SNAP标记物基于人DNA修复酶O(6)-烷基鸟嘌呤-DNA烷基转移酶。后者已通过引入突变而得到改变，其改变程度使得可以选出具有更小分子体积并对苄基鸟嘌呤具有极高亲和力的蛋白变体。SNAP标记物与苄基鸟嘌呤衍生物高度特异性反应，使苄基与和自身共价偶联的底物结合并切除鸟嘌呤。作为一种重组蛋白标记物，它能共价和化学计量地限定各种苄基鸟嘌呤修饰底物与融合蛋白的偶联。CLIP标记物是通过诱变从SNAP形成，其与苄基胞嘧啶衍生物而非苄基鸟嘌呤发生高度特异性反应。因此，可能在一个实验方法中同时差别性标记SNAP和CLIP标记物。SNAP技术(SNAP/CLIP质粒和底物)由新英格兰生物试验室公司(NEB)分销。
在"Journal of Biomedicine and Biotechnology",Vol.2010的ID658954,doi:10.1155/2010/658954的文章中Aliprandi等人披露了一种以VHH形式的重组抗SNAP的抗体。
有一种能同时检测CLIP和SNAP标记物的分析工具是最好了。
本发明的目的通过权利要求1所述的抗体得以实现。本发明的单克隆抗体特异性地结合SNAP标记物基序和CLIP标记物并包含具有氨基酸序列SEQ IDNo.3,4,5和8,9,10的CDR。特别是，本发明的抗体是鼠科抗体。
本发明的主题也是编码本发明抗体的核酸，特别是具有SEQ ID No.1或6所示核酸序列的核酸。
本发明抗体可通过本发明方法获得，其中借助SNAP标记物蛋白在非人类哺乳动物，特别是小鼠中实施免疫，以及从中获得杂交瘤细胞，通过结合试验鉴定同时识别SNAP和CLIP标记物的抗体细胞系。
本发明抗体同时检测SNAP和CLIP标记物的用途。Aliprandi等描述了能识别SNAP标记物的重组抗体。本发明的抗体可以用于染色组织切片。本发明的鼠科抗-SNAP抗体可以用于，特别是染色冷冻切片和石蜡切片。与Aliprandi等人已经发表过的抗体相比，本发明抗体的优越性在于其更高的价位，重组蛋白只能识别一个表位，而本发明抗体能识别两个表位。
图1：免疫组织化学；用HAI(抗-EGFR)和M2D11染色从小鼠获得的A431肿瘤冷冻切片。
图2：流式细胞术；图片显示在流式细胞术中M2D11和两种不同的SNAP融合蛋白结合。
图3：Western blot分析：两个图片显示，一方面在变性凝胶中M2D11与SNAP和CLIP-EGF两种蛋白结合。
图4：Western blot分析；此图片显示检测溶液中M2D11和SNAP蛋白结合的天然聚丙烯酰胺凝胶印迹。
本发明的抗体能同时检测SNAP标记物和CLIP标记物。本发明的抗体的优势在于可以在流式细胞术中检测SNAP融合蛋白。该抗体在ELISA(酶联免疫吸附测定)和Western blot中的灵敏度与Aliprandi等人描述的抗体相似。
除了描述的方法，免疫组织化学试验中测验本发明抗体。它可以用于检测冷冻切片和石蜡切片中的SNAP融合蛋白。
在特定的实施例中，抗体是单克隆抗体。特别是，抗体可以是鼠源的。鼠科抗体有优越性的原因在于鼠科IgG抗体属于分子生物学研究中最常用的抗体形式。因此，很多实验室的技术人员熟悉使用这类抗体和检测鼠科IgG抗体。
本发明抗体的重链可变区示于SEQ ID No.2，SEQ ID No.1涉及编码该区的核酸。
本发明抗体的轻链可变区示于SEQ ID No.7，SEQ ID No.6涉及编码该区的核酸。
本发明抗体重链的CDR见氨基酸序列SEQ ID No.3-5。本发明抗体轻链的CDR见氨基酸序列SEQ ID No.8-10。
本发明也涉及编码所述蛋白的核酸，尤其是SEQ ID No.1和6。
本发明还涉及制备本发明抗体的方法，其中借助SNAP标记物蛋白在非人类哺乳动物，特别是小鼠中实施免疫。从中获得杂交瘤细胞系以及通过相应的结合试验鉴定能同时识别SNAP和CLIP标记物的抗体细胞系。
本发明的抗体可以分别用于检测SNAP和CLIP标记物，也可以是二者的组合。
实施例
聚丙烯酰胺凝胶电泳和Western blot
在Laemmli缓冲液中使待分析的样本变性(或在不含SDS的天然样本缓冲液中)并在12％(w/v)SDS的聚丙烯酰胺凝胶和聚丙烯酰胺凝胶(160V,60min)上进行电泳。蛋白用考马斯染色法显色或转移到硝酸纤维素膜(Whatman,Schleicher&Schuell,Dassel,德国)(350mA,70分钟)显现。转移后，膜在室温下用1％(w/v)BSA阻断1小时。在PBS-T中洗涤三次后，蛋白印迹与一抗温育(1小时)。在进一步三次洗涤步骤后，用酶偶联的二抗(1小时)和对应的底物(10分钟)检测特异性结合。在对SNAP和CLIP蛋白的分析中，样本在变性前和BG或BC底物温育。结果在图3中显示。
图3.Western blot分析。两张图片3A，3B和3C显示，一方面，M2D11与两种蛋白SNAP和CLIP EGF在变性凝胶中结合。抗体显示与其它His6-标记蛋白(GFP-Ki4)没有交叉反应。此外，图3B显示抗体不与SNAP底物竞争与SNAP的结合。用BG生物素生物素化SNAP蛋白后，用M2D11进一步检测蛋白。另外，图3C显示抗体与不同的SNAP-scFv融合物(H22-SNAP,SNAP-2715)和与CLIP-scFv-SNAP融合蛋白结合。
图4：Western blot分析。该图显示检测溶液中M2D11和SNAP蛋白结合的天然聚丙烯酰胺凝胶印迹。图A部分显示通过Myc标记物标记蛋白表明有SNAP蛋白存在。可以明显看出，在溶液中蛋白也与M2D11结合，而游离蛋白只有超过1:4的比例能被检测到。在图B部分，抗体被额外检测到以致可以显示两种蛋白的共定位(colocalization)。
免疫组织化学
从源自具有A-431肿瘤的BALB/c小鼠(DSMZ No.ACC91)的EGFR阳性皮下肿瘤制备组织切片。杀死动物后，把肿瘤包埋入“荣格组织冷冻介质”(Leica Microsystems,Nussloch,德国)并冷冻于液氮。用Leica3050s kryostat制备8um的冷冻切片并干燥过夜。切片用丙酮固定10分钟，干燥并用Immunopen(Sigma Aldrich)勾画出轮廓。肿瘤细胞用EGFR特异性scFv融合蛋白425scFv SNAP(0.034mg/ml)作为一抗染色。用PBST三次洗涤后，用不同浓度的过氧化物酶标记抗体M2D11(储备液:657ng/μl)检测SNAP融合蛋白。两种抗体孵化步骤在室温下进行45分钟，洗涤步骤在室温下进行5分钟并振荡。用PBST洗涤两次和用TBST洗涤一次后，组织切片在3-氨基-9-乙基咔唑(AEC)溶液中温育直至染色可见。随后用苏木精进行复染，然后将切片安置在甘油凝胶中。结果显示于图1。
图1:免疫组织化学。用425(scFv)-SNAP(抗-EGFR)和M2D11染色从小鼠获得的A431肿瘤冷冻切片。
流式细胞术
用流式细胞仪(Becton&Dickinson)和CellQuest软件，通过流式细胞术分析M2D11的功能。就像检测SNAP融合蛋白与细胞特异性结合一样检测与不同细胞系细胞表面的非特异性结合。大约4*105细胞先在100ul的PBS中与1-2μgSNAP/CLIP融合蛋白温育，然后在100ul的PBS中与3.3ng M2D11在冰上温育30分钟。为作检测，细胞与GaM-PE(1:100,Dianova,汉堡,德国)在冰上温育30分钟。细胞随后用流式细胞术分析。在所有步骤之间，用标准细胞洗涤离心机进行PBS洗涤步骤。将细胞重悬在300ul PBS中以便测试。结果在图2中显示。
图2：流式细胞术；图片显示在流式细胞术中M2D11与两种不同的SNAP融合蛋白结合。在两种细胞系上，没有检测到或只检测到非常少的抗体与细胞表面的交叉反应。本文显示的Mono Mac1和A431细胞是例子。利用PC-3、CHO-K1、Kasumi、Mcf-7、L3.6pl、L540和FG细胞系额外进行了分析。

Claims (6)

1.一种与SNAP标记物基序和CLIP标记物特异性结合的单克隆抗体，所述单克隆抗体包含具有氨基酸序列SEQ ID No.3,4,5和8,9,10的CDR。
2.如权利要求1所述的抗体，其特征在于，所述抗体是鼠科抗体。
3.一种编码权利要求1或2所述抗体的核酸。
4.如权利要求3所述的核酸，其特征在于，所述核酸具有SEQ ID No.1或6所示的核酸序列。
5.一种制备权利要求1所述抗体的方法，其特征在于，借助SNAP标记物蛋白在非人类哺乳动物，特别是小鼠中实施免疫，从中获得杂交瘤细胞，通过结合试验鉴定同时识别SNAP和CLIP标记物的抗体细胞系。
6.如权利要求1或2所述抗体的用途，用于检测SNAP和/或CLIP标记物。
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