CN115057927A - 一种花生过敏原Ara h1特异性纳米抗体及其应用 - Google Patents
一种花生过敏原Ara h1特异性纳米抗体及其应用 Download PDFInfo
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Abstract
本发明提供了一种花生过敏原Ara h1特异性纳米抗体及其应用，所述的特异性纳米抗体为纳米抗体Nb95、纳米抗体Nb122、纳米抗体Nb152或纳米抗体Nb191中的至少一种。本发明所述的花生过敏原Arah1特异性纳米抗体可用于食物组分中过敏原特异性纳米抗体的制备和检测体系构建。
Description
技术领域
本发明属于食品领域，尤其是涉及一种花生过敏原Ara h1特异性纳米抗体及其应用。
背景技术
食物过敏，即免疫介导的全身不良免疫反应，现在被认为是一个全球健康问题，在食品安全领域越来越普遍。食物过敏可刺激免疫系统，诱发免疫球蛋白E(IgE)介导的超敏反应，释放过敏炎性介质，引发严重急性超敏反应，如皮疹、腹泻、腹痛，甚至过敏性休克，危及生命。但是，到目前为止，还没有完全有效的治疗食物过敏的方法，对易过敏的顾客唯一有效的方法就是避免摄入含有过敏原的食物。而避免食物过敏的首要任务是开发可靠的、灵敏度高的过敏原检测技术，以促进风险评估和食品中的致敏成分的标记。
在过去的几十年里，许多有效的技术被广泛应用于确保食品过敏原的标记和管理，如酶联免疫吸附试验(ELISA)、聚合酶链反应(PCR)和液相色谱-质谱(LC-MS)。其中，ELISA方法具有较高的敏感性和特异性，现已被广泛的应用于食品过敏原的检测。建立ELISA方法需要特异性的抗体，纳米抗体作为科学家们近年来研究的新型小分子抗体，因其具备分子质量小、穿透力强的优势，有较大的发展潜力。骆驼科动物自然产生的仅由重链组成的抗体称为重链抗体，其中由单个可变结构域组成的称为重链抗体可变区(VHH)，也被称为纳米抗体(Nb)。从生物学、免疫学再到医学各个领域的潜在应用来说，它可识别常规抗体不能识别的结构以及隐蔽表位。传统单克隆抗体、多克隆抗体的制备方法复杂、周期长、成本高，而纳米抗体制备简便、周期短、成本低，相比于常规单克隆抗体和多克隆抗体它还具有高稳定性(耐高温、耐酸碱)、易表达等优点。这些优良特性可使其具备常规抗体无法实现的分析或诊断优势。因而，纳米抗体成为检测食品中非法添加剂、农药残留及致敏成分的理想抗体。
发明内容
有鉴于此，本发明旨在克服现有技术中的缺陷，提出一种花生过敏原Ara h1特异性纳米抗体及其应用。
为达到上述目的，本发明的技术方案是这样实现的：
一种花生过敏原Ara h1特异性纳米抗体，所述的特异性纳米抗体为纳米抗体Nb95、纳米抗体Nb122、纳米抗体Nb152或纳米抗体Nb191中的至少一种；
所述的特异性纳米抗体包括3个互补决定区CDR1、CDR2、CDR3；
对于纳米抗体Nb95：所述的CDR1的氨基酸序列如SEQ ID NO.1所示，所述的CDR2的氨基酸序列如SEQ ID NO.2所示，所述的CDR3的氨基酸序列如SEQ ID NO.3所示；
对于纳米抗体Nb122：所述的CDR1的氨基酸序列如SEQ ID NO.4所示，所述的CDR2的氨基酸序列如SEQ ID NO.5所示，所述的CDR3的氨基酸序列如SEQ ID NO.6所示；
对于纳米抗体Nb152：所述的CDR1的氨基酸序列如SEQ ID NO.7所示，所述的CDR2的氨基酸序列如SEQ ID NO.8所示，所述的CDR3的氨基酸序列如SEQ ID NO.9所示；
对于纳米抗体Nb191：所述的CDR1的氨基酸序列如SEQ ID NO.10所示，所述的CDR2的氨基酸序列如SEQ ID NO.11所示，所述的CDR3的氨基酸序列如SEQ ID NO.12所示。
进一步，所述的特异性纳米抗体包括4个框架区FR1、FR2、FR3、FR4；
对于纳米抗体Nb95：FR1的氨基酸序列如SEQ ID NO.13所示，所述的FR2的氨基酸序列如SEQ ID NO.14所示，所述的FR3的氨基酸序列如SEQ ID NO.15所示，所述的FR4的氨基酸序列如SEQ ID NO.16所示；
对于纳米抗体Nb122：FR1的氨基酸序列如SEQ ID NO.17所示，所述的FR2的氨基酸序列如SEQ ID NO.18所示，所述的FR3的氨基酸序列如SEQ ID NO.19所示，所述的FR4的氨基酸序列如SEQ ID NO.20所示；
对于纳米抗体Nb152：FR1的氨基酸序列如SEQ ID NO.21所示，所述的FR2的氨基酸序列如SEQ ID NO.22所示，所述的FR3的氨基酸序列如SEQ ID NO.23所示，所述的FR4的氨基酸序列如SEQ ID NO.24所示；
对于纳米抗体Nb191：FR1的氨基酸序列如SEQ ID NO.25所示，所述的FR2的氨基酸序列如SEQ ID NO.26所示，所述的FR3的氨基酸序列如SEQ ID NO.27所示，所述的FR4的氨基酸序列如SEQ ID NO.28所示。
进一步，所述的纳米抗体Nb95的氨基酸序列如SEQ ID NO.29所示；
所述的纳米抗体Nb122的氨基酸序列如SEQ ID NO.30所示；
所述的纳米抗体Nb152的氨基酸序列如SEQ ID NO.31所示；
所述的纳米抗体Nb191的氨基酸序列如SEQ ID NO.32所示。
所述的花生过敏原Ara h1特异性纳米抗体的应用，所述的特异性纳米抗体在食物过敏原免疫检测中的应用。
所述的花生过敏原Ara h1特异性纳米抗体的应用，所述的特异性纳米抗体在食物过敏原表位鉴定中的应用。
所述的特异性纳米抗体在食品基质中过敏原免疫捕获中的应用。
所述的花生过敏原Ara h1特异性纳米抗体的应用，所述的特异性纳米抗体在基于抗体的过敏原纯化和示踪的应用。
过敏原蛋白引起的过敏反应主要是过敏原蛋白与IgE抗体分子的结合，避免过敏反应的最佳手段是避免过敏原的摄入。因此，基于特异性的纳米抗体能够开发食品中痕量过敏原的高灵敏快速检测体系。
相对于现有技术，本发明具有以下优势：
本发明所述的花生过敏原Ara h1特异性纳米抗体制备简便、周期短、成本低，且稳定性较高，可用于食物组分中过敏原特异性纳米抗体的制备和检测体系构建。
附图说明
图1为本发明实施例所述的花生过敏原Ara h1蛋白的电泳图；
图2为本发明实施例所述的花生过敏原Ara h1特异性纳米抗体筛选的柱状图；
图3为本发明实施例所述的花生过敏原Ara h1特异性纳米抗体蛋白电泳图；
图4为本发明实施例所述的花生过敏原Ara h1特异性纳米抗体免疫印迹图；
图5为本发明实施例所述的花生过敏原Ara h1特异性纳米抗体免疫捕获图；
图6为本发明实施例所述的花生过敏原Ara h1特异性纳米抗体的亲和力曲线图：6-A为纳米抗体Nb95，6-B为纳米抗体Nb122，6-C为纳米抗体Nb152，6-D为纳米抗体Nb191；
图7为本发明实施例所述的花生过敏原Ara h1Ara h1特异性纳米抗体对筛选图；
图8为本发明实施例所述的花生过敏原Ara h1Ara h1检测用纳米抗体浓度优化图；
图9为本发明实施例所述的花生过敏原Ara h1免疫检测方法构建图。
具体实施方式
除有定义外，以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂，如无特殊说明，均为常规生化试剂；所述实验方法，如无特殊说明，均为常规方法。
下面结合实施例来详细说明本发明。
实施例1花生蛋白Ara h1的提取
称取0.5g花生分离蛋白粉溶于10ml20mM Tris(pH＝8.0)中，并混合均匀。在4℃条件下，以12000rpm离心20min后取上清液。得到的上清液pH应保持在8.0左右。在将上清液通过0.22μm的水系膜，后测浓度。取上清液蛋白量控制在30mg，通过使用HiTrap Q FF阴离子交换柱，进行分级洗脱。洗脱液中NaCl浓度为300mM收集的样品，经过SDS-PAGE凝胶电泳进行表征，并切取蛋白条带通过胰蛋白酶消化后通过高分辨液质(LC-MS/MS)分析，确认获得花生过敏原蛋白Ara h1，如图1所示。
实施例2基于花生蛋白Ara h1的羊驼免疫和纳米抗体库的构建
将提取的Ara h1花生蛋白以每次0.3mg的注射量免疫羊驼，每隔一周免疫一次，共免疫6次，在第六次免疫完成后，间隔三天，采集该羊驼颈静脉血液。采用密度梯度离心法提取血液中的淋巴细胞，之后通过TRIzolTM试剂(Invitrogen)从淋巴细胞中提取总RNA，并分离沉淀RNA。以所提RNA为模板进行反转录获得cDNA。以cDNA为模板，进行巢式PCR扩增VHH片段。第一轮PCR以CALL001、CALL002为引物进行扩增，扩增产物主要有两个分子量大小不同的片段，通过1％琼脂糖凝胶电泳分离，使用QIAquick凝胶提取试剂盒(QIAGEN)切取约700bp处片段进行回收纯化。第二次PCR以PMCF和A6E为引物扩增1轮PCR产物，该引物带有NotI和PstI限制酶位点，可为之后进行酶切连接提供酶切位点。扩增产物通过1％琼脂糖凝胶电泳检验并通过PCR纯化试剂盒(QIAGEN)纯化。在NotI和PstI限制性内切酶(NEB)的作用下，纯化后的PCR产物与pMECS质粒载体进行双酶切，酶切产物通过DNA回收试剂盒(天根)纯化后，在T4 DNA连接酶(Thermo Scientific)的作用下进行连接形成重组质粒。重组质粒通过苯酚：氯仿：异戊醇(25：24：1)纯化后，用GenePulser XcellTM电穿孔仪(BIO-RAD)转化至大肠杆菌TG1感受态细胞(Lucigen)中。转化后的细胞涂布在具备氨苄青霉素抗性的LB平板上，置于37℃过夜培养。次日使用细胞刮刀对生长出的菌株进行收集，并重悬于添加了100％甘油的LB液体培养基中，于-80℃长期储存。同时，挑取48个随机菌落进行colonyPCR，通过1％琼脂糖凝胶电泳计算VHH片段正确插入率。通过对菌落浓度进行梯度稀释和平板计数，计算文库的库容量及多样性。
实施例3特异性纳米抗体的筛选和鉴定
对经过纯化的噬菌体进行三轮淘选。即通过在96孔板过夜包被抗原的方式，淘选出对抗原具备高亲和力的TG1细胞。首先将1ml含有纳米抗体文库的TG1细胞接种至300ml 2×TY培养基(含100μg/ml氨苄青霉素和1％葡萄糖)中，于37℃摇床中以220rpm培养2h，待其达到对数生长期时，加入～1012VCSM13辅助噬菌体侵染TG1细胞30min。侵染结束后，分装至50ml离心管后，4℃离心收集菌体。取1ml 2×TY培养基(含100μg/ml氨苄青霉素和100μg/ml卡那霉素)重悬菌体后接种于300ml 2×TY培养基(含100μg/ml氨苄青霉素和100μg/ml卡那霉素)中以37℃过夜培养。次日于4℃条件下离心收集上清，并与提前预冷的PEG/Nacl混匀以沉淀上清中的噬菌体。离心收集沉淀后通过1ml无菌PBS重悬，并确定其浓度是否达到1012pfu/ml。将～1011噬菌体加至过夜包被抗原的孔中(“+”)及过夜包被PBS的阴性对照孔(“-”)中，室温下孵育1h后，通过PBST多次洗涤以去除未与抗原特异性结合的噬菌体。结合的噬菌体用100μl三乙胺(100mM TEA，pH 11.0)洗脱，并用100μl Tris-HCl(1.0M，pH 7.4)中和后转移到一个无菌的离心管中。此时获得200μl特异性噬菌体，其中180μl用于重新扩大培养，10μl用于富集，10μl备份。进行富集时，取10μl噬菌体在每孔加入90μlPBS的96孔板中按照从上到下的顺序进行十倍梯度稀释，直至达到10-7。吸取10μl各稀释度的噬菌体加至90μl已达对数生长期的TG1细胞中，侵染30分钟。然后，取10μl各稀释度对应的TG1细胞液涂布于LB琼脂平板上，37℃过夜培养。180μl噬菌体进行重新扩大培养，为下一轮淘选准备，直至完成三轮淘选。
第三轮淘选结束后，分别从每轮淘选所获的平板中挑选单菌落，共计挑选200个，将其接种至添加10％(w/v)甘油，2％(w/v)葡萄糖和100μg/ml氨苄青霉素的2×TY培养基中，37℃过夜培养。接种1ml含0.1％(w/v)葡萄糖和100μg/ml氨苄青霉素2×TY培养基于无菌的2ml深孔板(Axygen)中，将各个单菌落的10μl培养液转移至其中并做好标记。将深孔板于37℃条件下振荡培养大约4小时，待OD600达到1时，向每孔中添加终浓度为1mM的IPTG后继续在37℃条件下振荡培养以诱导其表达纳米抗体。诱导完成后，离心收集菌体，并于-80℃过夜冻存，次日取出并用PBS重悬菌体，即通过冻融法获得周质裂解液。将该周质裂解液用于ELISA验证以挑选出特异性菌株。即向过夜包被1.5μg花生蛋白的阳性孔及包被PBS的阴性孔中各加入20μl周质裂解液及80μlPBS，在室温下孵育1小时。3％脱脂乳粉封闭1小时后分别孵育100μl以1:5000稀释的Mouse anti-His MAb(Invitrogen)作为一抗，以及1:5000稀释的带有碱性磷酸酶标记的Goat anti-Mouse MAb(Invitrogen)作为二抗。加入显色剂后每隔15分钟测定OD405，共测4次。如图2所示，选择阳性孔OD值至少为阴性孔的2倍的菌落(阳性菌落)。从阳性菌株中提取质粒并测序，挑选序列不同的特异性菌株。
实施例4特异性纳米抗体的表达与纯化
从序列不同的特异性菌株中提取质粒，并电转化E.coli WK6感受态细胞，涂布LB琼脂平板于37℃培养。挑取单菌落接种于330ml含0.1％(w/v)葡萄糖，100μg/ml氨苄青霉素和2mM MgCl2的TB培养基中，于37℃摇床中培养至OD600达到0.6左右，加入终浓度为1mMIPTG后于28℃摇床中过夜培养以诱导表达纳米抗体。次日，离心收集菌体，通过渗透压休克法获得含有特异性纳米抗体的周质提取物。将该周质提取物与HisPurTM Ni-NTA树脂(Thermo-Scientific)结合1小时后，通过PD-10柱(GE Healthcare)，并使用PBS洗涤三次以去除非特异性组分。纳米抗体通过500mM咪唑洗脱。收集纯化后的纳米抗体，通过尺寸排阻色谱(SEC)进一步纯化后经SDS-PAGE和免疫印迹分析鉴定，如图3-4所示。
实施例5特异性纳米抗体靶向过敏原的确证
利用免疫共沉淀(Co-IP)和高分辨液质(LC-MS/MS)对特异性纳米抗体的靶向蛋白进行确证。将花生蛋白与纳米抗体混合均匀后室温下孵育1小时，形成抗体-抗原复合物，然后加入预先处理过的HisPurTM Ni-NTA磁珠(Thermo-Scientific)于室温下孵育1小时，由于抗体带有His标签，抗体-抗原复合物会结合到磁珠上。孵育结束后，通过磁力架分离并收集磁珠，使用洗涤缓冲液(含50mM咪唑的PBST；pH 8.0)洗涤以去除吸附在磁珠上的非特异性组分。加入25μl洗脱缓冲液(含250mM咪唑的PBS；pH 8.0)，振荡孵育15分钟后通过磁力架收集溶液，该溶液含有纳米抗体及其靶向蛋白。经过SDS-PAGE分离纳米抗体和靶向蛋白，并切取靶向蛋白条带在胰蛋白酶消化后进行LC-MS/MS分析，分析结果同Uniprot中Ara h1序列比对确证获得纳米抗体的靶向蛋白为花生过敏原Ara h1，如图5所示。
实施例6纳米抗体亲和力测定
通过间接ELISA法测定纳米抗体亲和力。将100μl Ara h1蛋白4℃过夜包被在96孔板中。3％脱脂乳粉在室温下封闭1h后，分别加入相应浓度的梯度稀释纳米抗体，在室温下孵育1h。用anti-His MAb和HRP标记的Goat anti-Mouse MAb孵育后，以TMB为显色底物进行显色反应并在450nm测定吸光度，结果如图6与表1所示。纳米抗体的亲和力以平衡解离常数(Kd)即响应信号为最大值的一半时的纳米抗体浓度表示。
表1花生过敏原Ara h1特异性纳米抗体的性质
| Nb95 | Nb122 | Nb152 | Nb191 | |
| 分子量/kDa | 12.75 | 13.2 | 13.73 | 13.46 |
| 等电点 | 9.69 | 9.94 | 5.76 | 7.97 |
| 亲和力/nM | 625 | 981.7 | 6.1 | 77.9 |
实施例7纳米抗体的配对筛选
采用酶切、连接的方法对纯化的pHEN6c质粒进行重组，得到只带有His-标签的质粒，转化到大肠杆菌WK6细胞中进行表达和纯化。然后将所有的抗体进行两两配以筛选双抗夹心ELISA的合适抗体对，用只含His-标签的纳米抗体作为捕获抗体，含有HA-和His-标签的纳米抗体作为检测抗体，用1：5000稀释的anti-His MAb作为一抗，1：5000稀释的HRP标记的Goat anti-Mouse Mab作为二抗，并用TMB为底物进行显色并测定OD450，结果如图7所示。挑选合适的抗体对进行后续方法建立。
实施例8双抗夹心ELISA方法条件的优化
根据筛选的捕获和检测用纳米抗体对，进行纳米抗体浓度优化。将不同浓度的捕获抗体进行包被，通过与花生蛋白Ara h1孵育后，将不同浓度的仅带有His标签的检测抗体孵育，用1：5000稀释的anti-His MAb作为一抗，1：5000稀释的HRP标记的Goat anti-MouseMab作为二抗，并用TMB为底物进行显色并测定OD450，确定捕获抗体与检测抗体的应用浓度，结果如图8所示。
实施例9双抗夹心ELISA方法的构建
用100μl只含His-标签的纳米作为捕获抗体，在室温下于96孔板中包被1h后，用PBST洗涤5次，然后在室温下用3％脱脂乳粉封闭1h。将稀释在PBS中的100μl花生蛋白(50μg/ml)加入孔中，室温孵育1h，随后加入100μl His-和HA-标签的纳米抗体作为检测抗体，PBST洗涤后，将100μl 1:5000稀释的anti-His MAb和HRP标记的Goat anti-Mouse Mab分别作为一抗和二抗，分别于室温下孵育1h。最后，在加入TMB显色液后，用酶标仪测定450nm处的吸光度OD450。
实施例10双抗夹心ELISA方法标准曲线的建立
根据优化的条件，构建基于纳米抗体双抗夹心ELISA检测方法并测定花生蛋白Arah1的标准曲线。将花生蛋白Ara h1按一定的浓度比例稀释，以抗原浓度的对数值为横坐标，对应OD450值为纵坐标绘制曲线，结果如图9所示。根据绘制的曲线选取最佳的线性范围，检测限(LOD)为空白孔平均值加上三倍标准偏差(SD)所对应曲线上的浓度值，定量限(LOQ)为空白孔平均值加上十倍标准偏差(SD)所对应曲线上的浓度值。
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、改进等，均应包含在本发明的保护范围之内。
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<110> 南开大学
<120> 一种花生过敏原Ara h1特异性纳米抗体及其应用
<130> 2022.5.26
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Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys
85 90 95
Ala Cys Asp Asp Glu Phe Phe Phe Gly Gly Gly Gly Asn Pro Thr Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 32
<211> 126
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Arg Thr Leu Ser Pro Phe
20 25 30
Gln Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Phe Val Ala
35 40 45
Ser Ile Ser Arg Ile Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Ala Ile Ser Arg Asp Gly Ala Lys Asn Thr Val Thr Leu
65 70 75 80
Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Asp Glu Phe Gly Gly Ile Lys Leu Pro Gln Arg Ser Ser Thr
100 105 110
Val Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Claims (7)
1.一种花生过敏原Ara h1特异性纳米抗体，其特征在于：所述的特异性纳米抗体为纳米抗体Nb95、纳米抗体Nb122、纳米抗体Nb152或纳米抗体Nb191中的至少一种；
所述的特异性纳米抗体包括3个互补决定区CDR1、CDR2、CDR3；
对于纳米抗体Nb95：所述的CDR1的氨基酸序列如SEQ ID NO.1所示，所述的CDR2的氨基酸序列如SEQ ID NO.2所示，所述的CDR3的氨基酸序列如SEQ ID NO.3所示；
对于纳米抗体Nb122：所述的CDR1的氨基酸序列如SEQ ID NO.4所示，所述的CDR2的氨基酸序列如SEQ ID NO.5所示，所述的CDR3的氨基酸序列如SEQ ID NO.6所示；
对于纳米抗体Nb152：所述的CDR1的氨基酸序列如SEQ ID NO.7所示，所述的CDR2的氨基酸序列如SEQ ID NO.8所示，所述的CDR3的氨基酸序列如SEQ ID NO.9所示；
对于纳米抗体Nb191：所述的CDR1的氨基酸序列如SEQ ID NO.10所示，所述的CDR2的氨基酸序列如SEQ ID NO.11所示，所述的CDR3的氨基酸序列如SEQ ID NO.12所示。
2.根据权利要求1所述的花生过敏原Ara h1特异性纳米抗体，其特征在于：所述的特异性纳米抗体包括4个框架区FR1、FR2、FR3、FR4；
对于纳米抗体Nb95：FR1的氨基酸序列如SEQ ID NO.13所示，所述的FR2的氨基酸序列如SEQ ID NO.14所示，所述的FR3的氨基酸序列如SEQ ID NO.15所示，所述的FR4的氨基酸序列如SEQ ID NO.16所示；
对于纳米抗体Nb122：FR1的氨基酸序列如SEQ ID NO.17所示，所述的FR2的氨基酸序列如SEQ ID NO.18所示，所述的FR3的氨基酸序列如SEQ ID NO.19所示，所述的FR4的氨基酸序列如SEQ ID NO.20所示；
对于纳米抗体Nb152：FR1的氨基酸序列如SEQ ID NO.21所示，所述的FR2的氨基酸序列如SEQ ID NO.22所示，所述的FR3的氨基酸序列如SEQ ID NO.23所示，所述的FR4的氨基酸序列如SEQ ID NO.24所示；
对于纳米抗体Nb191：FR1的氨基酸序列如SEQ ID NO.25所示，所述的FR2的氨基酸序列如SEQ ID NO.26所示，所述的FR3的氨基酸序列如SEQ ID NO.27所示，所述的FR4的氨基酸序列如SEQ ID NO.28所示。
3.根据权利要求2所述的花生过敏原Ara h1特异性纳米抗体，其特征在于：所述的纳米抗体Nb95的氨基酸序列如SEQ ID NO.29所示；
所述的纳米抗体Nb122的氨基酸序列如SEQ ID NO.30所示；
所述的纳米抗体Nb152的氨基酸序列如SEQ ID NO.31所示；
所述的纳米抗体Nb191的氨基酸序列如SEQ ID NO.32所示。
4.权利要求1-3中任一项所述的花生过敏原Ara h1特异性纳米抗体的应用，其特征在于：所述的特异性纳米抗体在食物过敏原免疫检测中的应用。
5.权利要求1-3中任一项所述的花生过敏原Ara h1特异性纳米抗体的应用，其特征在于：所述的特异性纳米抗体在食物过敏原表位鉴定中的应用。
6.权利要求1-3中任一项所述的花生过敏原Ara h1特异性纳米抗体的应用，其特征在于：所述的特异性纳米抗体在食品基质中过敏原免疫捕获中的应用。
7.权利要求1-3中任一项所述的花生过敏原Ara h1特异性纳米抗体的应用，其特征在于：所述的特异性纳米抗体在基于抗体的过敏原纯化和示踪的应用。
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