CN112649615A - 一种检测不同大小的可溶性Aβ寡聚体的方法及应用 - Google Patents
一种检测不同大小的可溶性Aβ寡聚体的方法及应用 Download PDFInfo
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Abstract
本发明公开了一种检测不同大小的可溶性Aβ寡聚体的方法及应用。所述的方法是通过Co2+螯合将含有如SEQ ID NO.1所示氨基酸序列的SDP6纳米抗体结合到固相载体上，再将Co2+氧化为Co3+使纳米抗体稳定结合，然后吸附Aβ寡聚体，用PICUP(Photo‑Induced Cross‑Linking of unmodified protein)方法使纳米抗体和Aβ寡聚体共价结合，得到SDP6纳米抗体‑Aβ寡聚体复合物，然后检测复合物分子大小，再根据复合物分子量大小确定寡聚体大小。该方法利用纳米抗体的小分子特性以及与Aβ寡聚体特异性结合，光交联时趋于抗原抗体的共价连接，且共价键不会在变性电泳过程中被打开，因此能够鉴定不同大小的Aβ寡聚体，为进一步检测和研究不同大小Aβ寡聚体与AD发展进程相关性成为可能。
Description
技术领域
本发明涉及一种检测方法，特别涉及一种检测不同大小的可溶性Aβ寡聚体的方法及应用。
背景技术
阿尔茨海默症(Alzheimer’s disease，AD)是一种常见于70岁以上老年人的神经退行性疾病，严重危害人类的生命健康，其患病率在全球范围内日益增长，被世界卫生组织视为全球健康的首要问题。阿尔茨海默症的病理特征之一是淀粉样β蛋白(amyloid-β，Aβ)在患者大脑内的积累，因此Aβ可作为诊断阿尔茨海默症的生物标志物(Nature,2018,554(7691):249-254.)。Aβ是由淀粉样前体蛋白经β-分泌酶和γ-分泌酶水解生成一种由38-43个氨基酸组成的小的肽段，人体内Aβ的产生和清除的动态平衡失衡导致Aβ积累，逐步导致AD(Journal of the Neurological Sciences,2016,15(361):256-271.)。Aβ有单体、可溶性寡聚体、原纤维及纤维等多种结构，近期越来越多的研究表明，对神经元有损伤作用的可溶性Aβ寡聚体的毒性最大(International Journal of Alzheimer’s Disease,2011:603052.)，更适合作为诊断AD的关键生物标志物。而对于不同大小的可溶性Aβ寡聚体的毒害作用，仍存在着一些争议：有研究表明，Aβ二聚体通过阻断神经元和星形胶质细胞对谷氨酸的再摄取，使神经元过度激活，最终导致神经退化(Science,2019,365(6453):540-541.)；也有研究证明Aβ二聚体会损伤LTP(Long-term potentiation：长时程增强作用，与突触改变强度的能力相关，被视为构成学习与记忆基础的主要分子机制之一)且在皮摩尔浓度下具有高度突触毒性(Brain:a journal of neurology,2016,139(Pt 2):509-25.)；另外也有文章报道，Aβ三聚体和四聚体能与神经元结合，与Aβ引起的神经毒性关联最大(Journal of Neurochemistry,2016,136(3):594-608.)；还有文章提到，Aβ三聚体的结构在神经退行性变中有至关重要的作用，以及其组装成的高阶寡聚体也有神经毒性(Journalof the American Chemical Society,2017,139(2):966-975.)。因此，检测不同大小的可溶性Aβ寡聚体为进一步研究Aβ寡聚体不同大小对AD的危害程度至关重要。
现阶段Aβ寡聚体的检测方法主要方法有电泳法(Methods in MolecularBiology,2012,849:23-31.)、免疫共沉淀(American Journal of Pathlogy,2018,188(3):739-756.)和酶联免疫法(Cell Reports,2014,7(1):261-8；Journal of Neuropathlogy&Experimental Neurology,2011,70(5):360-76.)，它们主要的不足之处是会改变Aβ寡聚体的聚集状态且不具有特异性：例如电泳检测方法中SDS的存在以及电泳过程本身会改变Aβ的聚集(Amyloid,2005,12(2):88-95.)；免疫共沉淀检测中的洗脱过程中Aβ与抗体解离，Aβ会再次发生聚集；而酶联免疫测定则需要特异性识别不同Aβ寡聚体的抗体，但由于Aβ寡聚体的结构不稳定性，目前仍无法获得针对每种寡聚体的特异抗体。因此急需建立一种检测不同大小Aβ寡聚体的新方法。本发明提供一种能够稳定并鉴定出不同大小Aβ寡聚体的方法。
发明内容
为解决上述技术问题，本发明提供了一种检测不同大小的可溶性Aβ寡聚体的方法，能够区分不同大小的Aβ寡聚体，从而为进一步检测和研究不同大小Aβ寡聚体与AD发展进程相关性成为可能。
本发明通过如下技术方案实现：一种检测不同大小的可溶性Aβ寡聚体的方法，所述不同大小的可溶性Aβ寡聚体包括Aβ单体和Aβ二聚体；所述的方法是通过Co2+螯合将含有如SEQ ID NO.1所示氨基酸序列的SDP6纳米抗体结合到固相载体上，再将Co2+氧化为Co3+使纳米抗体稳定结合，然后吸附Aβ寡聚体，用PICUP(Photo-Induced Cross-Linking ofunmodified protein)方法使纳米抗体和Aβ寡聚体共价结合，得到SDP6纳米抗体-Aβ寡聚体复合物，然后检测复合物分子大小，再根据复合物分子量大小确定寡聚体大小。
进一步地，上述技术方案中，所述固相载体包括琼脂糖凝胶。
进一步地，上述技术方案中，SDP6纳米抗体-Aβ寡聚体复合物的分子量检测方法为Tricine-SDS-PAGE，并采用银染的方法对电泳胶进行染色。
一种检测不同大小的可溶性Aβ寡聚体的方法，包括以下步骤：
1)制备并纯化得到含有如SEQ ID NO.1所示氨基酸序列的SDP6纳米抗体；
2)将CoCl2溶液在琼脂糖凝胶孵育，制备鳌合Co2+的琼脂糖凝胶；向螯合Co2+的琼脂糖凝胶中加入步骤1)所得纯化的SDP6纳米抗体，孵育后加入双氧水使Co2+氧化为Co3+，再加入Aβ寡聚体，再用光催化交联的方法使固载在琼脂糖凝胶上的SDP6纳米抗体和Aβ寡聚体共价结合，得到SDP6纳米抗体-Aβ寡聚体复合物；
3)采用Tricine-SDS-PAGE方法检测不同大小的可溶性Aβ寡聚体。
进一步地，上述技术方案中，步骤1)中所述的Aβ寡聚体的制备方法，包括如下步骤：
(1)Aβ合成肽与1,1,1,3,3,3-六氟-2-丙醇在密封的容器中，超声、旋涡至Aβ全部溶解成单体，容器开盖风干，形成肽膜；
(2)向具有肽膜的容器中依次加入60mM氢氧化钠、PBS缓冲液和双蒸水，涡旋、超声至肽膜充分溶解并使肽单体的终浓度为100-250μM；
(3)向步骤(2)所得溶液中依次加入过硫酸铵、Tris(2,2-双吡啶)二氯钌(II)六水合物混匀后迅速放置于距光源一段距离，白炽灯光照一段时间后，加入1M的二硫苏糖醇终止反应，即得Aβ寡聚体，所述Aβ寡聚体包括Aβ单体和Aβ二聚体和微量Aβ三聚体。
进一步地，上述技术方案中，步骤(2)中所述氢氧化钠、PBS缓冲液和双蒸水的体积比为(0.5-2):9:9；步骤(2)中所述的肽单体和步骤(3)中所述的过硫酸铵、Tris(2,2-双吡啶)二氯钌(II)六水合物的浓度比为1:(2-2.5):(40-50)；肽单体、过硫酸铵、Tris(2,2-双吡啶)二氯钌(II)六水合物和二硫苏糖醇的体积比为18:(1-2.7):(1-2.7):(1-2.7)。
进一步地，上述技术方案中，步骤(3)所述距光源10-12cm处，白炽灯光照30-60s。
进一步地，上述技术方案中，步骤3)所述光催化交联的方法使固载在琼脂糖凝胶上的SDP6纳米抗体和Aβ寡聚体共价结合的方法为：加入过硫酸铵、Tris(2,2-双吡啶)二氯钌(II)六水合物混匀后迅速放置于距光源10-12cm处，白炽灯光照30-60s后，加入二硫苏糖醇终止反应，即得。
步骤3)所述方法，具体步骤为：取20μl琼脂糖凝胶于层析柱空管中，加入100mM的CoCl2溶液在琼脂糖凝胶中孵育30min后，用10-20倍琼脂糖凝胶体积的冲洗缓冲液冲洗凝胶，以除去未结合的Co2+；向层析柱中加入0.4mM纯化后的SDP6纳米抗体，在37℃下孵育30min；向层析柱中加入终浓度为10mM的H2O2避光孵育1h，用10-20倍琼脂糖凝胶体积的洗脱缓冲液冲洗凝胶；
所述的冲洗缓冲液为：15mM Tris-HCl，300mM NaCl，pH7.4，0.22μm过膜除菌，超声40min脱气；
所述的洗脱缓冲液(含EDTA)为：15mM Tris-HCl，300mM NaCl，20mM EDTA，150mM咪唑，0.22μm过膜除菌，超声40min脱气；
进一步地，上述技术方案中，步骤3)所述孵育的条件为37℃，孵育1h。
进一步地，上述技术方案中，步骤4)中所述的Tricine-SDS-PAGE方法检测琼脂糖凝胶上的不同大小的可溶性Aβ寡聚体时，采用银染的方法对电泳胶进行染色。
本发明还提供了所述的方法在检测大脑中不同大小淀粉样β蛋白中的应用。
本发明对比现有技术的有益效果是：本发明提供的不同大小Aβ寡聚体的检测方法选用的SDP6纳米抗体易获得且制备成本低；先固载纳米抗体再吸附抗原的操作步骤可以增加上样量或重复上样，从而提高检测灵敏度；选用Co3+介导固载可以减少非特异性吸附，且可以稳定纳米抗体，防止在交联的过程中与自身交联形成大分子物质产生沉淀；交联形成的共价连接的抗原抗体复合物不会在变性条件下分离；采用的Tricine-SDS-PAGE检测方法分辨率较普通的SDS-PAGE分辨率更高，银染检测方法的检测限较考马斯亮蓝小100倍，可以检测微量的Aβ寡聚体，且灵敏度也更高；由于纳米抗体分子较小，抗原抗体复合物的电泳检测分辨率高。总的来说，本发明提供的不同大小Aβ寡聚体的检测方法能够高效反应Aβ寡聚体的大小和数量，从而为后续研究不同大小Aβ寡聚体的危害提供了一种可行的方法。
附图说明
图1为本发明实施例1制备的Aβ寡聚体的琼脂糖凝胶电泳图；图中泳道1为未交联Aβ单体对照；泳道2为Aβ单体交联30s后终止反应所得寡聚体；泳道3为Aβ单体交联40s后终止反应所得寡聚体；泳道4为Aβ单体交联50s后终止反应所得寡聚体；泳道5为超低分子量marker。
图2为本发明实施例的SDP6纳米抗体-Aβ寡聚体复合物琼脂糖电泳图；图中泳道1为实施例2所得纯化后的SDP6纳米抗体；泳道2-5为实施例5所述通过光交联所得SDP6纳米抗体-Aβ寡聚体复合物，泳道6为低分子量marker。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1Aβ寡聚体的制备
(1)肽膜制备：称取4mg Aβ合成肽粉末(由上海肽仕生物科技有限公司合成)于离心管，加入1mL 1,1,1,3,3,3-六氟-2-丙醇(HFIP)并迅速盖上离心管盖防止HFIP挥发，超声、涡旋等方法使肽粉末充分溶解，然后分装为10管，每管100μL，打开离心管盖，过夜充分风干，形成肽膜。
(2)光催化交联法制备Aβ寡聚体(PICUP)：
①向形成肽膜的离心管中，加入100μL的溶解液(体积比10:45:45的60mM氢氧化钠、PBS缓冲液、双蒸水)，涡旋10min、超声10min使肽膜充分溶解并使Aβ肽的终浓度为200μM(可采用BCA法测蛋白浓度)。
②制备32μM Tris(2,2-双吡啶)二氯钌(II)六水合物(RuBpy)，640μM过硫酸铵(APS)，及1M二硫苏糖醇(DTT)。
③取18μL Aβ溶液于PCR管中，依次加入1μL APS、1μL RuBpy混匀后迅速放置于与光源10cm处，白炽灯光照50s后，迅速加入1μL DTT终止反应，得到Aβ寡聚体。(白炽灯光照30s和40s分别作为实验对照组，其余步骤相同，所得Aβ寡聚体均进行琼脂糖凝胶电泳检测验证)。
结果如图1所示，泳道1为未交联Aβ单体对照；泳道2为Aβ单体交联30s后终止反应所得寡聚体；泳道3为Aβ12-35单体交联40s后终止反应所得寡聚体；泳道4为Aβ单体交联50s后终止反应所得寡聚体，泳道5为marker蛋白。由于Aβ极易于聚集的特征，制备的Aβ单体样品中也存在少量二聚体；且在一定范围内，交联时间越长，寡聚体的含量和种类也越多。同时存在微量Aβ三聚体。
实施例2 SDP6纳米抗体的制备与纯化
按照中国发明专利ZL201610076553.4所述的方法利用噬菌体展示技术对羊驼噬菌体库进行多轮筛选富集具有Aβ特异性的噬菌体，通过对噬菌体培养制备抗体，并鉴定获得阳性克隆，通过测序获得其相应的编码序列，然后在大肠杆菌中表达获得可溶性的抗体片段，并进一步纯化得到氨基酸序列如SEQ ID NO.1所示的抗体片段。将氨基酸序列为SEQID NO.1的抗体片段命名为SDP6纳米抗体(本实施例所述SDP6纳米抗体的筛选、制备及纯化方法均通过ZL201610076553.4方法获得)。
实施例3 SDP6纳米抗体的固载
(1)配制以下溶液：
冲洗缓冲液：15mM Tris-HCl，300mM NaCl，pH7.4，0.22μm过膜除菌，超声40min脱气。
洗脱缓冲液(含EDTA)：15mM Tris-HCl，300mM NaCl，20mM EDTA，150mM咪唑，0.22μm过膜除菌，超声40min脱气。
(2)取20μL琼脂糖凝胶于1mL的层析柱空管中，接着加入100μL 100mM的CoCl2溶液在琼脂糖凝胶中孵育30min后，用10-20倍琼脂糖凝胶体积的冲洗缓冲液冲洗凝胶，以除去未结合的Co2+。
(3)分别向层析柱中加入20，40，60，80μL 0.4mM的实施例2所得纯化的SDP6纳米抗体在37℃下孵育30min使其固载于琼脂糖凝胶上。
(4)向层析柱中加入终浓度为10mM的H2O2避光孵育1h，使Co2+氧化为Co3+，接着用10-20倍琼脂糖凝胶体积的洗脱缓冲液冲洗凝胶，除去凝胶中未结合或结合不稳定的SDP6纳米抗体，将螯合了Co3+的琼脂糖凝胶从层析柱中取出备用。
实施例4吸附Aβ寡聚体
向实施例3所得螯合了Co3+的琼脂糖凝胶中加入实施例1制得的Aβ寡聚体，37℃，170rpm下孵育1h，再次加入Aβ寡聚体进行孵育，重复三次。得到吸附了Aβ寡聚体的琼脂糖凝胶。
实施例5光交联
将实施例4所得的吸附了Aβ寡聚体的琼脂糖凝胶中多余的悬液排除，加入2μL APS(浓度为640μM)、2μL RuBpy(浓度为32μM)混匀后迅速放置于与光源10cm处，白炽光光照50s后，迅速加入2μL DTT终止反应，使得固载在琼脂糖凝胶上的SDP6纳米抗体与Aβ寡聚体共价交联，得到固载有共价交联的SDP6纳米抗体-Aβ寡聚体复合物的琼脂糖凝胶。
实施例6 Tricine-SDS-PAGE检测
(1)按下表配制Tricine-SDS-PAGE所用的电泳胶：
其中3×凝胶缓冲液：称取18.17g Tris，0.15g SDS加入适量双蒸水中使其完全溶解，用HCl调节pH至8.45，定容至50ml。
阴极缓冲液：称取121.14Tris，加入适量双蒸水中使其完全溶解，用HCl调节pH至8.9，定容至1000ml。临用时稀释10倍。
阳极缓冲液：称取121.14Tris，179.17g Tricine，10g SDS(十二烷基硫酸钠)，加入适量双蒸水中使其完全溶解，用HCl调节pH至8.25，定容至1000ml。临用时稀释10倍。
(2)将实施例5所得固载有共价交联的SDP6纳米抗体-Aβ寡聚体复合物的琼脂糖凝胶用500μL洗脱缓冲液(不含EDTA：15mM Tris-HCl，300mM NaCl，150mM咪唑，0.22μm过膜除菌，超声40min脱气)清洗，再加入100μL冲洗缓冲液(15mM Tris-HCl，300mM NaCl，pH7.4，0.22μm过膜除菌，超声40min脱气)将其悬浮，与30μL的5×样品缓冲液(3.1mL 1mol/LTris-HCl(pH 6.8)；5mL 50％甘油；0.5mL 1％溴酚蓝；1.4mL蒸馏水)混合后煮沸10min，取6μL加入上样孔。电泳槽内部加满阴极缓冲液，外部加入适量阳极缓冲液。60V电压下样品在浓缩胶中移动，约40min后调电压100V，至溴酚蓝刚好移动至胶底部，电泳结束。
(3)采用银染的方法对电泳胶进行染色，使用快速银染试剂盒(上海碧云天生物技术有限公司，P0017S)经固定、清洗、增敏过程后，显色至条带清晰可见，终止显色后在凝胶成像仪系统中，拍照分析，结果如图2所示。
如图2所示，图中泳道1为实施例2所得纯化后的SDP6纳米抗体；泳道2-5为实施例5所述通过光交联所得SDP6纳米抗体-Aβ寡聚体复合物，泳道6为低分子量marker。可从图中看出可以检测得到SDP6纳米抗体与Aβ单体结合的复合物和SDP6纳米抗体与Aβ二聚体结合的复合物。
SEQUENCE LISTING
<110> 大连理工大学
<120> 一种检测不同大小的可溶性Aβ寡聚体的方法及应用
<130> 2020
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 130
<212> PRT
<213> 人工序列（Artifical Sequence）
<400> 1
Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Met Thr Phe Ser Ser
20 25 30
Tyr Val Met Thr Trp Tyr Arg Gln Ala Pro Gly Lys Lys Arg Glu Leu
35 40 45
Val Ala Ala Ile Thr Ser Ala Gly Gly Arg Thr Asn Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Asn Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Asn Ala Val Val Glu Ser Trp Asn Glu Asp Glu Tyr Asp Val Met
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys
115 120 125
Pro Gln
130
Claims (4)
1.一种检测不同大小的可溶性Aβ寡聚体的方法，其特征在于，所述的方法是通过Co2+螯合将含有如SEQ ID NO.1所示氨基酸序列的SDP6纳米抗体结合到固相载体上，再将Co2+氧化为Co3+使纳米抗体稳定结合，然后吸附Aβ寡聚体，用PICUP(Photo-Induced Cross-Linkingof unmodified protein)方法使纳米抗体和Aβ寡聚体共价结合，得到SDP6纳米抗体-Aβ寡聚体复合物，然后检测复合物分子大小，再根据复合物分子量大小确定寡聚体大小。
2.根据权利要求1所述的方法，其特征在于，所述固相载体包括琼脂糖凝胶。
3.根据权利要求1所述的方法，其特征在于，SDP6纳米抗体-Aβ寡聚体复合物的分子量检测方法为Tricine-SDS-PAGE，并采用银染的方法对电泳胶进行染色。
4.权利要求1-3中任一项所述的方法在检测大脑中不同大小淀粉样β蛋白中的应用。
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| CN104558172A (zh) * | 2015-01-04 | 2015-04-29 | 东南大学 | 一种针对β淀粉样蛋白的单域重链纳米抗体及其应用 |
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| US20150266947A1 (en) * | 2012-10-12 | 2015-09-24 | Arizona Board Of Regents On Behalf Of Arizona State University | Antibody based reagents that specifically recognize toxic oligomeric forms of tau |
| CN104558172A (zh) * | 2015-01-04 | 2015-04-29 | 东南大学 | 一种针对β淀粉样蛋白的单域重链纳米抗体及其应用 |
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