CN113264986B - 一种亲和肽m5稳定的金纳米颗粒及其制备方法和用途 - Google Patents
一种亲和肽m5稳定的金纳米颗粒及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种亲和肽M5稳定的金纳米颗粒及其制备方法和用途,本发明采用噬菌体展示技术筛选出一种亲和肽M5,相对于未经修饰的金纳米颗粒而言,经亲和肽M5修饰的金纳米颗粒显示出较好的稳定性,且细胞毒性低,可用作金纳米颗粒的稳定剂,为进一步研究金纳米颗粒在诊断、催化、治疗药剂递送等领域的应用提供了基础。
Description
技术领域
本发明属于功能性纳米材料技术领域,具体而言,涉及一种亲和肽M5稳定的金纳米颗粒及其制备方法和用途。
背景技术
金纳米颗粒(Gold nanoparticles,AuNPs)是指直径介于1~100nm之间的纳米金颗粒,由于其粒子的尺寸为纳米量级,使其具有不同于传统固体材料的特殊性质。纳米金在一些特定的晶面上存在着表面电子态,其费米能级恰好位于体能带结构沿该晶向的禁带之中,形成只能平行于表面方向运动的二维电子云,这种特点使得金纳米粒子的物理和化学性质明显不同于传统物质,主要表现在金纳米粒子具有表面效应、量子效应、体积效应和宏观隧道效应等。正是由于纳米金独特的催化效应、光学效应、磁学效应(光吸收和光散射)、电学效应和特殊的生物亲和效应,使得其在如诊断、催化、药物递送、传感器、光动力学治疗、电子学、探针等诸多领域有着广泛的应用。
目前,金纳米颗粒的制备方法主要可分为物理方法和化学方法,其中,物理方法是采用高能消耗的方式将块状金细化成为纳米级小颗粒,常见的物理方法包括机械研磨法、气相法、辐照分解、热分解、真空蒸镀法、软着陆法、激光消融法;化学方法是以金盐为原料,利用还原反应生成金纳米颗粒,在形成过程中通过控制粒子的生长从而控制其尺寸,常见的化学方法包括溶胶法、晶种生长法、反胶束法、相转移法、模板法、水相氧化还原法、湿化学合成法、电化学法、光化学法,化学方法是目前制备高质量的金纳米颗粒的主要方法,而且也是制备非球状金纳米颗粒的唯一方法,相对于物理方法而言,化学方法制备得到的金纳米颗粒粒径均匀、易于控制形貌。
在金纳米颗粒制备的过程中,纳米金粒子表面的活性使它们很容易团聚在一起,从而形成带有若干弱连接界面的尺寸较大的团聚体,即制备得到的金纳米颗粒很不稳定。因此,为了确保颗粒的稳定性,在制备过程中往往需要加入稳定剂,稳定剂在保证纳米金粒子在液相中稳定的同时,对于纳米金的生长控制、表面结构和性质及其应用具有至关重要的作用。目前,被广泛应用于合成金纳米颗粒的稳定剂有十六烷基三甲基溴化铵(CTAB)。由于羧基的存在,使得金纳米粒子显负电性,CTAB是一种阳离子表面活性剂,可以在水中形成带正电胶束,由于正负离子的相互吸引使CTAB插入聚集的金纳米粒子之间,因此,CTAB可使金纳米粒子稳定的存在于介质中。
相关研究表明CTAB具有极强的细胞毒性,使得经其修饰后的金纳米颗粒的应用受到了极大的限制,因此,目前该领域仍需要开发一些新型的无毒副作用或低毒性的稳定剂用于金纳米颗粒的制备中。鉴于此,本发明采用噬菌体展示技术筛选出一种亲和肽,相对于未经修饰的金纳米颗粒而言,所述亲和肽修饰的金纳米颗粒显示出较好的稳定性,且细胞毒性低,可用作金纳米颗粒的稳定剂,为进一步研究金纳米颗粒在诊断、催化、治疗药物递送等领域的应用提供了基础。
发明内容
本发明针对现有技术存在的问题,即常用作金纳米颗粒稳定剂的CTAB具有极强的细胞毒性,提供了一种亲和肽M5稳定的金纳米颗粒及其制备方法和用途,经实验验证表明,所述亲和肽M5修饰的金纳米颗粒具有较好的稳定性,可用作金纳米颗粒的稳定剂,为进一步研究金纳米颗粒的应用提供了基础。
本发明的上述目的通过以下技术方案得以实现:
本发明的第一方面提供了一种亲和肽。
进一步,所述亲和肽包括如SEQ ID NO.1所示的序列或其变体;
优选地,所述亲和肽特异性结合冠状病毒;
更优选地,所述亲和肽特异性结合冠状病毒的主蛋白酶Mpro;
最优选地,所述冠状病毒为SARS-CoV-2。
进一步,所述SEQ ID NO.1为在SEQ ID NO.2的C端加上一个-NH2修饰基团。
进一步,所述SEQ ID NO.2为KQEGAWVIIGLL。
进一步,所述变体是指在所述亲和肽的基础上,修饰上一种或多种氨基酸残基但是仍然保留亲和肽的生物活性的多肽。
进一步,所述变体可采用任何方法进行合成,所述方法包括(但不限于):易错PCR、重排、寡核苷酸-定向诱变、装配PCR、有性PCR诱变、体内诱变、盒式诱变、递归全体诱变、指数全体诱变、位点特异性诱变、基因再组装、GSSM或这些方法的任意组合。
本发明的第二方面提供了本发明第一方面所述的亲和肽在制备亲和肽稳定的金纳米颗粒中的应用。
进一步,所述制备的过程包括如下步骤:
(1)采用柠檬酸钠溶液、氯金酸溶液制备金纳米颗粒;
(2)在本发明第一方面所述的亲和肽的C端加一个半胱氨酸,通过Au-S键与步骤(1)制得的金纳米颗粒结合,得到亲和肽稳定的金纳米颗粒。
进一步,所述步骤(1)中柠檬酸钠溶液的浓度为2.2mM,氯金酸溶液的浓度为25mM。
进一步,所述步骤(1)中柠檬酸钠溶液先取150mL,置于水浴中进行搅拌;
优选地,所述水浴的温度为90℃;
优选地,所述搅拌的条件为400rpm磁力搅拌。
进一步,在上述溶液中加入氯金酸溶液,反应后加入柠檬酸钠溶液和氯金酸溶液,反应后再次加入柠檬酸钠溶液和氯金酸溶液;
优选地,所述加入氯金酸溶液的体积为1mL;
优选地,所述加入柠檬酸钠溶液和氯金酸溶液的体积分别为1mL;
优选地,所述反应的时间均为30min。
进一步,上述反应后,冷却至室温,即得金纳米颗粒溶液。
本发明第三方面提供了一种亲和肽稳定的金纳米颗粒。
进一步,所述亲和肽包括如SEQ ID NO.1所示的序列或其变体;
优选地,所述亲和肽特异性结合冠状病毒;
更优选地,所述亲和肽特异性结合冠状病毒的主蛋白酶Mpro;
最优选地,所述冠状病毒为SARS-CoV-2。
进一步,所述变体是指在所述亲和肽的基础上,修饰上一种或多种氨基酸残基但是仍然保留亲和肽的生物活性的多肽。
进一步,所述变体可采用任何方法进行生产,所述方法包括(但不限于):易错PCR、重排、寡核苷酸-定向诱变、装配PCR、有性PCR诱变、体内诱变、盒式诱变、递归全体诱变、指数全体诱变、位点特异性诱变、基因再组装、GSSM或这些方法的任意组合。
本发明第四方面提供了一种亲和肽稳定的金纳米颗粒的制备方法。
进一步,所述方法包括如下步骤:
(1)采用柠檬酸钠溶液、氯金酸溶液制备金纳米颗粒;
(2)在本发明第一方面所述的亲和肽的C端加一个半胱氨酸,通过Au-S键与步骤(1)制得的金纳米颗粒结合,得到亲和肽稳定的金纳米颗粒。
进一步,所述步骤(1)中柠檬酸钠溶液的浓度为2.2mM,氯金酸溶液的浓度为25mM。
进一步,所述步骤(1)中柠檬酸钠溶液先取150mL,置于水浴中进行搅拌;
优选地,所述水浴的温度为90℃;
优选地,所述搅拌的条件为400rpm磁力搅拌。
进一步,在上述溶液中加入氯金酸溶液,反应后加入柠檬酸钠溶液和氯金酸溶液,反应后再次加入柠檬酸钠溶液和氯金酸溶液;
优选地,所述加入氯金酸溶液的体积为1mL;
优选地,所述加入柠檬酸钠溶液和氯金酸溶液的体积分别为1mL;
优选地,所述反应的时间均为30min。
进一步,上述反应后,冷却至室温,即得金纳米颗粒溶液。
本发明第五方面提供了本发明第三方面所述的亲和肽稳定的金纳米颗粒在目标物检测方面的应用;
优选地,所述目标物包括金属离子、有机分子、寡核苷酸、蛋白质。
金纳米颗粒(Gold nanoparticles,AuNPs)有着十分显著的表面等离子体共振效应(SPRE),AuNPs的聚集会导致溶液颜色从红色变为蓝色,当聚集的AuNPs再分散时,溶液的颜色又会从蓝色变为红色。AuNPs的这种由于团聚状态和分散状态的改变而引起的颜色变化,可被用来直接或间接地检测待测目标物。通过在AuNPs表面组装上可特异性识别待测目标物的组分,然后通过特异性识别过程使AuNPs之间的距离减小或增大,造成AuNPs发生聚集与分散状态之间的转换,同时伴随着溶液颜色的变化,紫外-可见吸收光谱也会出现吸收峰的变化,进而实现对待测目标物的检测。
本发明第六方面提供了本发明第三方面所述的亲和肽稳定的金纳米颗粒在制备催化剂方面的应用。
金纳米催化剂是指以金纳米粒子为催化活性组分的催化剂,其催化反应活性和金纳米粒子的尺寸密切相关,当金纳米粒子尺寸小于5nm时,金纳米催化剂能够展现出优异的催化反应活性,目前,金纳米催化剂已经在一氧化碳氧化、丙烯环氧化、醇醛的选择性氧化等众多反应中展示出独特的催化反应性能,但是金纳米粒子在催化反应、低温催化反应过程中容易发生聚集,导致其稳定性较差,极大地限制了金纳米催化剂的应用。而本发明所述的亲和肽用作金纳米颗粒的稳定剂制备得到的亲和肽稳定的金纳米颗粒,不仅能解决其稳定性差的问题,而且相对于传统的稳定剂而言,细胞毒性低。
本发明第七方面提供了本发明第三方面所述的亲和肽稳定的金纳米颗粒在制备药物递送载体方面的应用。
药物递送载体是通过纳米颗粒与细胞的相互作用设计得到的靶向性的药物递送载体,靶向给药能够使药物在病变的部位聚集,避免了对正常组织的毒性。以金纳米颗粒为药物递送的载体,将药物包封在颗粒内部或修饰在颗粒表面,实现安全有效的靶向性药物治疗,其优点主要体现在:(1)药物装载到纳米颗粒内部或表面,可防止降解及灭活;(2)通过实体瘤高通透性、滞留效应(Enhanced permeability and retention effect,EPR)和特异性修饰增加药物靶向性;(3)降低毒副作用;(4)可量产。
本发明第八方面提供了本发明第三方面所述的亲和肽稳定的金纳米颗粒在制备生物传感器方面的应用。
金纳米颗粒的一个重要光学性质是表面等离子体共振吸收特性。其在分散态和聚集态具有不同的表面等离子体共振吸收而构建的比色传感体系,采用紫外吸收光谱法或目视法对靶标分子进行定性和定量分析,其检测原理是利用了金纳米颗粒表现出来的一种特性,即附着于高折射率玻璃表面的金膜能够吸收激光,并在金膜表面产生电子波(表面等离子),只有在特定入射角以及特定入射光线波长才有可能出现这种现象,并且这种现象高度依赖于金膜,将待分析物附着到金膜上的感受器就可以产生可测量的信号。
本发明的第九方面提供了本发明第三方面所述的亲和肽稳定的金纳米颗粒在制备成像造影剂方面的应用。
相对于人体组织,金纳米颗粒对X射线有较强的吸收,并且是传统的碘剂化合物的2.7倍,因而能够大大提高CT成像的对比度,可用于成像造影。金纳米颗粒造影增强效果一般与聚集在单位体积组织中金的总表面积成正比。由于粒径越小的金纳米颗粒,比表面积越高,从而粒子表面散射越强,将会有更好的CT造影增强效果。通过改变核心和壳体的相对大小,金纳米颗粒的壳体的光学吸收可以被调整到近红外光谱范围(690~900nm),在近红外区可以做强吸收剂或散射体,能够进行荧光成像和光热治疗。
除非另有定义,本发明上下文中的所使用的所有的技术和科学术语具有本领域普通技术人员所理解的相同含义。本发明的说明书中所使用的术语只是为了描述具体的实施例,不是旨在于限制本发明,此外,对部分术语解释如下。
本发明中使用的术语“特异性结合”,是指两个分子形成在生理条件下相对稳定的复合体。特异性结合的特点在于高亲和力和低至中容量。非特异性结合通常具有低亲和力和中至高容量。通常,当亲和常数K。高于106M-1或优选高于108M-1时,认为结合是特异性的。必要时,通过改变结合条件减少非特异性结合而基本上不影响特异性结合。上述条件为本领域所公知,且本领域技术人员使用常规技术就可选择合适的条件。所述条件通常用抗体浓度、溶液离子强度、温度、供结合时间、非相关分子(例如:血清白蛋白、乳酪蛋白)浓度等来限定。
本发明中使用的术语“肽”,是指两个或两个以上的氨基通过肽键(肽键(Peptidebond)是指一个氨基酸的羧基与另一个氨基酸的氨基缩合,除去一分子水形成的酰胺键)共价连接形成的聚合物。在本发明中术语“多肽”和术语“亲和肽”可互换使用,是指由多个氨基酸组成的肽。
本发明中使用的术语“生物传感器”,是指利用生物要素与物理化学检测要素组合在一起对被分析物进行检测的装置,包括三个部分:敏感的生物元件(生物材料如生物组织、微生物、细胞器、细胞感受器、酶、抗体、核酸等,生物派生材料或者类生物材料)、检测元件(用如光学、压电、电化学、温度或者电磁等物理化学方式工作)、连接二者的换能器。
本发明的优点和有益效果:
(1)本发明首次筛选出亲和肽M5,并将其应用于金纳米颗粒的稳定中,所述亲和肽M5修饰的金纳米颗粒具有较好的稳定性,可用作金纳米颗粒的稳定剂。
(2)相对于目前本领域常用的具有极强的细胞毒性的稳定剂CTAB而言,本发明提供的亲和肽M5用作稳定金纳米颗粒的稳定剂,细胞毒性非常低。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1显示的是筛选过程简要示意图、筛选得到的噬菌体蓝色斑点结果图和部分测序相关结果展示图;
图2显示的是M5多肽与靶蛋白Mpro结合解离动力学曲线图;
图3显示的是M5多肽修饰的金纳米颗粒和未经修饰的金纳米颗粒对BSA蛋白的响应评价的结果图,其中,A图:M5多肽修饰的金纳米颗粒,B图:未经修饰的金纳米颗粒。
具体实施方式
下面结合具体实施例,进一步阐述本发明,仅用于解释本发明,而不能理解为对本发明的限制。本领域的普通技术人员可以理解为:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1 SARS-CoV-2的病毒主蛋白酶Mpro的亲和肽M5的制备
1、实验材料
Ph.D.-12噬菌体展示肽库试剂盒购自New England Biolabs,Inc.。相互作用动力学分析和亲合力测定使用ForteBio Octet仪器。实验试剂和实验操作方法均按照产品的使用说明进行。
2、实验方法
通过噬菌体展示技术获得SARS-CoV-2的病毒主蛋白酶Mpro的亲和肽,使用M13噬菌体,pIII展示系统的多肽文库,该文库包括了109个不同的多肽序列。将“吸附-洗脱-扩增”的过程重复3-5轮,得到能够与靶标蛋白结合的噬菌体。提取噬菌体的DNA,完成测序,分析得到了能够与靶标蛋白结合的多肽序列,并进一步合成相关的高亲和肽序(具体筛选过程示意图见图1),详细实验步骤如下:
噬菌体展示实验
第一天
(1)用0.1M NaHCO3溶液(pH=8.6)制备得到10-100μg/mL Mpro蛋白分子溶液;
(2)在平皿中加入1.5mL的上述步骤(1)制备得到的溶液,并反复旋转直至表面完全湿润;
(3)在湿盒中,4℃温和振荡孵育过夜或4℃保存于湿盒中备用;
第二天
(4)将ER2738接种至10mL LB+Tet(四环素)的培养基中,该培养物将在步骤(11)中用于滴定,可在5-10小时内使用,如果要在同一天扩增洗脱的噬菌体(请参阅步骤(12)),还应在装有ER2738的25mL锥形瓶中接种20mL LB培养基,在37℃剧烈振摇下孵育两种培养物,孵育滴定培养直至达到所需要求;仔细监测20mL培养物,以使其不会超出对数早阶段(OD600=0.01-0.05),以用于步骤(12);
(5)封闭:将平皿反扣在洁净的纸巾上,去除包被液,加满封闭液(BSA)(封闭液配制:取7mL 0.1M NaHCO3溶液(pH=8.6),加入35mg的BSA,得到5mg/mL的BSA溶液,即为封闭液),4℃孵育至少1h。平皿中加入2mL的封闭液,4℃摇晃2h;
(6)封闭完成后,丢弃封闭液。用TBST(TBS+0.1%[v/v]Tween-20)将每块板快速洗涤6次,反复旋转,以确保孔底及孔侧面均被洗涤,洗涤要迅速,避免平皿干燥;
(7)用1mL TBST稀释1×1011噬菌体(10μL,100X稀释),移液到涂层板上,并在室温下轻轻摇动10-60min。
(8)将其正面朝下拍在干净的纸巾上,倒掉无结合的噬菌体;
(9)按照步骤(6)用TBST将板清洗10次,每次使用一块干净的纸巾,以防止交叉污染;
(10)洗脱已结合的噬菌体:1mL洗脱缓冲液(0.2M甘氨酸-盐酸(pH=2.2))洗脱结合的噬菌体,轻缓地摇动不超过10min,将洗脱液转入微量离心管中,用150μL 1M的Tris-HCl(pH=9.1)溶液中和;
(11)取少量洗脱液(20μL)测定滴度;
(12)通过将洗脱液添加到步骤(4)中的20mL ER2738培养物中来扩增其余的洗脱液,37℃剧烈振摇孵育4.5h;
(13)将培养物转移到离心管中,4℃,12000g离心10min后,将上清液转移至新的试管中,然后相同条件下再离心;
(14)将80%的上层清液转移至新管中,并向其中加入1/6体积的20%PEG/2.5MNaCl溶液,4℃条件下沉淀噬菌体,至少2小时或过夜;
第三天
(15)4℃14000rpm离心15min,弃掉上清液,短暂地重新旋转试管,并用移液管除去残留的上清液;
(16)用1mL TBS悬浮沉淀物并转入微量离心管中,4℃14000rpm离心5min使残留的细胞沉淀;
(17)将上清液转移至新的微量离心管中,并通过加入1/6体积的20%PEG/2.5MNaCl溶液进行沉淀,在冰上孵育15-60min,4℃,14000rpm离心10min,弃去上清液,再次离心,并用微量移液器除去残留的上清液;
(18)用200μL TBS悬浮沉淀物,微量离心1min,以沉淀任何残留的不溶物,将上清液转移到新管中,即为扩增的洗脱液;
(19)测量噬菌体的滴度;
(20)包被平皿进行第二轮筛选;
第四天和第五天
(21)在步骤(19)中,从滴定板中计数蓝色噬菌斑,并确定噬菌体滴度,滴度应在1013-1014pfu/mL左右,计算对应于1×1011-2×1011pfu的噬菌体体积,如果滴度太低,在筛选时可采用对应于109pfu的噬菌体体积;
(22)进行第二轮淘选:使用计算量的第一轮扩增洗脱液作为输入噬菌体,并将洗涤步骤中的Tween 20浓度提高到0.5%(v/v);
(23)测定第二轮筛选扩增洗脱液的滴度;
(24)包被平皿进行第三轮筛选;
第六天
(25)进行第三轮淘选:使用与第一轮(步骤(7))相同的输入滴度,使用第二轮扩增的洗脱液,重复步骤(4)-(10),在洗涤步骤中再次使用0.5%Tween;
(26)按照步骤(11)在LB/IPTG/Xgal板上滴定未扩增的第三轮洗脱液(无须扩增第三轮洗脱液,测定未扩增的第三轮洗脱液的滴度。如果不进行第四轮筛选则不必扩增第三轮的筛选洗脱物。滴度测定时的蓝色噬斑可用于测序:平板的孵育时间不得长于18h。将剩余的洗脱物保存于4℃的条件下,可至少保存一周。
噬菌体滴度测定
(1)5-10mL LB培养基中接种ER2738克隆,并摇动孵育4-8h(对数中期,OD600约为0.5);
(2)在细胞生长时,将顶层琼脂放入微波炉中融化,分装至无菌培养管中,每管3mL,在45℃的条件下保存备用;
(3)在37℃的条件下,按预期稀释度预热一块LB/IPTG/Xgal板至少1h,直至准备使用;
(4)在LB中制备10至103倍的噬菌体系列稀释液,最终体积为1mL;
(5)当步骤(1)中的培养物达到对数中期时,分装至微量离心管中,每管200μL;
(6)将10μL每种噬菌体稀释液添加至每个试管中,每管仅加入一种稀释液,迅速涡旋,室温条件下孵育1-5min;
(7)一次将一种感染的感染细胞转移至含有45℃顶层琼脂的培养管中,短暂涡旋;
(8)立即将步骤(7)的培养物倒入预热的LB/IPTG/Xgal板上,轻轻倾斜并旋转平板,以均匀铺展顶部琼脂;
(9)冷却平板5min,37℃倒置培养过夜;
(10)在大约有100个噬菌斑的板上计数噬菌斑,将每个数字乘以该板的稀释因子,以每10μL噬菌斑形成单位(pfu)的噬菌体滴度。
提取DNA测序
(1)LB培养ER2738过夜,1:100稀释,1mL/管,每管接种一个噬菌斑;
(2)枪头穿刺蓝色噬菌斑,至以上培养管中;
(3)37℃振摇孵育4.5-5h;
(4)将培养物转移至1.5mL的离心管中,14000rpm离心30s;
(5)吸取上清至新的离心管中,14000rpm离心30s;
(6)吸取80%的上清至新的离心管中,4℃的条件下保存;
(7)取500μL,加入200μL PEG/NaCl,混匀,室温下静置10-20min。
(8)4℃,14000rpm离心10min,弃上清,再次离心,弃上清;
(9)用100μL碘化物重悬,剧烈震荡,加入250μL的无水乙醇,室温下静置10-20min;
(10)4℃,14000rpm离心10min,弃上清,加入500μL 70%的乙醇,再次离心,4℃,14000rpm离心10min;
(11)弃上清,晒干;
(12)用30μL TE Buffer重悬后,-20℃的条件下冻存,送测序。
3、实验结果
通过噬菌体展示技术筛选得到的Mpro蛋白的亲和肽M5及其序列见表1。
表1通过噬菌体展示技术筛选得到的Mpro蛋白的亲和肽M5及其序列
实施例2亲和肽M5与靶标蛋白的亲和力和相互作用动力学检测
1、实验方法
通过生物膜干涉技术(Bio-layer Interferometry,BLI)检测亲和肽M5与靶标蛋白Mpro之间相互作用的亲和力大小,并研究多肽与靶标蛋白之间的结合和解离的动力学过程。将M5多肽在C端修饰Biotin,固定在SA修饰的传感器上,绘制其与不同浓度的Mpro相互作用时的结合和解离动力学曲线,通过结合和解离动力学曲线计算得到它们相互作用的解离平衡常数KD,详细实验步骤如下:
(1)将表面修饰有链霉亲和素(Streptavidin,SA)的生物传感器浸入PBS缓冲液中进行平衡,至少10min;
(2)将传感器浸入PBS缓冲液中得到基线;
(3)将传感器浸入已知浓度的固化溶液(M5多肽溶液)中,溶液中生物素化的多肽结合到生物传感器表面,使其表面膜层发生变化。该步骤使得多肽结合到SA生物传感器上。
(4)将固化后的传感器浸入PBS缓冲液中再做基线。
(5)将固化后的生物传感器浸入含有Mpro蛋白的样品溶液中,由于多肽与Mpro蛋白相互结合而导致膜层厚度的增加。此步骤中Mpro蛋白浓度为900nM、300nM、100nM。
(6)将已结合待测抗体的传感器浸入缓冲液中进行解离,Mpro蛋白从生物传感器表面脱落导致膜层厚度的降低。
(7)通过对实验过程中生物传感器生物膜层厚度的实时监控,拟合得到亲和肽M5与Mpro蛋白相互作用的动力学常数。
2、实验结果
M5多肽与靶蛋白Mpro结合解离动力学曲线见图2,亲和肽M5与靶蛋白相互作用的结合解离动力学和亲和力分析的结果表明,应用噬菌体展示技术筛选出的亲和肽M5与Mpro蛋白有较强的亲和力,KD为(7.24±0.40)E-10M。
实施例3亲和肽M5修饰的金纳米颗粒的制备及其稳定性的验证
1、实验方法
(1)金纳米颗粒的制备
将150mL柠檬酸钠溶液(2.2mM)加入到250mL圆底烧瓶中,置于90℃的水浴中,400rpm磁力搅拌,加入1mL氯金酸溶液(25mM),反应30min后再加入1mL 60mM的柠檬酸钠溶液和1mL 25mM的氯金酸溶液,反应30min后再加入1mL 60mM的柠檬酸钠溶液和1mL 25mM的氯金酸溶液,反应30min后冷却至室温,即为金纳米颗粒溶液;
(2)亲和肽M5修饰的金纳米颗粒的制备
在亲和肽M5的C端加一个半胱氨酸(KQEGAWVIIGLLC-NH2),通过Au-S键与金纳米颗粒结合,具体实验步骤如下:
在制备好的金纳米颗粒溶液中加入以上多肽,室温孵育30min后,16500rpm离心5min,弃上清,去除未结合的多肽分子,在所得的沉淀中加入600μL超纯水,超声重悬(超声过程要间隔两到三次);
(3)经亲和肽M5修饰的金纳米颗粒稳定性的验证
取上述亲和肽M5修饰的金纳米颗粒和未经修饰的金纳米颗粒各200μL,在所述溶液中分别加入不同浓度的BSA溶液,进行紫外可见近红外吸收光谱测量。
2、实验结果
实验结果显示,未经修饰的金纳米颗粒中加入BSA后,由于BSA破坏了其双电层结构,因此,SPR峰的位置发生了移动,表明未经修饰的金纳米颗粒的稳定性较差(见图3B),而经M5多肽修饰的金纳米颗粒在加入不同浓度的BSA后,SPR峰的位置不发生变化(见图3A),表明M5多肽具有稳定金纳米颗粒的作用,可用作金纳米颗粒的稳定剂。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
序列表
<110> 中国医学科学院基础医学研究所
<120> 一种亲和肽M5稳定的金纳米颗粒及其制备方法和用途
<141> 2021-05-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Lys Gln Glu Gly Ala Trp Val Ile Ile Gly Leu Leu
1 5 10
Claims (5)
1.一种亲和肽,其特征在于,所述亲和肽的序列如SEQ ID NO.1所示。
2.权利要求1所述的亲和肽在制备亲和肽稳定的金纳米颗粒中的应用。
3.一种亲和肽稳定的金纳米颗粒,其特征在于,所述亲和肽的序列如SEQ ID NO.1所示。
4.一种亲和肽稳定的金纳米颗粒的制备方法,其特征在于,所述方法包括如下步骤:
(1) 采用柠檬酸钠溶液、氯金酸溶液制备金纳米颗粒;
(2) 在权利要求1所述的亲和肽的C端加一个半胱氨酸,通过Au-S键与步骤(1)制得的金纳米颗粒结合,得到亲和肽稳定的金纳米颗粒。
5.根据权利要求4所述的方法,其特征在于,所述步骤(1)中柠檬酸钠溶液的浓度为2.2mM,氯金酸溶液的浓度为25 mM。
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