CN113041357B - 一种针对新型冠状病毒的核酸适体纳米颗粒及其制备方法和应用 - Google Patents

一种针对新型冠状病毒的核酸适体纳米颗粒及其制备方法和应用 Download PDF

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CN113041357B
CN113041357B CN202110190184.2A CN202110190184A CN113041357B CN 113041357 B CN113041357 B CN 113041357B CN 202110190184 A CN202110190184 A CN 202110190184A CN 113041357 B CN113041357 B CN 113041357B
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杨朝勇
宋彦龄
孙淼
宋婷
卢瑶
黄佳傲
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Abstract

本发明公开了一种针对新型冠状病毒的核酸适体纳米颗粒及其制备方法和应用，其中，制备方法包括如下步骤：1)将一种或多种新型冠状病毒的核酸适体末端分别接poly A或T尾，并将核酸适体和对应的Poly T或A混合，形成序列末端稳定的核酸适体的混合物；2)将序列末端稳定的核酸适体混合物偶联到纳米颗粒表面，核酸适体纳米颗粒。

Description

一种针对新型冠状病毒的核酸适体纳米颗粒及其制备方法和 应用
技术领域
本发明涉及一种针对新型冠状病毒的核酸适体纳米颗粒及其制备方法和应用。
背景技术
2019新型冠状病毒(SARS-CoV-2或2019nCoV)是在人体中发现的冠状病毒新毒株，是目前已知的第7种可以感染人的冠状病毒。人感染了新冠病毒后常见体征有发热、咳嗽、气促和呼吸困难等，在较严重的病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。不仅如此，新冠病毒还具有广泛的传染能力。据美国约翰斯·霍普金斯大学发布的疫情统计显示，自2020年1月新冠疫情爆发以来，截止到2020年11月底，全球累计新冠确诊病例超过六千万，全球平均每日新增确诊病例二十万；每18秒就有1人因新冠病毒死亡，累计死亡人数已达到142万。
新型冠状病毒的感染是通过病毒表面的刺突糖蛋白(S蛋白)与细胞表面受体血管紧张素转化酶2(ACE2)的结合而启动的，其中S蛋白介导了受体结合与膜融合过程。S蛋白是三聚体，由两个功能性亚基S1和S2组成，其中S1亚基包含有负责与ACE2结合的受体结合域(RBD)，而S2亚基则负责细胞膜融合过程。S蛋白既有封闭构象也有开放构象，以混合结构共存。当ACE2结合一个开放位点后，S蛋白构象就会变得开放，并开始一系列有利于与受体细胞结合的构象变化。一旦S蛋白在所有三个结合位点上与ACE2结合，它的中央核心S2就会暴露出来，从而SARS-CoV-2与细胞膜融合。
由于冠状病毒广泛的宿主性以及自身基因组的结构特征，使其在进化过程中很容易发生突变。目前已知的新冠病毒突变中，D614G突变—S1的第614位天冬氨酸(D)突变成甘氨酸(G)占比74％，已成为主流的突变病毒毒株。与未突变的病毒相比，突变后的S蛋白开放构象大大增加，与ACE2结合的概率显著提高，从而表现出更强地感染人体细胞的能力。有研究表明，与未突变的病毒相比，其感染能力约为未突变病毒的九倍。
现在对于新型冠状病毒的感染没有特异性的治疗方法，大多根据患者的临床情况进行诊疗。因此，针对新冠病毒适体抑制剂的研发刻不容缓。
发明内容
本发明的主要目的，在于提供一种针对新型冠状病毒的核酸适体纳米颗粒的制备方法。
本发明解决其技术问题的所采用的技术方案是：
一种针对新型冠状病毒的核酸适体纳米颗粒的制备方法，包括如下步骤：
1)将一种或至少两种新型冠状病毒的核酸适体末端分别接poly A或T尾形成核酸适体’，并将核酸适体’和对应的Poly T或A混合，形成序列末端稳定的核酸适体的混合物；
2)将序列末端稳定的核酸适体混合物偶联到纳米颗粒表面，获得核酸适体纳米颗粒。
在本发明优选实施例中，所述的纳米颗粒包括金属纳米颗粒、脂质体纳米颗粒、细胞外囊泡纳米颗粒、外泌体纳米颗粒中的至少一种。
在本发明优选实施例中，所述的纳米颗粒的粒径为2-500nm。优选为3-50nm。
在本发明优选实施例中，核酸适体种类为三种。
在本发明优选实施例中，核酸适体的序列分别为CoV2-1C、CoV2-4C和CoV2-6C3(见表1)。
在本发明优选实施例中，还包括CoV2-1C、CoV2-4C、CoV2-6C、CoV2-1、CoV2-C、CoV2-6C3序列的突变体或截短序列。
在本发明优选实施例中，所述的突变体CoV2-1C、CoV2-4C、CoV2-6C、CoV2-1、CoV2-C和CoV2-6C3所示序列具有80％以上同源性的序列。
本发明还提供所述的制备方法制备得到的核酸适体纳米颗粒在制备新冠病毒SARS-CoV-2感染的检测试剂、预防形病毒感染药物或治疗药物、或中和环境中的新冠病毒中的用途。
本发明还提供针对新型冠状病毒的核酸适体纳米颗粒，其中，所述的核酸适体纳米颗粒包括纳米颗粒，以及偶联在纳米颗粒表面的一种或至少两种核酸适体，所述的核酸适体具有与S蛋白或受体结合域的结合表位。
本所述的球形核酸适体在制备预防和/或治疗新冠病毒SARS-CoV-2感染药物中的用途。
本技术方案与背景技术相比，具有如下优点：
本发明采用纳米颗粒，将一种或至少两条不同结合表位的核酸适体与纳米颗粒偶联，可以应对该纳米粒子圆周范围内所有的受体结合域突变带来的构象变化。因此，相较于原来的以核酸适体结构去阻断局域内ACE2-受体结合域的结合，本发明构建的核酸适体纳米颗粒能够应对由于不同的突变带来的S蛋白三聚体的不同构象变化，能够成为紧急应对可能发生的新冠病毒突变株的快速、有效且通用方法。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为验证三条体结合是否相互影响。
图2为核酸适体金纳米颗粒形貌、RBD亲和力及稳定性表征。
图3为核酸适体金纳米颗粒抗假病毒侵染能力表征。
图4为核酸适体金纳米颗粒D614G突变加病毒侵染能力表征。
具体实施方式
为了更好地解释本发明，以下结合具体实施例进一步阐明本发明的主要内容，但本发明的内容不仅仅局限于以下实施例。
1)核酸适体预处理：所用的CoV2-1C、CoV2-4C和CoV2-6C3组成为5’端修饰有巯基，在此之后加有11个T，中间为特定核酸适体序列，3’端在测定亲和力时带有FAM标签(所用序列列于表1中)。将3.92nmole CoV2-1C溶于39μL无菌无酶水中；3.00nmole CoV2-4C溶于30μL无菌无酶水中；4.10nmole CoV2-6C溶于41μL无菌无酶水中，配置成为100μM储备液。并配置TM缓冲溶液(20mM Tris,50mM MgCl2,pH 8.0)用于溶解11.80nmole的11个A的序列PolyA-11(100μM)。
2)核酸适体与纳米金偶联：将100μM的CoV2-1C、CoV2-4C和CoV2-6C3各3μL与9μL100μM的PolyA-11混合形成核酸适体’。并将此核酸适体’与PolyA-11摩尔比为1:1的混合物在95℃杂交10min，使11个A的PolyA-11与核酸适体'5’末端的11个T杂合，形成序列末端稳定CoV2-1C、CoV2-4C和CoV2-6C的混合物。将恢复至室温的CoV2-1C、CoV2-4C和CoV2-6C混合物投入5nm的AuNPs(2.5nM,400μL),置于-20℃冰箱2小时，使CoV2-1C、CoV2-4C和CoV2-6C混合物中的巯基与AuNPs表面的Au充分反应，以此制备出所需的球形核酸适体。
3)纯化处理：在室温下解冻后，将偶联有核酸适体的AuNPs转移至100kDa的超滤管中，8000g离心20min，去除未连接在AuNPs表面的多余核酸适体。将得到的红色沉淀用PBST(含0.1％Tween的PBS)溶解，并置于4℃保存、备用。
4)CoV2-1C、CoV2-4C和CoV2-6C3在RBD上结合表位竞争验证：首先，验证所用的核酸适体CoV2-1C、CoV2-4C和CoV2-6C3能够结合RBD，并且结合表位不存在位点竞争。把5’端标记FAM的CoV2-1C当作靶适体，向其中分别加入等摩尔量的没有荧光标记的CoV2-4C和CoV2-6C3，以此类推，在5’端标记FAM的CoV2-4C中分别加入等摩尔量的没有荧光标记的CoV2-1C和CoV2-6C3；在5’端标记FAM的CoV2-6C3中分别加入等摩尔量的没有荧光标记的CoV2-1C和CoV2-4C，观察加入的其他两个核酸适体序列对所选靶序列结合RBD能力的影响。如图1A所示，序列之间的结合竞争<25％，几乎不存在3种不同核酸适体序列之间的两两位点竞争。
5)CoV2-1C、CoV2-4C和CoV2-6C3混合物抑制RBD-ACE2结合能力表征：不同核酸适体序列抑制RBD-ACE2结合的能力不同，CoV2-6C3>CoV2-4C>CoV2-1C。将CoV2-1C、CoV2-4C和CoV2-6C混合后，抑制能力较抑制效果最好的CoV2-6C3相比还有所提高(图1B)。
6)球形核酸适体性能表征：通过透射电子显微镜(TEM)及动态光散射(DLS)表征偶联核酸适体前后AuNPs粒度，偶联核酸适体后的AuNPs粒径变大，表明偶联在AuNPs表面的核酸适体增大了原有AuNPs的粒度(图2A-D)。并且，较游离的CoV2-1C、CoV2-4C和CoV2-6C3，球形核酸适体提高了对RBD的结合能力，结合亲和力降低至5.46±0.72pM(图2E-F)。球形核酸适体较游离的CoV2-1C、CoV2-4C和CoV2-6C3相比，抗酶切稳定性也大幅度地提高(图2G-H)。
球形核酸适体在假病毒水平的抑制能力表征：将核酸适体偶联到AuNPs形成球形核酸适体，可有效提高游离序列的中和效率。甚至，只具备结合RBD能力，而不能有效抑制RBD-ACE2结合的序列(CoV2-1C)，在偶联到AuNPs上后，也可实现其抑制能力体现(1C-AuNPs)。较其他游离核酸适体及单独核酸适体偶联的AuNPs相比，3种不同序列混合偶联的AuNPs(Cocktailed AuNPs)中和假病毒侵染ACE2过表达的293T细胞的中和率高达96.16±0.92％，其IC50低至207.70fM(图3A-B)。除此之外,利用冷冻电镜直观地表征了CocktailedAuNPs结合在了假病毒的刺突上(图3C)。
8)球形核酸适体对新型冠状病毒突变株D614G的结合和抑制性能表征:对于最近席卷全球的新型冠状病毒突变株D614G，CoV2-1C、CoV2-4C和CoV2-6C仍能很好的结合D614G型的S糖蛋白(图4A)。并且，Cocktailed AuNPs对于突变株D614G假病毒的中和率高达94.52±0.97％，优于商品化的中和抗体(图4B)
表1
Figure GDA0003757795120000061
由于S蛋白是新冠病毒打开人类细胞之门的“钥匙”，因此，本发明以S蛋白上受体结合域(RBD)为靶标，经体外筛选技术SELEX(指数富集配体系统进化)，得到了有效抑制新冠病毒的三条核酸适体。靶向RBD的核酸适体具有结合能力强、作用效率高、生成成本低、批间差异小、稳定且易保存等优点，是阻止新冠病毒侵染人体细胞中和试剂研发的一个突破口。
本发明引入了纳米金颗粒(AuNPs)作为构建载体，以弥补核酸适体位阻小的缺点，增强核酸适体与RBD间的相互作用，提高核酸适体作为中和试剂的抑制效率。将AuNPs与三条不同RBD结合表位的核酸适体偶联形成球形核酸适体，此球形核酸适体不仅在尺寸上能够同时占据S蛋白开放构象的三个RBD位点，而且AuNPs本身也能覆盖暴露的S2蛋白中央核心部分，成功阻止S蛋白和ACE2结合。球形核酸适体显著提高了核酸适体阻碍新冠病毒入侵正常人体细胞的能力，并且能够有效应对D614G变异株病毒。
以上所述，仅为本发明较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
序列表
<110> 厦门大学
<120> 一种针对新型冠状病毒的核酸适体纳米颗粒及其制备方法和应用
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<213> 人工序列(Artificial Sequence)
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tttttttttt tatccagagt gacgcagcat ttcatcgggt ccaaaagggg ctgctcggga 60
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Claims (6)

1.一种针对新型冠状病毒的核酸适体纳米颗粒的制备方法，包括如下步骤：
1)将一种或多种新型冠状病毒的核酸适体末端分别接Poly A或T尾形成核酸适体’，并将核酸适体’和对应的Poly T或A混合，形成序列末端稳定的核酸适体的混合物；所述的核酸适体’的序列为CoV2-6C3，或为CoV2-1C、CoV2-4C以及CoV2-6C3的组合；其序列分别如下
CoV2-6C3：SH C6-TTTTTTTTTTTCGCAGCACCCAAGAACAAGGACTGCTTAGGATTGCGATAGGTTCGG-(FAM)；
CoV2-1C：SH C6-TTTTTTTTTTTCAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGA CA-(FAM)
CoV2-4C：SH C6-TTTTTTTTTTTATCCAGAGTGACGCAGCATTTCATCGGGTCCAAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT-(FAM)；
2)将序列末端稳定的核酸适体混合物偶联到金属纳米颗粒表面，获得核酸适体纳米颗粒。
2.根据权利要求1所述的一种针对新型冠状病毒的核酸适体纳米颗粒的制备方法，其特征在于：所述的纳米颗粒的粒径为3-50nm。
3.根据权利要求1所述的一种针对新型冠状病毒的核酸适体纳米颗粒的制备方法，其特征在于：核酸适体种类为一种或多种。
4.根据权利要求1-3任一项所述的制备方法制备得到的核酸适体纳米颗粒在制备感染新冠病毒SARS-CoV-2的检测试剂、预防新冠病毒SARS-CoV-2病毒感染药物或治疗药物、或中和环境中的新冠病毒中的用途。
5.针对新型冠状病毒的核酸适体纳米颗粒，其中，所述的核酸适体纳米颗粒包括纳米颗粒，以及偶联在金属纳米颗粒表面的一种或多种核酸适体，所述的核酸适体具有与S蛋白或受体结合域的结合表位，所述核酸适体末端分别接Poly A或T尾形成核酸适体’，并将核酸适体’和对应的Poly T或A混合，形成序列末端稳定的核酸适体的混合物，所述的核酸适体’的序列为CoV2-6C3，或为CoV2-1C、CoV2-4C以及CoV2-6C3的组合；其中，序列分别如下：
CoV2-6C3：SH C6-TTTTTTTTTTTCGCAGCACCCAAGAACAAGGACTGCTTAGGATTGCGATAGGTTCGG-(FAM)；
CoV2-1C：SH C6-TTTTTTTTTTTCAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGA CA-(FAM)
CoV2-4C：SH C6-TTTTTTTTTTTATCCAGAGTGACGCAGCATTTCATCGGGTCCAAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT-(FAM)。
6.根据权利要求5所述的核酸适体纳米颗粒在制备预防和/或治疗新冠病毒SARS-CoV-2感染药物中的用途。
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