CN116042629A - 一种特异性识别ace2蛋白的适配体 - Google Patents
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Abstract
本发明涉及一种特异性识别ACE2蛋白的适配体,其核苷酸序列如SEQ ID NO.1所示序列。本发明得到的特异性识别ACE2蛋白的ssDNA适配体具有亲和力强、特异性高、稳定性高等优势,可竞争性结合ACE2蛋白,从而中和病毒,在抗病毒方面具有潜在的应用前景。
Description
本申请是申请号为:202110983737.X,申请日为:2021年8月25日,申请名称为:特异性识别ACE2蛋白的ssDNA适配体及其应用的分案申请。
技术领域
本发明涉及生物技术领域,尤其涉及一种特异性识别ACE2蛋白的适配体。
背景技术
SARS-CoV-2是一种单股正链RNA病毒,其外膜表面S蛋白呈蘑菇状,使病毒形如皇冠状。SARS-CoV-2感染宿主细胞过程中,其S蛋白受体结合结构域(RBD)识别并结合细胞表面血管紧张素转化酶2(ACE2)的肽酶结构域,介导病毒入侵人体细胞。S蛋白RBD与ACE2蛋白的相互作用是病毒传播的重要决定因素之一。因此,设计靶向S蛋白或ACE2蛋白的药物和抗体,或者设计另一种小分子来抑制S蛋白和ACE2蛋白的相互作用,有可能为新型冠状病毒药物的研制提供新线索。
目前,已有科研和医疗机构在研潜在的抗新型冠状病毒药物,包括一些中成药、活性天然产物和小分子抑制剂:(1)目前全球临床应用最多且获美国FDA批准的新型冠状病毒治疗药物瑞德西韦(Remdesivir)是一种核苷类似物,具有抗病毒活性。但其是用于抗埃博拉病毒感染的药物,并非新型冠状病毒特效药,虽然迄今不能认定瑞德西韦完全无用,但已有数据无法证明该药能显著提高患者的治疗效果,且瑞德西韦可能会产生严重副作用及其较高的成本和资源消耗,世界卫生组织并不推荐使用该药。(2)云南农大发现茶叶有效成分EGCG能够强力结合新型冠状病毒S蛋白,并阻断其与ACE2受体结合;第二军医大学通过将人ACE2的胞外域连接到人免疫球蛋白IgG1的Fc区,生成了重组的ACE2-Ig蛋白,可中和新型冠状病毒。但植物有效成分提取和重组蛋白制备,都存在操作繁琐、制备时间长及成本较高的问题。(3)哌加他尼(Macugen)是一种抗血管内皮生长因子的适配体,2004年12月经美国食品与药品监督管理局批准作为可用于治疗年龄相关的黄斑变性疾病的药物。已有核酸适配体在药物开发上的成功应用范例。但常规的核酸适配体筛选通常要进行十几轮,再进行克隆测序,不仅耗时耗力,且传统的测序通量很低,得到的适配体不一定是最佳的。
因此,目前仍旧没有临床疗效确切的抗病毒药物。核酸适配体能与靶标特异性结合,有望用于SARS-CoV-2的检测和治疗。
发明内容
为解决上述技术问题,本发明提供了一种特异性识别ACE2蛋白的适配体,具有特异性高、稳定性高、合成方便、易标记功能基团等特点,可特异性结合新型冠状病毒S蛋白的受体ACE2蛋白,阻断新型冠状病毒的结合感染,是新型冠状病毒特效药的有力备选。
本发明的第一个目的是提供一种特异性识别ACE2蛋白的适配体,其核苷酸序列为SEQ ID NO.1所示序列,具体序列为:
5’-TTAGCAAAGTAGCGTGCACTTTTGACCGCCCTACCCCCAGTGTCATCTAATCCCCCCACCCGACCATTCGGAAGTACCGTACCATTGC-3’。
进一步地,ssDNA适配体的3’端或5’端修饰有功能基团或分子。
进一步地,功能基团或分子为同位素、电化学标记物、酶标记物、荧光基团、生物素、亲和配基或巯基。功能基团或分子用于提高适配体的稳定性、提供检测信号,或者用于连接适配体与其他物质形成组合物。
本发明的第二个目的是提供上述ssDNA适配体在检测ACE2蛋白中的应用。
本发明的第三个目的是提供上述ssDNA适配体在分离富集ACE2蛋白中的应用。
本发明的第四个目的是提供一种检测ACE2蛋白的产品,所述产品含有上述适配体。本领域技术人员可知晓,检测产品的形式可为组合物、试纸、试剂盒、芯片、各类传感器等。
本发明的第四个目的是要求保护上述ssDNA适配体在制备抗病毒感染药物中的应用。
进一步地,上述药物用于预防或治疗病毒感染。
进一步地,上述药物为抗新型冠状病毒感染药物。
ACE2蛋白作为病毒受体,本发明提供的特异性识别ACE2蛋白的ssDNA适配体可与病毒竞争性结合ACE2受体,从而阻断病毒的结合感染,如ssDNA适配体可与新型冠状病毒S蛋白竞争性结合ACE2蛋白,实现抗新型冠状病毒治疗。
本发明的一种抗新型冠状病毒感染的药物,包括上述ssDNA适配体。
进一步地,上述药物还包括抗新型冠状病毒的物质。
进一步地,抗新型冠状病毒的物质为抑制新型冠状病毒增殖的物质或促进细胞抗新型冠状病毒作用的物质。
进一步地,抗新型冠状病毒的物质为天然植物提取物、动物提取物、重组蛋白、抗体、化学合成高分子或化学合成小分子。
借由上述方案,本发明至少具有以下优点:
本发明为新型冠状病毒的治疗提供了可在体外筛选、可高通量获得、筛选周期短、合成方便且稳定性良好、亲和力高、易修饰和标记的高特异性适配体序列,同时,本发明的适配体可单独使用或携带相关药物,对新型冠状病毒疾病的治疗具有潜在开发前景。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。
图1为人ACE2蛋白的纯化结果;
图2为第0、3、5、9、10、12轮筛选后核酸适配体的吸光图;
图3为ACE2 Aptamer A1的二级结构图谱;
图4为ACE2 Aptamer A2的二级结构图谱;
图5为Aptamer n.c.的二级结构图谱;
图6为N Aptamer 2的二级结构图谱;
图7为酶联核酸适配体方法测定核酸适配体与蛋白之间相互作用的示意图;
图8为ACE2 Aptamer A1/A2与ACE2蛋白相互作用的特异性结果;
图9为ACE2 Aptamer A2与ACE2蛋白相互作用的亲和力结果;
图10为ACE2适配体Aptamer A2与RBD-ACE2交互位点预测;
图11为ACE2特异性核酸适配体抑制SARS-CoV-2假病毒感染Vero细胞结果;
图12为ACE2 Apt-siRNA结构模式图(A)及其靶向ACE2阳性细胞干扰SARS-CoV-2病毒基因表达的模式图(B)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1
1、人ACE2蛋白的表达和纯化
(1)将构建好的带谷胱甘肽S转移酶(GST)标签的重组PET28a目的基因表达质粒转化至大肠杆菌BL21菌株中,用IPTG诱导表达GST-ACE2重组蛋白。
(2)采用谷胱甘肽琼脂糖凝胶偶联物(Glutathione Sepharose)进行GST标签蛋白纯化,通过SDS-PAGE和BCA测量纯化的蛋白质。结果如图1所示,纯化后的ACE2蛋白约为120kD。
2、筛选靶向人ACE2蛋白的单链DNA核酸适配体
使用指数富集配体系统进化技术(SELEX)及高通量测序技术,应用生物信息学分析,得到靶向人ACE2蛋白的单链DNA核酸适配体。具体步骤如下:
(1)构建随机ssDNA库
随机ssDNA库包含两个引物区域和一个45个碱基的随机区域:5′-GCAATGGTACGGTACTTCC-N45-CAAAAGTGCACGCTACTTTGCTAA-3′(N45代表45个核苷酸,在每个位置上等摩尔地掺入了A,G,C和T),使用5′-FAMTM标记的正向引物(5′-FAM-GCAATGGTACGGTACTTCC-3′)和5′-磷酸化的反向引物(5′-P-TTAGCAAAGTAGCGTGCACTTTTG-3′)PCR扩增随机DNA库,PCR反应条件为95℃5min,40循环(95℃1min,37℃30s,58℃40s),58℃5min。苯酚萃取和乙醇沉淀ssDNA。
(2)酶切随机ssDNA库
使用lambda外切酶切割纯化后的PCR产物,37℃反应30min后,75℃加热10min终止反应,苯酚萃取和乙醇沉淀ssDNA,得到足量的随机ssDNA库用于后续筛选实验。
(3)SELEX技术富集靶向ACE2蛋白的ssDNA核酸适配体
将随机ssDNA库90℃加热10min后,立即在冰上冷却10min,制备形成二级结构的ssDNA核酸适配体库。将ssDNA核酸适配体库与Ni-NTA磁珠在Binding buffer(50mM Tris-HCl pH 8.0,150mM NaCl,1.5mM MgCl2,2mM DTT和1%(w/v)BSA)中在室温下摇动预孵育30min,沉淀并丢弃非特异性DNA-磁珠复合物。然后将得到的上清液与含有合适浓度ACE2蛋白的Ni-NTA磁珠在结合缓冲液中室温孵育1h,用Elution buffer洗脱并收集与ACE2蛋白结合的ssDNA核酸适配体。用苯酚/氯仿/异戊醇处理洗脱的上清液,乙醇沉淀ssDNA核酸适配体。再次使用PCR扩增ssDNA核酸适配体后,纯化PCR产物,并用lambda外切酶酶切后,苯酚萃取和乙醇沉淀的ssDNA用于下一轮筛选。使用相同程序重复十次。每轮筛选所用的ACE2蛋白质浓度为:100μL结合缓冲液中的1μg(第1轮),0.5μg(第2-5轮),0.25μg(第6轮)和0.125μg(第7-12轮)。第十二轮之后,通过PCR扩增ssDNA,对PCR产物测序,分析测序数据寻找ssDNA序列,测序文件里面提取ssDNA的序列,然后计数,按照counts数量对其排序,从而获得丰度最高的适配体ACE2 Aptamer A1,ACE2Aptamer A2。单链DNA适配体采用常规单链DNA化学合成的方法合成,5’端加生物素标记,使用RNAfold软件预测靶向ACE2蛋白的ssDNA核酸适配体最稳定的二级结构。图2显示了SELEX筛选靶向ACE2蛋白的ssDNA适配体库的富集情况,如图所示,在第12轮时结合的蛋白明显增多。
(1)ACE2 Aptamer A1的核苷酸序列为:
5’-bio-TTAGCAAAGTAGCGTGCACTTTTGTACCACCTTCCCCCGAACAACGTTTTCCCCCCAACCCAACCCCTAGGAAGTACCGTACCATTGC-3’
预处理:在90℃下热变性10分钟,并立即在冰上复性,形成如图3所示的二级结构。
(2)ACE2 Aptamer A2的核苷酸序列(SEQ ID NO.1)为:
5’-bio-TTAGCAAAGTAGCGTGCACTTTTGACCGCCCTACCCCCAGTGTCATCTAATCCCCCCACCCGACCATTCGGAAGTACCGTACCATTGC-3’
预处理步骤同(1),形成如图4所示的二级结构。
(3)为了证明核酸适配体的特异性,我们合成了一个阴性对照适配体(Aptamern.c.),核苷酸序列为:
5’-bio-GCAATGGTACGGTACTTCCGGATGCGGAAACTG-3’
预处理步骤同(1),形成如图5所示的二级结构。
(4)使用新型冠状病毒N蛋白的适配体N Aptamer 2(发明专利202010256645.7中的N Aptamer 2序列)为阳性对照,核苷酸序列为:
5’-bio-GCAATGGTACGGTACTTCCGGATGCGGAAACTGGCTAATTGGTGAGGCTGGGGCGGTCGTGCAGCAAAAGTGCACGCT-3’
预处理步骤同(1),形成如图6所示的二级结构。
3、通过ELAA检测ACE2 Aptamer A1/A2与ACE2蛋白相互作用的特异性
采用酶联核酸适配体方法(ELAA)验证ACE2 Aptamer A1和ACE2 Aptamer A2可以特异结合人ACE2蛋白。示意图如图7所示,检测步骤如下:
使用1ug人ACE2蛋白,包被空白ELISA酶标板,室温1小时(或者4度过夜),PBST洗涤3遍,1% BSA室温封闭1小时;100nM Biotin-ssDNA适配体(ACE2 Aptamer A1),90℃10分钟,立即放冰上;每孔加入100μL ssDNA适配体室温1h,轻轻震荡,PBST洗涤3遍,加入辣根过氧化物酶标记的链霉亲和素(avidin-HRP,1:1000),室温1h,轻轻震荡,PBST洗涤3遍,加入显色底物TMB 100μL,室温15min避光反应;加入100μL 2M的浓硫酸终止;OD450检测吸光度。
将ACE2 Aptamer A1替换为ACE2 Aptamer A2或Aptamer n.c.,其余步骤同上。
将人ACE2蛋白替换为SARS-CoV-2N蛋白,ACE2 Aptamer A1替换为N Aptamer 2,其余步骤同上。
结果如图8所示,其中,Aptamer n.c.为阴性对照,N Aptamer 2为阳性对照;柱状图代表三次独立实验的均值±标准差;统计学分析通过one-way ANOVA完成;****表示P<0.0001。结果表明,单链DNA适配体ACE2 Aptamer A1/A2都能够特异性的与ACE2蛋白结合,其中ACE2 Aptamer A2结合效果更好。阳性对照适配体N Aptamer 2与SARS-CoV-2N蛋白特异性结合,阴性对照适配体Aptamer n.c.与所有测试蛋白均无特异性结合,也进一步证明了实验结果可靠,ACE2 Aptamer A1/A2与ACE2蛋白的结合是特异性的。
4、通过ELAA检测ACE2 Aptamer A2与ACE2蛋白相互作用的亲和力
参照上述3、中ELAA检测步骤,将ACE2蛋白与倍比浓度增加的5'-生物素化的ACE2Aptamer A2或Aptamer n.c.一起孵育,添加avidin-HRP二抗后,计算ACE2蛋白-适配体复合物的量,并绘制以X为适配体浓度、Y为吸光度值的曲线,拟合Michaelis-Menten方程,计算出平衡解离常数(Kd)。
结果如图9。ACE2 Aptamer A2的Kd为(5.41±1.23)nM;阴性对照Aptamer n.c.的Kd为(0.27±0.13)nM,可见ACE2 Aptamer A2与靶标ACE2蛋白亲和力强。
本发明的筛选方法省去了构建克隆、转化提质粒鉴定过程,直接PCR扩增ssDNA分析排序,寻找最佳适配体,测序通量高。
实施例2
1、ACE2适配体Aptamer A2交互位点与RBD-ACE2交互位点存在重合
为了阐明上述筛选到的ACE2适配体抑制SARS-CoV-2假病毒感染的分子机制,我们使用UNAFOLD(Nucleic Acids Res 2003,31(13):3406-3415)预测了ACE2 Aptamer A2的二级结构,3d RNA(Int J Mol Sci 2019,20(17):4116)预测其三级结构。将ACE2蛋白(PDBID6M0J:A)作为受体,3d RNA构建好三级结构的ACE2 Aptamer A2作为配体输入HDOCK(NatProtoc 2020,15(5):1829-1852)进行分子对接,预测ACE2与ACE2 Aptamer A2的三维结构。与RCSB PDB数据库(http://www.rcsb.org)上SARS-CoV-2RBD与ACE2蛋白结构(PDBID6M0J)相比较,发现ACE2 Aptamer A2与ACE2的交互位点与RBD-ACE2交互位点存在重合(见图10,其中,A为ACE2与SARS-CoV-2RBD的三维结构(PDBID 6M0J),B为HDOCK预测的ACE2与ACE2 Aptamer A2的三维结构)。我们推测ACE2 Aptamer A2可能通过阻断ACE2上的RBD交互界面,阻断RBD结合ACE2从而抑制了SARS-CoV-2假病毒入侵。
2、检测ACE2特异性核酸适配体对SARS-CoV-2假病毒感染的抑制作用
为了探讨ACE2特异性核酸适配体ACE2 Aptamer A2对SARS-CoV-2S蛋白假病毒感染的影响,我们使用对照或ACE2的适配体处理Vero细胞1h,感染SARS-CoV-2假病毒(MOI=2)24h后,荧光显微镜观察到ACE2 Aptamer A2处理的Vero细胞的绿色荧光较弱(图11为感染SARS-CoV-2S蛋白假病毒24h的Vero细胞的代表性荧光图片)。从图11中可知,相比对照组,ACE2适配体ACE2 Aptamer A2显著抑制了SARS-CoV-2假病毒感染Vero细胞。
3、ACE2 Apt-siRNA的设计和合成
我们还为ACE适配体偶联小核酸药物的用于抑制新型冠状病毒基因表达的应用提供了解决方案,如下图3。
ACE2 Apt-siRNA(图3),具备如下特性:1)其Aptamer实现靶向ACE2阳性细胞;2)Aptamer与ACE2的结合位点在ACE2酶活性位点、RBD-ACE2交互位点之外,当SARS-CoV-2RBD结合ACE2介导细胞内吞,可确保ACE2Apt-siRNA进入靶细胞,在Dicer酶作用下形成RNA诱导的沉默复合物(RISC),实现RNA干扰(RNAi)。我们预测,ACE2 Apt-siRNA可以靶向ACE2阳性细胞并干扰这类细胞SARS-CoV-2变异株的基因表达(图3)。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种特异性识别ACE2蛋白的适配体,其特征在于:所述适配体的核苷酸序列如SEQID NO.1所示。
2.根据权利要求1所述的适配体,其特征在于:所述适配体的3′端或5′端修饰有功能基团或分子。
3.根据权利要求2所述的适配体,其特征在于:所述功能基团或分子为同位素、电化学标记物、酶标记物、荧光基团、生物素、亲和配基或巯基。
4.权利要求1-3任一项所述的适配体在制备检测ACE2蛋白产品中的应用。
5.权利要求1-3任一项所述的适配体在制备分离富集ACE2蛋白产品中的应用。
6.一种检测ACE2蛋白的产品,其特征在于:含有权利要求1-3任一项所述的适配体。
7.权利要求1-3任一项所述的适配体在制备抗病毒感染药物中的应用。
8.根据权利要求7所述的应用,其特征在于:所述抗病毒感染药物为抗新型冠状病毒感染药物。
9.一种抗病毒感染药物,其特征在于:含有权利要求1-3任一项所述的适配体。
10.根据权利要求9所述的抗病毒感染药物,其特征在于:所述抗病毒感染药物为抗新型冠状病毒感染药物。
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