CN116240214A - 一种靶向心衰标志物的核酸适配体及其应用 - Google Patents
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Abstract
本发明公开了一种靶向心衰标志物的核酸适配体及其应用,通过SELEX筛选和亲和性检测筛选获得专一靶向GDF15蛋白的核酸适配体,并经过结构预测和剪切修饰,优化适配体结构,并通过酶联亲和性检测,验证特定的剪切修饰适配体GDF15‑7‑+11A、GDF15‑7‑+11T以及GDF15‑7‑截短2等与靶标具有更好的亲和性和特异性。
Description
技术领域
本发明属于生物检测领域,具体是一种靶向心衰标志物的核酸适配体及其应用。
背景技术
生长分化因子-15(Growth/differentiation factor 15,GDF15)是转化生长因子-β超家族(TGFβsuper family)的一员。目前,GDF15的应用主要集中在心力衰竭的病情评估和心梗、脑梗等疾病的预后,例如,US20100167331A1中公开了通过检测GDF15以诊断患有稳定型冠心病的受试者经皮心脏介入治疗(PCI)是否成功;US7955854B2中公开了通过检测GDF15以诊断表现出心房颤动的受试者是否为心力衰竭;CN102652261B中公开了通过检测GDF15以预测心脏手术患者中的肾衰竭风险;CN106257287B中公开了生长分化因子15评估高血压患者首发脑卒中的新应用。
因此方便、快速的检测体内GDF15表达量,以对心衰等相关疾病进行预测或风险评估是迫切需要解决的问题。基于此,本发明提供靶向针对血液样本中GDF15的核酸适配体,以期能够实现GDF15的快速检测。
发明内容
基于此,本发明提出了一种靶向GDF15的核酸适配体。
本发明提出的一种特异性结合GDF15蛋白的核酸适配体,所述核酸适配体的核苷酸序列包括以下序列中的至少一种:
(1)以下一个或多个序列:
GDF15-3:5’-TCATCAACTTCGGACCGCACCCAGGAGCAAAGCATACATTGC-3’,SEQ ID NO:1;
GDF15-7:5’-TTAAAATCGGAACCTCATGGCCAACAGGAACGTTACATTGC-3’,SEQ ID NO:2;
GDF15-8:5’-TTAAATCGCATGGCCAGGAACTTTCGGCATCGCAATTACATGC-3’ ,SEQ ID NO:3;
GDF15-12: 5’-TTAACTCATGGCCATCTAACTTTCTATCCAGATCATTTCCATGC-3’ ,SEQ IDNO:4;
GDF15-14: 5’-TAAACCGGCCAACTCAGCAACCGCAACAACCAAAGCATGC-3’ ,SEQ ID NO:5。
GDF15-7-截短1:5’-AATCGGAACCTCATGGCCAACAGGAACGTTACA-3’ ,SEQ ID NO:6;
GDF15-7-截短2:5’-AAATCGGAACCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:7;
GDF15-7-截短3:5’-ATCGGAACCTCATGGCCAACAGGAACGTTAC-3’,SEQ ID NO:8。
GDF15-7-D28C:5’-AAATCGGAACCTCATGGCCAACAGGAAGTTACAT-3’,SEQ ID NO:9;
GDF15-7-D27A:5’-AAATCGGAACCTCATGGCCAACAGGACGTTACAT-3’,SEQ ID NO:10;
GDF15-7-+11A:5’-AAATCGGAACACTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:11;
GDF15-7-+11T:5’-AAATCGGAACTCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:12;
GDF15-7-+11C:5’-AAATCGGAACCCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:13。
(2)与SEQ ID NO:1-13中任意一个所示的DNA序列具有60%以上同源性且特异性结合GDF15蛋白的DNA序列,优选可以是70%以上、80%以上、85%以上、90%以上、92%以上、94%以上、96%以上、98%以上、99%以上;
(3)由SEQ ID NO:1-13中任意一个所示的DNA序列转录且特异性结合GDF15蛋白的RNA序列。
另外,本领域技术人员应该理解,作为对上述技术方案的改进,可对上述核酸适配体的核苷酸序列上的某一位置进行修饰,例如,磷酸化、甲基化、氨基化、巯基化、用硫取代氧、用硒取代氧或连接同位素化等,条件是这样修饰后得到的核酸适配体序列具有合乎需要的性质,例如,可以具有与修饰前的母体核酸适配体序列相等或更高的结合高表达GDF15蛋白的亲和力,或虽然亲和力没有明显提升但是具有更高的稳定性。
在另一方面,本发明还提供核酸适配体的缀合物。本领域技术人员应该理解,作为对上述技术方案的改进,可在上述核酸适配体的核苷酸序列上连接荧光标记物,例如放射性物质、治疗性物质、生物素、地高辛、纳米发光材料、小肽、siRNA或酶标记等,条件是这样修饰后得到的核酸适配体序列具有合乎需要的性质,例如,可以具有与修饰前的母体核酸适配体序列相等或更高的结合高表达GDF15蛋白的亲和力,或虽然亲和力没有明显提升但是具有更高的稳定性。
换言之,以上不论是经部分取代或经过修饰后的核酸适配体序列,都具有原核酸适配体基本相同或类似的分子结构、理化性质和功能,都可应用于与高表达GDF15蛋白的结合。
作为一个总的发明构思,本发明还提供了一种核酸适配体的衍生物,其是将所述核酸适配体或者所述核酸适配体的缀合物的核苷酸序列的骨架改造成硫代磷酸酯骨架而得,或者是将所述核酸适配体或者所述核酸适配体的缀合物改造成的肽核酸,条件是所述衍生物都具有与原核酸适配体基本相同或类似的分子结构、理化性质和功能,且都与高表达GDF15蛋白结合。
本发明所使用的术语“硫代磷酸酯骨架”具有本领域普通技术人员一般理解的含义,其是指RNA和DNA核酸适配体的磷酸二酯骨架的非桥键氧原子可以被一个或两个硫原子取代,分别产生具有硫代磷酸酯或二硫代磷酸酯键的硫代磷酸酯骨架。已知这样的硫代磷酸酯骨架具有增加的对其靶标的结合亲和力,以及对核酸酶降解的增强的抗性。
做为一个总的发明构思,本发明还提供了所述的核酸适配体或所述的核酸适配体的缀合物或所述的核酸适配体的衍生物在由以下各项组成的组中的任意一项中的用途:
(1)纯化GDF15蛋白或检测GDF15蛋白;
(2)表达GDF15的细胞、组织或活体定位成像;
(3)捕获表达GDF15的细胞或外泌体。
作为一个总的发明构思,本发明还提供了所述的核酸适配体或所述的核酸适配体的缀合物或所述的核酸适配体的衍生物在制备用于靶向肿瘤的药物中的用途。
在一个实施方案中,本发明的核酸适配体、其缀合物或其衍生物,优选本发明的核酸适配体可以用于GDF15蛋白纯化或检测,检测受试者肿瘤组织中GDF15的表达量。
作为一个总的发明构思,本发明还提供了一种试剂盒,包含所述的核酸适配体或所述的核酸适配体的缀合物或所述的核酸适配体的衍生物;优选地,所述试剂盒包含SEQIDNO:1-13所示的任意一个或两个核酸适配体、其缀合物或其衍生物;更优选地,所述试剂盒包含SEQID NO:1-13所示的任意一个或两个核酸适配体。
作为一个总的发明构思,本发明还提供了所述的核酸适配体或所述的核酸适配体的缀合物或所述的核酸适配体的衍生物在制备试剂盒中的应用,其中所述试剂盒用于选自由以下各项组成的组中的任意一项:
(1)纯化GDF15蛋白或检测GDF15蛋白;
(2)表达GDF15的细胞、组织或活体定位成像;
(3)捕获表达GDF15的细胞或外泌体。
本发明使用体外指数富集的配基系统进化(SELEX)技术,筛选得到了特异性结合高表达GDF15蛋白的核酸适配体。具体的,本发明人设计并合成了一个随机单链DNA文库和相应的引物,用来筛选具有高度特异性、分子量小、化学性质稳定、易于保存和标记的能够结合高表达GDF15蛋白的核酸适配体,由此筛选得到一些特异性结合GDF15蛋白的核酸适配体,并检测了它们与GDF15蛋白的结合能力。
与现有技术相比,本发明的有益效果是:
通过SELEX筛选和亲和性检测筛选获得专一靶向GDF15蛋白的核酸适配体,并经过结构预测和剪切修饰,优化适配体结构,并通过酶联亲和性检测,验证特定的剪切修饰适配体GDF15-7-+11A、GDF15-7-+11T以及GDF15-7-截短2等与靶标具有更好的亲和性和特异性。
附图说明
图1为25KDa的GDF15蛋白表达纯化。
图2为模拟GDF15-7(SEQ ID NO.2)和GDF15-8(SEQ ID NO.3)与GDF15蛋白空间构象的相互作用。(A)为GDF15-7的模拟空间构象;(B)为GDF15-8的模拟空间构象。
图3为核酸适配体GDF15-3、GDF15-7、GDF15-8、GDF15-12、GDF15-14特异性检测识别GDF15蛋白。
图4为GDF15-7(SEQ ID NO.2)空间结构。
图5为优化剪切核酸适配体结构。(A)为GDF15-7-D28C;(B)为GDF15-7-D27A;(C)为GDF15-7-+11A;(D)为GDF15-7-+11T;(E)为GDF15-7-+11C。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1. GDF15蛋白的表达
将人源GDF15基因构建入带谷胱甘肽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-GDF15 重组蛋白。
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GDF15蛋白约为25kD。
实施例2 SELEX技术靶向GDF15蛋白筛选核酸适配体
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 MgCl2,2mMDTT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GDF15蛋白的Ni-NTA磁珠在结合缓冲液中室温孵育1h,用Elutionbuffer洗脱并收集与GDF15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GDF15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20个最优结果中而获得丰度最高的适配体GDF15-3、GDF15-7、GDF15-8、GDF15-12、GDF15-14,序列如下。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GDF15蛋白的ssDNA核酸适配体最稳定的二级结构。
GDF15-3:5’-TCATCAACTTCGGACCGCACCCAGGAGCAAAGCATACATTGC-3’ ,SEQ ID NO:1;
GDF15-7:5’-TTAAAATCGGAACCTCATGGCCAACAGGAACGTTACATTGC-3’ ,SEQ ID NO:2;
GDF15-8: 5’-TTAAATCGCATGGCCAGGAACTTTCGGCATCGCAATTACATGC-3’ ,SEQ IDNO:3;
GDF15-12: 5’-TTAACTCATGGCCATCTAACTTTCTATCCAGATCATTTCCATGC-3’ ,SEQ IDNO:4;
GDF15-14: 5’-TAAACCGGCCAACTCAGCAACCGCAACAACCAAAGCATGC-3’ ,SEQ ID NO:5。
实施例3核酸适配体亲合力检测
1、抗原的包被:用50mmol/L的碳酸盐包被缓冲液稀释GDF15蛋白至浓度为10μg/mL,按100 μL/孔的量加入至96孔酶标板,4℃放置过夜;
2、第二天弃去包被液,用PBST洗涤三次,每孔加入150μL、1%的BSA封闭2h;
3、PBST洗涤三次,每孔加入不同浓度的的核酸适配体,浓度分别为500、250、200、100、5、2.5、1.25、1 nmol/L,37℃孵育2h;
4、PBST洗涤三次,每孔加入稀释的加入辣根过氧化物酶标记的链霉亲和素(avidin-HRP,1:1000),37 ℃孵育1h;
5、PBST洗涤5次,加入100μL TMB底物避光显色20min,然后加入2mol/L的浓硫酸终止反应,在450 nm处测量吸光度,测定结果使用软件Prism 8分析。
结果如下表1所示:
表1 优选适配体亲和性Kd值
如上结果所示,5条优选的适配体均能与GDF15蛋白具有较强的亲和性,基于上述结果,进一步对GDF15-7和GDF15-8两条适配体序列进行优化剪切,以期获得靶向GDF15蛋白的亲和性更高的适配体序列。
实施例4核酸适配体的特异性检测
1、抗原的包被:用50mmol/L的碳酸盐包被缓冲液溶解GDF15蛋白以及其他抗原(BSA、活化素βA、神经营养因子NRTN、GDF5、GDF11)至浓度为10μg/mL,按100 μL/孔的量加入至96孔酶标板中,4℃放置过夜;
2、第二天弃去包被液,用PBST洗涤三次,每孔加入150μL 1%的BSA封闭2h;
3、PBST洗涤三次,每孔加入浓度200nmol/L的核酸适配体,37 ℃孵育2h;
4、PBST洗涤3次,每孔加入100μL 1:500稀释的SM-HRP二抗,37 ℃孵育1 h。
5、PBST洗涤5次,加入100μL TMB底物避光显色20min,然后加入2 mol/L的H2SO4终止反应,在450nm处测量吸光度,测定结果使用软件Prism 8分析;
结果见图3,从图中可以看出核酸适配体GDF15-3、GDF15-7、GDF15-8、GDF15-12、GDF15-14能够特异性识别GDF15蛋白,对其他靶标,因不具备相应的结合位点,因而几乎不具备亲和性。
实施例5人工优化剪切
将GDF15-7(SEQ ID NO.2)和GDF15-8(SEQID NO.3)的二级结构通过mfold网站预测得到,在此基础上利用RNAComposer获得适配体的RNA 3D结构,再通过BIOVIA DiscoveryStudio Visualizer软件将RNA 3D结构转化为DNA 3D结构,并进行能量最小化。利用ChemOffice对从PubChem获得的GDF15的化学结构进行能量最小化。最后,使用HDOCK网站对两条适配体和GDF15蛋白的结合进行分子对接,通过BIOVIA Discovery StudioVisualizer软件分析获得的适配体-靶标相互作用模型。如图2A和图2B所示,模拟GDF15-7(SEQ ID NO.2)和GDF15-8(SEQ ID NO.3)与GDF15蛋白的相互作用。
如图4所示,GDF15-7(SEQ ID NO.2)的高级结构主要由顶部茎环结构、中部颈环1、中部颈环2和底部B-DNA螺旋组成。在适配体与靶标相互作用模型中,可以发现位于两个中部颈环之间的G28和A29核苷酸以不同形式的键和作用与GDF15蛋白结合(图2A)。因此,推测GDF15-7通过顶部颈环深入GDF15蛋白中部,而中部颈环及躯干部,即G28、A29、T15、C16及中部颈环能够与GDF15蛋白表位契合,实现其特异性结合。
由此设计下述序列,1、剪切B-DNA螺旋,缩短适配体长度;2、扩大中部颈环,尝试对G28、A29及临近核酸进行突变,使核酸空间结构更契合结合表位;3、适当调整其他茎环结构。
得到的适配体序列如下:
GDF15-7-截短1:5’-AATCGGAACCTCATGGCCAACAGGAACGTTACA-3’ ,SEQ ID NO:6;
GDF15-7-截短2:5’-AAATCGGAACCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:7;
GDF15-7-截短3:5’-ATCGGAACCTCATGGCCAACAGGAACGTTAC-3’,SEQ ID NO:8;
GDF15-7-D28C:5’-AAATCGGAACCTCATGGCCAACAGGAAGTTACAT-3’,SEQ ID NO:9;
GDF15-7-D27A:5’-AAATCGGAACCTCATGGCCAACAGGACGTTACAT-3’,SEQ ID NO:10;
GDF15-7-+11A:5’-AAATCGGAACACTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:11;
GDF15-7-+11T:5’-AAATCGGAACTCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:12;
GDF15-7-+11C:5’-AAATCGGAACCCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:13;
突变和插入位点命名以GDF15-7-截短2为基础进行核苷酸排序。
实施例5优化剪切后的核酸适配体亲合力检测
检测方法与实施例3完全相同,结果如表2所示:
表2 优化剪切后的适配体亲和性Kd值
首先,对GDF15-7进行截短,适当的截短可以使适配体空间结构与靶标更契合,提高其亲和性,但是B-DNA序列也起到了维持适配体空间结构稳定的作用,过度的截短反而可能其构象,使得亲和性变差。如表2 所述,在尝试的多个截短适配体中,GDF15-7-截短2能够较好的保持与GDF15蛋白的亲和性,其他截短有可能极大地改变空间构象结构。因此在GDF15-7-截短2适配体基础上,进一步对适配体序列进行修饰,并检测其亲和性。
如上表2所示,在GDF15-7-截短2适配体基础上分别针对结合靶标附近的C28、A27核苷酸(GDF15-7截短2为基础进行核苷酸排序)进行删除,发现极大的影响了适配体的空间构象,影响适配体的靶向性和亲和性。因而调整剪切策略,将适配体上中部颈环2进行扩大调整,获得GDF15-7-+11A、GDF15-7-+11T、GDF15-7-+11C,再进行亲和性检测,发现当第11位(GDF15-7截短2为基础进行核苷酸排序)插入碱基A和T时,可能影响到中部颈环2的结构,使G28、A29、T15、C16部分(GDF15-7为基础进行核苷酸排序,GDF15-7截短2为基础进行核苷酸排序则分别为G25、A26、T12、C13)与GDF15的靶标表位更加契合,能够提高其亲和性和靶向性。适配体结构参见图5A-5E。
综上,通过SELEX筛选和亲和性检测筛选获得专一靶向GDF15蛋白的核酸适配体,并经过结构预测和剪切修饰,优化适配体结构,并通过酶联亲和性检测,验证特定的剪切修饰适配体GDF15-7-+11A、GDF15-7-+11T以及GDF15-7-截短2等与靶标具有更好的亲和性和特异性。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.一种靶向GDF15蛋白的核酸适配体,其特征在于,所述适配体序列为:
GDF15-3:5’-TCATCAACTTCGGACCGCACCCAGGAGCAAAGCATACATTGC-3’,SEQ ID NO:1;
GDF15-7:5’-TTAAAATCGGAACCTCATGGCCAACAGGAACGTTACATTGC-3’,SEQ ID NO:2;
GDF15-8:5’-TTAAATCGCATGGCCAGGAACTTTCGGCATCGCAATTACATGC-3’ ,SEQ ID NO:3;
GDF15-12: 5’-TTAACTCATGGCCATCTAACTTTCTATCCAGATCATTTCCATGC-3’ ,SEQ ID NO:4;
GDF15-14: 5’-TAAACCGGCCAACTCAGCAACCGCAACAACCAAAGCATGC-3’ ,SEQ ID NO:5。
2.根据权利要求1所述的核酸适配体,其特征在于,所述适配体序列为:
GDF15-7:5’-TTAAAATCGGAACCTCATGGCCAACAGGAACGTTACATTGC-3’,SEQ ID NO:2;
GDF15-8:5’-TTAAATCGCATGGCCAGGAACTTTCGGCATCGCAATTACATGC-3’ ,SEQ ID NO:3。
3.根据权利要求1所述的核酸适配体,其特征在于,所述适配体序列为:
GDF15-7-截短1:5’-AATCGGAACCTCATGGCCAACAGGAACGTTACA-3’ ,SEQ ID NO:6;
GDF15-7-截短2:5’-AAATCGGAACCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:7;
GDF15-7-截短3:5’-ATCGGAACCTCATGGCCAACAGGAACGTTAC-3’,SEQ ID NO:8。
4.根据权利要求1所述的核酸适配体,其特征在于,所述适配体序列为:
GDF15-7-D28C:5’-AAATCGGAACCTCATGGCCAACAGGAAGTTACAT-3’,SEQ ID NO:9;
GDF15-7-D27A:5’-AAATCGGAACCTCATGGCCAACAGGACGTTACAT-3’,SEQ ID NO:10;
GDF15-7-+11A:5’-AAATCGGAACACTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:11;
GDF15-7-+11T:5’-AAATCGGAACTCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:12;
GDF15-7-+11C:5’-AAATCGGAACCCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:13。
5.根据权利要求1所述的核酸适配体,其特征在于,所述适配体序列为:
GDF15-7-截短1:5’-AATCGGAACCTCATGGCCAACAGGAACGTTACA-3’ ,SEQ ID NO:6;
GDF15-7-截短2:5’-AAATCGGAACCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:7;
GDF15-7-+11A:5’-AAATCGGAACACTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:11;
GDF15-7-+11T:5’-AAATCGGAACTCTCATGGCCAACAGGAACGTTACAT-3’,SEQ ID NO:12。
6.根据权利要求1-5任一所述的核酸适配体,其特征在于,所述核酸适配体的核苷酸序列被修饰且修饰后的核酸适配体特异性结合GDF15蛋白,所述修饰选自磷酸化、甲基化、氨基化、巯基化、用硫取代氧、用硒取代氧和同位素化中的至少一种。
7.一种核酸适配体的缀合物,其特征在于,其是在权利要求1-5任一所述核酸适配体的核苷酸序列上连接其他用于标记、检测、诊断或治疗的物质而得,且所述核酸适配体的缀合物特异性结合GDF15蛋白。
8.如权利要求7所述的缀合物,其特征在于,所述其他用于标记、检测、诊断或治疗的物质为荧光标记物,优选为FAM、放射性物质、治疗性物质、生物素、地高辛、纳米发光材料、小肽、siRNA和酶标记中的至少一种。
9.根据权利要求1~6任一所述的核酸适配体及权利要求7或8所述的缀合物在制备靶向GDF15蛋白试剂中的应用。
10.一种试剂盒,其特征在于,包含权利要求1~6任一所述的核酸适配体及权利要求7或8所述的缀合物。
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