CN116675774A - 抗c-MET纳米抗体、编码核酸及其应用 - Google Patents
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Abstract
本发明公开了抗c‑MET纳米抗体、编码核酸及其应用,纳米抗体的氨基酸序列如SEQ ID NO:1~SEQ ID NO:7任一所示,所述纳米抗体包含框架区FR和互补决定区CDR;所述纳米抗体具有高水溶性、高耐性、高稳定性等特点,能够特异性靶向c‑MET蛋白,亲和力高。
Description
本申请为申请号:202210328624.0,申请日:2022-03-30,发明名称:抗c-MET纳米抗体、编码核酸及其应用的分案申请。
技术领域
本发明属于基因工程技术领域,特别是涉及一种抗c-MET纳米抗体、编码核酸及其应用。
背景技术
Hamers-Casterman等在骆驼血清中发现了缺失轻链和重链恒定区CH1,只包含一个重链可变区和其他两个重链恒定区(CH2、CH3)的纳米抗体(nanobody,Nb),又被称为VHH(variable domain of heavy chain of heavy chain antibody,VHH),纳米抗体由于其高水溶性、穿透力强等特性,在基因工程中得到广泛应用。
c-MET是一种表达于多种细胞表面的受体酪氨酸激酶,在肝癌、肺癌、结肠癌、乳腺癌、胰腺癌、卵巢癌、前列腺癌、胃癌和胶质母细胞瘤等肿瘤细胞存在高表达,且c-MET的高表达与促进癌症的发生和发展直接相关,这使c-MET成为癌症靶向治疗的重要靶点,抗体药物由于其靶向性好、副作用小的优势,在肿瘤相关抗原的靶向治疗中发挥着强大的功能,但现有抗c-MET单克隆抗体制备技术复杂,生产成本高;单克隆抗体的热稳定差且在体免疫原性强,不适合用于肿瘤诊断和成像研究的靶向分子。
发明内容
本发明实施例的目的在于提供一种抗c-MET纳米抗体,较传统抗体具有高水溶性、高耐性、高稳定性、高抗原结合性、低免疫原性以及较强的组织穿透力,所述纳米抗体的原核表达量高,能够特异性靶向c-MET蛋白,亲和力高。
本发明实施例的目的还在于提供一种抗c-MET纳米抗体的编码核酸及其应用。
本发明所采用的技术方案是,抗c-MET纳米抗体,所述纳米抗体包含SEQ ID NO:8所示的FR1、SEQ ID NO:9所示的CDR1、SEQ ID NO:10所示的FR2、SEQ ID NO:11所示的CDR2、SEQ ID NO:12所示的FR3、SEQ ID NO:13所示的CDR3、SEQ ID NO:14所示的FR4;
或:SEQ ID NO:15所示的FR1、SEQ ID NO:16所示的CDR1、SEQ ID NO:17所示的FR2、SEQ ID NO:18所示的CDR2、SEQ ID NO:19所示的FR3、SEQ ID NO:20所示的CDR3、SEQID NO:21所示的FR4;
或:SEQ ID NO:22所示的FR1、SEQ ID NO:23所示的CDR1、SEQ ID NO:24所示的FR2、SEQ ID NO:25所示的CDR2、SEQ ID NO:26所示的FR3、SEQ ID NO:27所示的CDR3、SEQID NO:28所示的FR4;
或:SEQ ID NO:29所示的FR1、SEQ ID NO:30所示的CDR1、SEQ ID NO:31所示的FR2、SEQ ID NO:32所示的CDR2、SEQ ID NO:33所示的FR3、SEQ ID NO:34所示的CDR3、SEQID NO:35所示的FR4;
或:SEQ ID NO:36所示的FR1、SEQ ID NO:37所示的CDR1、SEQ ID NO:38所示的FR2、SEQ ID NO:39所示的CDR2、SEQ ID NO:40所示的FR3;
或:SEQ ID NO:41所示的FR1、SEQ ID NO:42所示的CDR1、SEQ ID NO:43所示的FR2、SEQ ID NO:44所示的CDR2、SEQ ID NO:45所示的FR3、SEQ ID NO:46所示的CDR3、SEQID NO:47所示的FR4;
或:SEQ ID NO:48所示的FR1、SEQ ID NO:49所示的CDR1、SEQ ID NO:50所示的FR2、SEQ ID NO:51所示的CDR2、SEQ ID NO:52所示的FR3、SEQ ID NO:53所示的CDR3、SEQID NO:54所示的FR4。
进一步的,所述纳米抗体的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6或SEQ ID NO:7所示。
抗c-MET的纳米抗体的制备方法,包括以下步骤:
使用c-MET抗原免疫羊驼,提取羊驼外周淋巴血中的总RNA,通过RT-PCR扩增抗体,将扩增产物插入噬菌体得到噬菌体展示库;
使用c-MET蛋白从噬菌体展示库中淘选抗c-MET的纳米抗体,使用ELISA筛选阳性单克隆抗体,对其进行测序得到纳米抗体序列。
一种编码核酸,用于编码权利要求1或2所述的纳米抗体。
一种编码核酸,具有SEQ ID NO:55~SEQ ID NO:61任一所示的核酸序列。
一种表达载体,包含上述的编码核酸。
一种宿主细胞,包含上述的编码核酸或表达载体。
纳米抗体在c-MET分子检测试剂、活体成像探针和/或治疗性抗体的用途。
本发明的有益效果是:本发明实施例使用重组c-MET蛋白免疫羊驼,随后利用该羊驼外周血淋巴细胞建立了针对于c-MET的纳米抗体基因库,试验中将c-MET偶联在酶标板上,以此形式的抗原利用噬菌体展示技术筛选免疫性的纳米抗体基因库(羊驼重链抗体噬菌体展示基因库),从而获得了针对c-MET特异性的纳米抗体基因,将此基因转至大肠杆菌中,建立了能在大肠杆菌中高效表达的纳米抗体株,所述纳米抗体的原核表达量高,具有高水溶性、高耐性、高稳定性、高抗原结合性、低免疫原性以及较强的组织穿透力,且能够特异性靶向c-MET蛋白,亲和力高,所述纳米抗体与IR-808染料偶联后,用于活体成像探针及食管鳞癌细胞EC109的在体成像,效果良好。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是抗c-MET纳米抗体多序列比对结果图,表明这7种纳米抗体的同源性较高。
图2是实施例中血清免疫后的单克隆phage ELISA检测结果图。
图3中:A是低浓度咪唑洗脱1-7蛋白的SDS-PAGE电泳图,B是高浓度咪唑洗脱1-7蛋白的SDS-PAGE电泳图。
图4是实施例中1-7蛋白的ELISA检测结果图。
图5是实施例中1-7蛋白用于食管鳞癌细胞(EC109)的荧光共聚焦显微镜成像图。
具体实施方式
下面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
构建针对c-MET蛋白的纳米抗体噬菌体展示库。
将c-MET抗原与弗式佐剂混合后免疫羊驼,共免疫3次,首次免疫后7天进行第二次免疫,免疫间隔为14天,按照重量比确定每次免疫剂量为1mg。
将免疫前羊驼血清作为阴性对照,免疫后1周分别采集血清样本,用于检测抗体产生情况,免疫结束后抽取50mL羊驼外周淋巴血淋巴细胞,提取其中的总RNA,通过RT-PCR技术扩增抗体的VHH区,并将克隆产物通过同源重组方法插入噬菌体,获得噬菌体展示库,库容量为2.12×1013CFU/mL。
实施例2
亲和淘选以及淘选后的文库扩增。
(1)第一轮亲和淘选
1)用碳酸盐缓冲液(50mM,pH=9.6)将c-MET蛋白稀释到100μg/mL、50μg/mL、4μg/mL,每孔加入100μL包被酶标板孔,每个靶标做5组重复,在4℃下包被过夜,弃包被液,用PBST洗涤3次;
2)每孔中加入200μL 3%的OVA封闭液,37℃下封闭1h,弃封闭液,用PBST洗涤3次;
3)每孔中加入100μL噬菌体文库(2.12×1013CFU/mL),37℃下孵育1h,弃掉未结合的噬菌体,用0.1%的PBST洗涤5次,再用PBS洗涤5次;
4)加入100μL、pH=2.2的Gly-HCl洗脱液,室温孵育8min,洗脱下特异性结合的噬菌体,重复一次,将洗脱液转移至1.5mL的无菌离心管中,迅速加入10μL Tris-HCl中和缓冲液;
5)取10μL上述溶液进行梯度稀释,测定滴度,计算淘选回收率,其余洗脱物混合后用于噬菌体展示库扩增。
(2)文库扩增
1)淘选洗脱液与处于对数生长前期的E.coli TG1培养物5mL混匀,37℃静置30min,200rpm振荡培养30min;1000G离心20min,弃上清,用500μL 2×YT重悬涂布于200mm2×YT-GA平板;
2)10mL 2×YT液体培养基刮菌,取500μL悬液加入50mL 2×YT液体培养基中,37℃振荡培养30min;按cell:phage=1:10的比例加入M13KO7辅助噬菌体,37℃静置30min后,200rpm振荡培养30min;用离心管分装培养物,28℃、4000G离心10min,沉淀物用50mL 2×YT-AK液体培养基重悬,30℃、200rpm振荡培养过夜;
3)将过夜培养物在4℃、12000G下离心30min,取上清转移到新离心管中,加入1/5体积的PEG/NaCl,混匀后置于4℃下4h以上;
4)4℃、12000G离心30min,弃上清,将沉淀重悬于1mL PBS中,加入1/5体积的PEG/NaCl,混匀后置于4℃下2h以上;
5)4℃、15000G离心5min,弃上清,将沉淀重悬于200μL PBS中,即为扩增产物,测滴度,用于下一轮淘选。
(3)第二轮亲和淘选
1)用碳酸盐缓冲液(50mM,pH=9.6)将c-MET蛋白稀释到50μg/mL和10μg/mL,每孔加入100μL包被酶标板孔,每个靶标做5组重复,在4℃下包被过夜,弃包被液,用PBST洗涤3次;
2)每孔中加入200μL 3%的牛血清白蛋白(BSA)封闭液,37℃下封闭1h,弃封闭液,用PBST洗涤3次;
3)每孔中加入100μL噬菌体文库(8.35×1013CFU/mL),37℃下孵育1h,弃掉未结合的噬菌体,用0.1%的PBST洗涤10次,再用PBS洗涤10次;
4)加入100μL、pH=2.2的Gly-HCl洗脱液,室温孵育8min,洗脱下特异性结合的噬菌体,重复一次,将洗脱液转移至1.5mL的无菌离心管中,迅速加入10μL Tris-HCl中和缓冲液;
5)取10μL上述溶液进行梯度稀释,测定滴度,计算淘选回收率,其余洗脱物混合后用于第三轮亲和淘选。
(4)第三轮亲和淘选
1)用碳酸盐缓冲液(50mM,pH=9.6)将c-MET蛋白稀释到10μg/mL和2μg/mL,每孔加入100μL包被酶标板孔,每个靶标做5组重复,在4℃下包被过夜,弃包被液,用PBST洗涤3次;
2)每孔中加入200μL 3%的OVA封闭液,37℃下封闭1h,弃封闭液,用PBST洗涤3次;
3)每孔中加入100μL噬菌体文库(4.1×1013CFU/mL),37℃下孵育1h,弃掉未结合的噬菌体,用0.1%的PBST洗涤15次,再用PBS洗涤15次;
4)加入100μL pH=2.2的Gly-HCl洗脱液,室温孵育8min,洗脱下特异性结合的噬菌体,重复一次,将洗脱液转移至1.5mL的无菌离心管中,迅速加入10μL Tris-HCl中和缓冲液;
5)取10μL上述溶液进行梯度稀释,测定滴度,计算淘选回收率。
三轮淘选的回收率如表1所示,结果表明,噬菌体展示库中阳性噬菌体的富集度逐渐增加,说明噬菌体展示库中能与c-MET抗原特异性结合的噬菌体得到了有效的富集。
表1三轮淘选的投入量、洗脱量及回收率
实施例3
特异性噬菌体克隆的鉴定及分析。
(1)噬菌体的救援
1)从第一轮淘选洗脱物滴度的平板上,用灭菌枪头随机挑取50个单克隆接种于1mL 2×YT-AK中,37℃、200rpm振荡培养14h;
2)取200μL上述培养物,按cell:phage=1:10的比例加入M13KO7噬菌体,37℃静置30min,然后于200rpm振荡培养1h;
3)补加500μL体积的2×YT-AK,37℃振荡培养过夜;
4)第二天12000rpm离心2min,取上清用于阳性克隆鉴定。
(2)阳性噬菌体克隆的鉴定
1)将c-MET蛋白用碳酸盐缓冲液(50mM,pH=9.6)稀释至终浓度为2μg/mL,按100μL/孔加入酶标板孔中,每组三组重复,4℃包被过夜,弃包被液,PBST洗涤3次;
2)每孔加入200μL封闭液,37℃封闭1h,弃去孔内液体,每孔用200μL PBST洗涤3次;
3)每孔加入50μL噬菌体培养液上清和50μL 5%脱脂奶粉,37℃孵育1h,弃去孔内液体,每孔用200μL PBST洗涤5次;
4)加入辣根过氧化物酶标记的抗c-MET抗体(用脱脂奶粉按1:10000稀释),按100μL/孔加入酶标孔中,37℃孵育1h,弃去孔内液体,每孔用200μL PBST洗涤6次;
5)每孔加入100μL TMB显色液显色,37℃孵育20min,加入100μL终止液终止反应,于450nm下测吸收值,将(实验孔OD450nm-空板对照OD450nm)÷(阴性对照OD450nm-空板对照OD450nm)≥2.1的作为阳性克隆。
(3)阳性噬菌体克隆的序列分析
从获得的阳性克隆中挑选出21个克隆株,其P/N值≥5,送至生物技术公司进行DNA测序,并使用ELISA检测其特异性,如图2所示,筛选得到7种特异结合性高的纳米抗体1-1、1-7、1-23、1-58、1-79、2-88、3-28,所述纳米抗体均包含框架区FR和互补决定区CDR,所述纳米抗体的序列号及其编码核酸的序列号如表2所示。
纳米抗体中疏水性残基被亲水性残基所取代,导致其天然可溶性较高的;纳米抗体在化学或热变性后的有效重折叠使纳米抗体的稳定性较好;纳米抗体由4个保守序列和3个互补决定区组成(Complementarity-determining region,CDR),其CDR3含有16~18个氨基酸残基,与人VH基因家族3(VH3)的序列具有高度同源性,但是比VH区的空间构象更长且可形成凸环结构,使纳米抗体更容易与抗原结合;纳米抗体的分子量只有15kDa,具有较强的组织穿透力,且能结合到较难结合的表位;纳米抗体缺乏Fc结构域,避免了由Fc结构域与髓系细胞表达的受体结合,导致的非靶向摄取(被肝脏、脾脏和健康组织摄取)。
表2纳米抗体、编码核酸等的序列号
纳米抗体1-1、1-7、1-23、1-58、1-79、2-88、3-28的序列对比如图1所示,各氨基酸序列之间的同源性较高,可达到80%以上;对于本发明所提及的编码核酸,本领域技术人员应当理解,实际包括互补双链的任意一条,或者两条;为了方便,在本发明描述中,虽然多数情况下只给出了一条链,但实际上也公开了与之互补的另一条链,另外,本发明中的基因序列包括DNA形式或RNA形式,公开其中一种,意味着另一种也被公开。
实施例4
在原核表达系统中表达和纯化抗c-MET纳米抗体。
1)将测序分析后获得的纳米抗体1-7的VHH片段克隆到pET28a原核表达载体中,转化到BL21(DE3)感受态细胞;
2)涂布LB平板(含50mg/L卡那霉素);
3)挑选单个菌落接种在5mL含有卡那霉素的LB培养液中,37℃摇床培养14h;
4)接种3mL的菌液至300mL LB培养基中,37℃、200rpm振荡培养约2h,培养到OD值达到0.6-0.8时,加入IPTG(终浓度0.3mM),15℃振荡培养24h;
5)离心收菌,菌体经超声破碎以获得抗体粗提液,12000G离心30min取上清;
6)上清经镍柱离子亲和层析纯化纳米抗体1-7,采用咪唑梯度洗脱法,如图3所示,其中M为Marker,A中各咪唑洗脱液的浓度分别为10mmol/L、30mmol/L、50mmol/L、100mmol/L、250mmol/L,用于洗去杂带,B中咪唑洗脱液的浓度为500mmol/L,从图3的B可以看出纳米抗体1-7的纯度达90%以上,计算纳米抗体1-7的原核表达量,每升菌可纯化约13mg,说明纳米抗体1-7的获取时间较短,生产成本较低。
重复上述过程,在原核表达系统中对测序获得的纳米抗体1-1、1-23、1-58、1-79、2-88、3-28进行表达和纯化,获得各纳米抗体的SDS-PAGE电泳,基于此获得各纳米抗体的纯度及原核表达量,所述纳米抗体1-1的原核表达量为8mg/L菌、纳米抗体1-23的原核表达量为6mg/L菌、纳米抗体1-58的原核表达量为4mg/L菌、纳米抗体1-79的原核表达量为5mg/L菌、纳米抗体2-88的原核表达量为7mg/L菌、纳米抗体3-28的原核表达量为8mg/L菌。
实施例5
利用间接ELISA法检测c-MET纳米抗体的亲和力。
1)包被:ELISA 96孔板每孔用100μL的2μg/mL c-MET抗原(PBS稀释)4℃包被过夜,弃去孔内液体,每孔用200μL PBST洗涤3次(每次5min,轻轻摇动),洗涤后控干;
2)封闭:每孔加入200μL封闭液,37℃封闭1h;弃去孔内液体,每孔用200μL PBST洗涤3次(每次5min,轻轻摇动);
3)加样:每孔加入100μL PBS稀释的纳米抗体1-7(线性浓度0、1.17nM、5.86nM、29.32nM、146.60nM、733.01nM、1466.02nM、2932.04nM、5864.07nM、11728.14nM、23456.28nM),37℃孵育1h;
弃去孔内液体,每孔用200μL PBST洗涤3次(每次5min,轻轻摇动);
4)加酶标抗体:每孔加入100μL PBS稀释(1:50000)的缀合有HRP的抗His-Tag抗体,37℃孵育1h;弃去孔内液体,每孔用200μL PBST洗涤6次(每次5min,轻轻摇动);
5)加底物显色液:每孔加入100μL TMB底物37℃避光反应20min,加入100μL、1M的H2SO4终止反应,立即读取OD450nm;
6)数据分析:OD450nm减去空白对照后,以纳米抗体浓度(nM)的对数为横坐标,OD450nm为纵坐标做曲线得到如图4所示的亲和力拟合曲线,将OD450nm达到其最大值一半时的纳米抗体浓度作为亲和力Kd(单位nM),Kd=256.6(±18.63)nM。
重复上述过程可获取各纳米抗体的亲和力拟合曲线,对其进行计算,可得纳米抗体1-1、1-23、1-58、1-79、2-88、3-28的亲和力分别为125.3nM、131.6nM、121.1nM、364.0nM、131.7nM、127.1nM。
实施例6
c-MET纳米抗体1-7与IR-808染料偶联c-MET纳米抗体近红外荧光分子探针。
1)取21.3mg MES溶于5mL ddH2O中,调节pH=6,过滤除菌,取3mg IR-808染料溶于150μL DMSO溶液,配制为母液(终浓度0.026M),取8.06mg EDC溶于400μL ddH2O中,配制为母液(终浓度0.26M),取6mg NHS溶于200μL ddH2O中,配制为母液(终浓度0.52M);
2)活化IR-808染料:取上述MES溶液970μL、EDC溶液10μL、NHS溶液10μL、IR-808染料10μL于1.5mL离心管中,在磁力搅拌器上300rpm、22℃反应15min,反应后将溶液冷冻干燥;
3)利用未活化的IR-808染料绘制标准曲线,计算活化后的IR-808染料的浓度;
4)以摩尔比1:5偶联c-MET纳米抗体1-7与活化后的IR-808染料,反应条件为:在PBS缓冲液中,20℃、300rpm反应2h,反应结束后用SDS-PAGE和IVIS仪器确定c-MET纳米抗体1-7与IR-808染料偶联成功,利用紫外分光光度计计算c-MET纳米抗体探针的浓度。
实施例7
c-MET纳米抗体近红外荧光分子探针用于食管鳞癌细胞(EC109)荧光共聚焦显微镜成像。
1)胰酶消化下来EC109细胞后,离心弃培养基,用1mL培养基悬浮细胞,取10μL计数,按每共聚焦小皿接种1×105个细胞铺皿,贴壁培养过夜;
2)从细胞培养箱取出细胞后,PBS清洗细胞1次;
3)每皿加入2mL培养基稀释的抗c-MET纳米抗体近红外荧光分子探针(浓度梯度0μM、2.99μM、5.98μM),4℃避光孵育2h,PBS清洗细胞5次;
4)每皿加入2mL 4%多聚甲醛室温固定细胞20min,PBS清洗细胞3次;
5)每皿加入1mL 0.3% Triton-X100冰上通透细胞5min,PBS清洗3次;
6)每皿加入100μL PBS稀释(1:500)的缀合有Alexa Fluor 488的抗His-Tag抗体,37℃避光孵育1h,PBS清洗细胞5次;
7)每皿加入1mL DAPI室温染色25min,PBS清洗细胞2次,再加1mL PBS去拍荧光共聚焦显微镜。
检测结果如图5所示,图5中三行示图分别为荧光分子探针浓度为0、2.99μM、5.98μM时的荧光共聚焦显微结果,DAPI染色为细胞核,缀合His-tag的Alex488染料指示带有His标签的抗c-MET纳米抗体与高表达c-MET蛋白的EC109细胞膜表面结合,Merge指示细胞核和细胞膜上高表达c-MET蛋白与荧光分子探针的结合情况,由图5可知纳米抗体1-7制得的荧光分子探针能够高亲和、特异性靶向c-MET靶标,且荧光强度随抗体浓度的依赖性升高。
本说明书中的各个实施例均采用相关的方式描述,各个实施例之间相同相似的部分互相参见即可,每个实施例重点说明的都是与其他实施例的不同之处。尤其,对于系统实施例而言,由于其基本相似于方法实施例,所以描述的比较简单,相关之处参见方法实施例的部分说明即可。
以上所述仅为本发明的较佳实施例而已,并非用于限定本发明的保护范围。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等,均包含在本发明的保护范围内。
Claims (7)
1.抗c-MET的纳米抗体,其特征在于,所述纳米抗体包含SEQ ID NO:29所示的FR1、SEQID NO:30所示的CDR1、SEQ ID NO:31所示的FR2、SEQ ID NO:32所示的CDR2、SEQ ID NO:33所示的FR3、SEQ ID NO:34所示的CDR3、SEQ ID NO:35所示的FR4;
或:SEQ ID NO:48所示的FR1、SEQ ID NO:49所示的CDR1、SEQ ID NO:50所示的FR2、SEQID NO:51所示的CDR2、SEQ ID NO:52所示的FR3、SEQ ID NO:53所示的CDR3、SEQ ID NO:54所示的FR4。
2.根据权利要求1所述的抗c-MET的纳米抗体,其特征在于,所述纳米抗体的氨基酸序列如SEQ ID NO:4或SEQ ID NO:7所示。
3.一种编码核酸,其特征在于,用于编码权利要求1或2所述的纳米抗体。
4.根据权利要求3所述的一种编码核酸,其特征在于,具有SEQ ID NO:58、SEQ ID NO:61任一所示的核酸序列。
5.一种表达载体,其特征在于,包含权利要求3或4所述的编码核酸。
6.一种宿主细胞,其特征在于,包含权利要求3或4所述的编码核酸,或权利要求5所述的载体。
7.权利要求1~2任一项所述纳米抗体在制备c-MET分子检测试剂、活体成像探针和/或治疗性抗体中的用途。
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