CN114456261B - 抗sars-cov-2病毒s蛋白rbd结构域的纳米抗体及其用途 - Google Patents
抗sars-cov-2病毒s蛋白rbd结构域的纳米抗体及其用途 Download PDFInfo
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Abstract
本申请涉及纳米抗体领域,尤其涉及一种抗SARS‑COV‑2病毒S蛋白RBD结构域的纳米抗体及其用途,所述纳米抗体具有如SEQ ID NO:1所示氨基酸序列,且具有如SEQ ID NO:2‑SEQ ID NO:4所示氨基酸序列所示的三个互补决定区,纳米抗体能识别SARS‑COV‑2病毒S蛋白的RBD结构域,纳米抗体阻断病毒与细胞受体ACE2的结合,所述纳米抗体与病毒受体ACE2蛋白竞争,使抗体具有抑制病毒感染的能力。
Description
技术领域
本申请涉及纳米抗体领域,尤其涉及一种抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体及其用途。
背景技术
SARS-CoV-2病毒是一种新型高致病性冠状病毒,该病毒与2002年出现的SARS病毒有79.5%的序列相似性。在基因组水平上,SARS-CoV-2与蝙蝠冠状病毒的同源性为96%。SARS-CoV-2潜伏期较长,可达14天,也因此感染初期不明显,从而使该病毒更容易播散,造成蔓延、爆发态势。为抑制疫情的进一步蔓延,快速诊断试剂盒的开发,疫苗的研制和中和抗体的制备尤为重要。
冠状病毒由双层脂质的囊膜组成,包含刺突蛋白、包膜蛋白、膜蛋白以及核衣壳蛋白。其中,膜蛋白介导病毒与宿主ACE2蛋白的相互作用,SARS-CoV-2与SARS-CoV使用相同的入侵受体。序列比对发现,SARS-CoV-2与SARS-CoV的氨基酸序列同源性为80%。 S蛋白分为S1和S2两个亚基,其中S1的功能是与宿主受体结合,S2的主要功能是促进病毒和细胞膜融合。S1的RBD结构域与ACE2蛋白结合的高亲和力导致病毒能在人群中快速传播。S蛋白暴露于病毒表面,含有大量的抗原决定簇,可以产生针对病毒的保护性抗体。
纳米抗体(Nanobody)是由比利时科学家于1993年在自然杂志中首次报道,在羊驼外周血液中存在一种天然缺失轻链的抗体,该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区。单独克隆和表达的VHH保留抗体的稳定性和抗原结合活性。纳米抗体分子量约为15KD,是目前发现的最小的抗原结合单位。
因此,亟需开发新的用于针对SARS-COV-2的纳米抗体及其试剂盒。
发明内容
本申请提供了一种抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体及其用途,以解决针对SARS-COV-2病毒的纳米抗体的技术问题。
第一方面,本申请提供了一种抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体,所述纳米抗体具有如SEQ ID NO:1所示氨基酸序列,且具有如SEQ ID NO:2-SEQ ID NO: 4所示氨基酸序列所示的三个互补决定区;所述纳米抗体能识别SARS-COV-2病毒S蛋白的RBD结构域。
可选的,所述纳米抗体还包括:如SEQ ID NO:1所示氨基酸序列,经替换、缺失和/或增加N个氨基酸得到的具有相同功能的抗体,其中,N为正整数。
可选的,所述纳米抗体的N端和/或C端连接有标签序列。
可选的,所述纳米抗体与RBD结合的EC50为0.4213ng/ml-1.872ng/ml。
第二方面,本申请提供了一种编码第一方面所述纳米抗体的核酸分子。
可选的,所述核酸分子的核苷酸序列如SEQ ID NO:5所示。
可选的,所述生物材料包括重组DNA、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
第三方面,本申请提供了一种重组抗体,所述重组抗体由第一方面所述的抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体和人源fc片段组成,所述人源fc片段的氨基酸序列如SEQ ID NO:6所示。
第四方面,本申请提供了一种编码第一方面所述纳米抗体的核酸分子。
可选的,所述核酸分子的第一方面所述的SARS-COV-2病毒S蛋白RBD结构域的纳米抗体以及第三方面所述的重组抗体在制备抑制SARS-COV-2病毒感染的药物、制备 SARS-COV-2病毒检测试剂或试剂盒中的用途。
第四方面,本申请提供了一种SARS-COV-2病毒检测试剂或试剂盒,包括第一方面所述的SARS-COV-2病毒S蛋白RBD结构域的纳米抗体或者第三方面所述的重组抗体。
本申请实施例提供的上述技术方案与现有技术相比具有如下优点:
本申请实施例提供的抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体,能够与新型冠状病毒SARS-COV-2的S蛋白RBD区特异性结合,阻断病毒与细胞受体ACE2的结合,所述纳米抗体与病毒受体ACE2蛋白竞争,使抗体具有抑制病毒感染的能力。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分,示出了符合本发明的实施例,并与说明书一起用于解释本发明的原理。
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的介绍,显而易见地,对于本领域普通技术人员而言,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1是F11纳米抗体与RBD蛋白结合曲线;
图2是F11纳米抗体对ACE2蛋白与RBD蛋白结合的抑制曲线图;
图3是F11纳米抗体的凝胶电泳图;
图4是F11纳米抗体应用于胶体金检测试剂盒时的灵敏度结果。
具体实施方式
为使本申请实施例的目的、技术方案和优点更加清楚,下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本申请的一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本申请保护的范围。
下面将结合实施例、对比例及实验数据对本发明的方法进行详细说明。
实施例1纳米抗体的制备
1、拯救噬菌体表面展示纳米抗体库
将构建好的纳米抗体库以噬菌粒的形式保存在宿主菌中,在淘选过程开始前,应当先拯救文库,使其成为噬菌体展示的抗体库。具体方法如下:
将1.5mL带E-tag标签的抗体库接种到300mL 2YT-AG培养基中,至OD600nm约 0.3-0.4;37℃振荡培养约1.5h至OD600nm=0.5-0.6;按细菌:phage=1:5加入helper phage(M13K07),37℃振荡培养约1h;4000rpm 15℃离心15min,去除培养基;加入200 mL 2YT-AK(100μg/mL Amp,50μg/mL Kan)培养基重悬细菌,37℃培养2h;10000 rpm离心20min去除沉淀;上清加入40mL PEG/NaCl沉淀phage,冰浴过夜;10000rpm 离心20min,去掉上清;用0.6mL 2YT培养基重悬phage,4℃备用。
注:若需要大量制备phage,更换kan抗性的培养基以后,培养时间从两小时延长至过夜培养。获得的噬菌体经过梯度稀释,感染TG1菌,涂布SOBAG平板,通过菌落计数计算噬菌体库滴度。
2、淘选单链抗体
本实验利用His Bind Resin结合抗原蛋白,从噬菌体展示的抗体库中淘选抗体,具体过程如下:
活化His Bind Resin:取200μL His Bind Resin于1.5mL离心管中,1000g离心1min,去掉保存溶液;加入200μL的ddH2O清洗树脂一次,1000g离心1min,去掉上清,重复此步骤一次;加入200μL离子化缓冲液,重悬树脂,静置10min,离心去掉上清;加入200μL结合缓冲液,重悬树脂,静置10min;取40μL树脂加入1.5mL离心管中,离心去掉上清。
3、淘选识别目的蛋白的单链抗体:
取纯化后的蛋白35μL(约10μg),加入165μL PBS中,混匀后加入装有活化后的树脂的EP管中,旋转混匀1h,1000g离心1min,去掉上清;加入200μL漂洗缓冲液,重悬树脂,1000g离心1min,去掉上清,此步骤重复一次。取拯救后的噬菌体展示抗体库溶液300μL,加入0.3μL Triton X-100,用微量移液器轻轻混匀;加入40μL 未活化的树脂,轻微旋转反应1h;1000g离心1min,取上清加入40μL已包被抗原蛋白的树脂,轻微旋转反应2h;1000g离心1min,去掉上清;加入500μL漂洗缓冲液 (含0.1%Triton X-100),重悬树脂,轻微振荡漂洗5min,1000g离心1min,去掉上清,此步骤重复5次;加入500μL漂洗缓冲液含(0.1%Tween-20),重悬树脂,轻微振荡漂洗5min,1000g离心1min,去掉上清,此步骤重复5次;最后一次漂洗后,将树脂转移到新的EP管中,1000g离心1min,去掉上清;加入200μL洗脱缓冲液,轻微旋转洗脱20min;1000g离心1min,取上清,加入5mL TG1菌液中,37℃感染1h;将感染的菌液涂布SOBAG平板,30℃倒置培养过夜;次日,用2YT-AG培养基刮下平板上的菌落,并拯救成噬菌体进行下一轮淘选。
4、挑选识RBD蛋白的阳性克隆:
从SOBAG平板上随机挑取单菌落接种到96孔细菌培养板中,每孔加入200μL 2YT-AG,37℃培养过夜;吸取25μL菌液到新的细菌培养板中,加入175μL 2YT-AG 培养基,37℃培养3h;3500rpm离心10min,去上清,菌沉淀重悬于200μL 2YT-AI(100 μg/mL Amp,1mM IPTG)培养基中,30℃诱导过夜;3500rpm离心10min,上清4℃保存备用;将纯化的目的蛋白加入到ELISA板中,4℃包被过夜;倾掉包被液后,以PBS 洗3次,4%PBSM(PBS含4%脱脂牛奶)封闭1h;以PBS洗1次后,每孔加入50μ L以上制备好的单链抗体上清和50μL 4%PBSM,于37℃反应1h;用PBS和PBST各洗3次后,每孔加入100μL anti-E/HRP conjugation(以4%PBSM按1:5000稀释), 37℃保温1h;
用PBST和PBS洗涤三次,加入100μL TMB底物溶液,避光反应15min,加入25 μL2mol/L H2SO4终止反应,用酶标仪测定OD450nm值。
5、阳性克隆测序和序列分析
将上述淘选得到的、经ELISA鉴定为阳性的单克隆送金开瑞测序,测序的通用引物为 S1:5’-GACCATGATTACGCCAAGC-3’,用DNAstar和Clustalw1.8分析抗体的重链与轻链可变区序列。
最终获得了2株不同的纳米抗体,分别为:A11和F11纳米抗体,其中,F11的氨基酸序列如SEQ ID NO:1所示,F11的核苷酸序列如SEQ ID NO:5所示;余下的A11的氨基酸序列如SEQ ID NO:8所示,A11的核苷酸序列如SEQ ID NO:9所示。
纳米抗体:A11和F11的CDR区的氨基酸序列如下表1所示:
表1。
6、抗体基因重组与表达
根据测序结果,设计抗体FR1和FR4引物,扩增抗体基因;抗体基因经sfiI和NotI双酶切,插入抗体表达载体pSecTag2A-Fc2载体中;用质粒抽提试剂盒提取质粒;转染前将所有试剂置于室温10分钟,以下操作使用6孔培养皿;将3μg质粒DNA稀释到250 μL无血清的DMEM培养基,用移液枪吹吸3-4次;将5μL PEI试剂稀释到250μL无血清的DMEM培养基,用移液枪吹吸3-4次;
注:无血清的DMEM培养基是稀释液,不能使用含血清的培养基进行DNA和将稀释好的PEI转染试剂一次性全部加入到已稀释好的质粒DNA中,立即用移液枪吹吸3-4次;室温放置10-15分钟,以形成PEI-DNA复合物;转染前18-24小时进行细胞的计数并铺板,以便在转染时,使贴壁细胞的汇合度大约80%左右;弃去孔中原培养基,加入1ml新鲜的 DMEM完全培养基;
制备PEI-DNA复合物;将PEI-DNA混合液均匀滴入细胞培养基中,轻轻做十字运动,让PEI-DNA复合物分散均匀;培养皿放于5%CO2,37℃恒温培养箱中,72h后收集细胞,检测蛋白表达量。按照相同的比例转化100ml细胞,纯化重组表达的抗体,表达后的抗体的如图3所示,其中,从左到右的第一列为蛋白marker;第二列为制备的重组抗体纯化过程中的洗脱液,第三列为重组抗体纯化过程中的流穿液,第四列为重组抗体浓缩液,由图 3可知,F11抗体得到了有效表达,且可纯化、浓缩后获取相关抗体。
实施例2纳米抗体与RBD蛋白结合情况
将实施例1筛选获得的2株不同的纳米抗体分别与RBD结合,检测抗体与RBD结合的EC50值,RBD蛋白按照2μg/ml包被微孔板,2株纳米抗体的EC50值如
图1为F11抗体与RBD蛋白的结合曲线,由图1可知,RBD蛋白按照2μg/ml包被微孔板,梯度稀释F11抗体,检测信号随着抗体数量增加而增加,抗体与RBD结合的EC50 为0.4212-1.872ng/ml,说明抗体可以特异性识别RBD蛋白。
实施例3纳米抗体对ACE2蛋白与RBD蛋白结合的抑制情况
将RBD蛋白按照2μg/ml包被微孔板,将实施例1筛选获得的2株不同的纳米抗体分别与1ug/ml的藕连HRP的ACE2蛋白混合,测定OD450nm数值
由表2的数据可知,所述2株抗体和ACE2蛋白均存在竞争关系,IC50的范围为2.433-2.763nM,该抗体可能具有抑制病毒感染的能力,有望用于治疗抗体开发。
图2是F11纳米抗体对ACE2蛋白与RBD蛋白结合的抑制曲线图;OD450nm数值随着F11纳米抗体的浓度升高而降低,说明该纳米抗体和ACE2蛋白存在竞争关系。
实施例4重组抗体的构建与表达
1、重组表达载体的构建
根据测序结果,用引物(F:CGGCCCAGCCGGCCATGGCC,R:GGACTAGTGCGGCCGCTGAGGAGACGGTGACCTG)扩增抗体基因;抗体基因经sfiI和NotI双酶切,插入抗体表达载体pSecTag2A-fc,用质粒抽提试剂盒提取质粒。其中所述pSecTag2A-fc载体为用 pSecTag2A载体(Thermo Fisher,V90020)改造,在pSecTag2A载体的hind III和BamH I 酶切位点中插入了人源IgG1 Fc基因片段,所述人源fc基因片段的氨基酸序列如SEQ ID NO:6所示,所述人源fc基因片段的核酸序列如SEQ ID NO:7所示。
2、融合蛋白的表达
转染前将所有试剂置于室温10分钟,以下操作使用6孔培养皿;将3μg质粒DNA 稀释到250μL无血清的DMEM培养基,用移液枪吹吸3-4次;将5μL PEI试剂稀释到250 μL无血清的DMEM培养基,用移液枪吹吸3-4次;注意:无血清的DMEM培养基是稀释液,不能使用含血清的培养基进行DNA和将稀释好的PEI转染试剂一次性全部加入到已稀释好的质粒DNA中,立即用移液枪吹吸3-4次;室温放置10-15分钟,以形成PEI-DNA 复合物;转染前18-24小时进行细胞的计数并铺板,以便在转染时,使贴壁细胞的汇合度大约80%左右;弃去孔中原培养基,加入1ml新鲜的DMEM完全培养基;制备PEI-DNA 复合物;将PEI-DNA混合液均匀滴入细胞培养基中,轻轻做十字运动,让PEI-DNA复合物分散均匀;培养皿放于5%CO2,37℃恒温培养箱中,72h后收集细胞,检测蛋白表达量。按照相同的比例转化100ml细胞,纯化重组表达的抗体。取上清进行亲和层析纯化和 SDS-PAGE电泳分析,纯化的纳米抗体用间接竞争ELISA初步测定其活性。通过优化诱导表达条件(如宿主菌、表达载体、诱导培养时间、温度以及IPTG浓度等),可以进一步提高目的蛋白表达量,为大量制备纳米抗体提供了途径。
实施例5纳米抗体用于SARS-COV-2病毒的检测
S蛋白的RBD结构域是病毒结合受体的重要区域,筛选识别RBD区域的纳米抗体,可以中和SARS-CoV-2病毒感染,开发治疗抗体。同时,纳米抗体经过分子生物学改造,生产带有人源fc片段的重组抗体,用作诊断试剂盒的质控抗体。高质量的纳米抗体,可以直接开发检测病毒检测试剂盒。
一、SARS-COV-2病毒的ELISA检测试剂盒,
1、SARS-COV-2病毒的ELISA检测试剂盒,包括:
包被有所述的抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体的ELISA酶标板;
阴性对照;
重组表达SARS-COV-2病毒S蛋白;
酶标记的酶标二抗;
样品稀释液、包被缓冲液、封闭液、ELISA酶标板洗涤液、抗体稀释液、显色液和终止液。
2、SARS-COV-2病毒的ELISA检测试剂盒的使用方法
(1)包被酶标板:取实施例4中纯化的重组抗体,用包被液稀释至适当的含量,96孔酶标板中每孔加100μL,置2-8℃吸附24小时。空去包被液,包被用洗液洗板3次。抗原蛋白包被浓度确定,先用包被液将纯化的重组PCV3 Cap蛋白稀释至蛋白含量为5μg/ml, 10μg/ml,20μg/ml,30μg/ml,40μg/ml,加至96孔酶标板的微孔中,每孔加100μL,置2-8℃吸附24小时。从中选择一个优化的包被浓度,即10μg/ml。
包被液:
0.05M pH 9.5 碳酸缓冲液
Na2CO3 1.50g
NaHCO3 2.94g
戊二醛 0.10ml
调pH至9.5,定容至1000ml。
包被用洗液:
0.01M pH7.2 磷酸钾缓冲液
K2HPO4·3H2O 1.56g
KH2PO4 0.38g
调pH至7.2,定容至1000ml。
(2)封闭:空干包被洗液,加包被用封闭液150μL/孔,置2-8℃过夜。去除封闭液后自然干燥24小时。
包被用封闭液:
调pH至7.2,定容至1000ml。
(3)抗体的孵育
重组表达SARS-COV-2病毒S蛋白溶液,加入包被板孔中,100μL/孔。将酶标板置于37℃孵育30min。空净重组表达蛋白溶液后用洗涤液洗板5次。
洗涤液:(10倍浓度)
调pH至7.2,定容至1000ml。
4、酶标二抗的孵育:用磷酸缓冲液将辣根过氧化物酶标记的酶标二抗稀释至工作浓度,向酶标板孔中加入100μL/孔,置37℃,30min。
5、显色:加入50μL底物液A,50μL底物液B,轻摇混合,37℃反应10min。
底物溶液A:柠檬酸4.2g,醋酸钠13.6g,过氧化氢脲0.5g,调pH至5.0,定容至1000ml。
底物溶液B:四甲基联苯胺1.0g,甲醇100ml,二甲亚砜50ml,聚乙烯吡咯烷酮16.0g,调pH至5.0,定容至1000ml。
6、终止:显色结束后加终止液100μL/孔。终止液:2mol/L H2SO4。
7、读板:在450nm波长下,读取酶标板光吸收值。
8、该试剂盒的判定标准为:
试验有效性判定:阳性对照孔平均值≥0.506;阴性对照平均值≤0.254;
临界值(标准阳性样本阈值)=阴性样本OD450平均值+3×标准差=0.254+3×0.021=0.317
阴性判定:样品OD值<临界值者(0.317)为阴性;
阳性判定:样品OD值≥临界值者(0.317)为阳性。
9、该试剂盒的性能指标:
(1)特异性:除重组表达SARS-COV-2病毒S蛋白溶液外,其他检测样品均为阴性。这些数据表明,本发明提供的试剂盒与其他血清抗体之间不存在交叉反应。
(2)灵敏性:重组表达SARS-COV-2病毒S蛋白溶液稀释至0.0075ng/L时仍能检出。
(3)稳定性:将试剂盒置于37℃不少于2天与4℃存放的试剂盒同步检测20份样品,其符合率为100%。
(3)精密度:取浓度呈梯度的7种重组表达SARS-COV-2病毒S蛋白溶液标本,分别稀释,分别同批测定10次,批内变异系数为均低于4%;同样7份血清,隔天再测定10 次,变异均低于5%;符合试剂盒精密度要求。
二、纳米抗体F11用作SARS-COV-2病毒抗体的胶体金检测试剂盒的质控
取448ng、112ng、28ng、7ng、3.5ng、1.85ng、0.875ng的F11抗体分别稀释于80-100μL PBS稀释液中,加入SARS-CoV-2成品检测卡检测。
图4是纳米抗体F11用作质控抗体应用于胶体金检测试剂盒时的灵敏度结果,由图4 可知,所有检测卡C线正常,T线随抗体梯度稀释信号减弱,表明灵敏度高,抗体特异性良好。
需要说明的是,在本文中,诸如“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者任何其他变体意在涵盖非排他性地包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上所述仅是本发明的具体实施方式,使本领域技术人员能够理解或实现本发明。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其他实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所申请的原理和新颖特点相一致的最宽的范围。
序列表
<110> 武汉华美生物工程有限公司
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Claims (9)
1.一种抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体,其特征在于,所述纳米抗体的氨基酸序列如SEQ ID NO:1所示,所述纳米抗体能识别SARS-COV-2病毒S蛋白的RBD结构域。
2.根据权利要求1所述的纳米抗体,其特征在于,所述纳米抗体的N端和/或C端连接有标签序列。
3.一种编码权利要求1或2所述抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体的核酸分子。
4.根据权利要求3所述的核酸分子,其特征在于,所述核酸分子的核苷酸序列如SEQ IDNO:5所示。
5.一种含有权利要求3或4所述核酸分子的重组DNA、质粒载体、噬菌体载体、工程菌或转基因细胞系。
6.一种重组抗体,其特征在于,所述重组抗体由权利要求1或2所述的抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体和人源Fc片段组成,所述人源Fc片段的氨基酸序列如SEQ IDNO:6所示。
7.权利要求1或2所述的抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体或者权利要求6所述的重组抗体在制备抑制SARS-COV-2病毒感染的药物用途。
8.权利要求1或2所述的抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体或者权利要求6所述的重组抗体在制备SARS-COV-2病毒检测试剂盒中的用途。
9.一种SARS-COV-2病毒检测试剂盒,其特征在于,包括权利要求1或2所述的抗SARS-COV-2病毒S蛋白RBD结构域的纳米抗体或者权利要求6所述的重组抗体。
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Denomination of invention: Nanoantibodies against the RBD domain of the S protein of SARS-COV-2 virus and their applications Granted publication date: 20230707 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: CUSABIO BIOTECH Co.,Ltd. Registration number: Y2024980009781 |