CN117088972A - 一种靶向SARS-CoV-2RBD蛋白的鲨源纳米抗体和应用 - Google Patents
一种靶向SARS-CoV-2RBD蛋白的鲨源纳米抗体和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向SARS‑CoV‑2RBD蛋白的鲨源纳米抗体和应用。纳米抗体核苷酸序列如SEQ ID NO:4,氨基酸序列如SEQ ID NO:8;制备是将RBD蛋白免疫条纹斑竹鲨,提取外周血淋巴细胞总RNA并反转录为cDNA;再将扩增得到的VNAR片段与扩增的pR2噬菌粒通过无缝克隆连接,转化TG1感受态细胞,构建噬菌体抗体文库;从文库中筛选SARS‑CoV‑2RBD蛋白特异性的纳米抗体序列并重组表达,获得靶向的鲨源纳米抗体。本发明的纳米抗体分子量小,可利用配对的纳米抗体检测SARS‑CoV‑2RBD抗原,可应用于SARS‑CoV‑2RBD蛋白的免疫印迹、酶联免疫吸附实验以及COVID‑19制药应用。
Description
本发明申请是申请号为2022101043013、申请日为2022-01-28的分案申请。
技术领域
本发明属于生物技术领域的一种SARS-CoV-2抗体及其制备方法,具体涉及一种靶向SARS-CoV-2RBD的鲨源纳米抗体及其制备方法。
背景技术
SARS-CoV-2属于冠状病毒,其通过表面突刺蛋白(spike)的受体结合区域(RBD)与上皮细胞表面的血管紧张素转换酶2(ACE2)结合后进入细胞,完成侵染。
从康复患者体内分离的全人源抗体被证实具有很好的抗病毒作用,这些传统的单克隆抗体由2条重链和2条轻链组成,具有分子量大,生产工艺复杂,不易加工改造等局限性。相对于传统的抗体,驼类或软骨鱼类(如鲨鱼)体内存在一种仅由2条重链组成的抗体,命名为重链抗体,其抗体可变区仅由2个相同的重链可变区组成,该区域被称为单域抗体(sdAb)。单域抗体蛋白直径小于10纳米,因此又被称为纳米抗体。羊驼和鲨鱼的单域抗体分别称为VHH和VNAR。单域抗体比传统的scFv或Fab具有居多优势,其分子量小、穿透性强、稳定性及可溶性更高,其发挥功能不依赖糖基化修饰等。与传统抗体相比,单域抗体通常具有一个扩展的CDR3,可以形成一个凸出的表面结构来识别抗原表位,因此可以利用其帮助识别传统抗体难以识别的隐藏抗原表位。目前在单域抗体方面研究开发最好的是羊驼单域抗体,其在探索抗原受体起源,研发疫苗、治疗药物、诊断试剂和生物技术研究工具等方面已取得较好进展。同理,可将鲨源单域抗体开发成诊断试剂、治疗性抗体药物等。目前尚无抗SARS-CoV-2RBD的鲨源纳米抗体序列专利。
发明内容
为了解决背景技术中存在的问题,本发明提供了能以高亲和力结合新冠病毒(SARS-CoV-2)受体结合区域(RBD)的鲨源重链抗体可变区序列(VNAR),该可变区序列又称为纳米抗体,其能够用于预防、治疗和/或诊断SARS-CoV-2感染,能够用于相关制药应用。
本发明的技术方案如下:
一、一种靶向SARS-CoV-2RBD的鲨源纳米抗体:
所述鲨源纳米抗体的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2或SEQ ID NO.3或SEQ ID NO.4所示。
所述鲨源纳米抗体氨基酸序列如SEQ ID NO.5或SEQ ID NO.6或SEQ ID NO.7或SEQ ID NO.8所示,将抗体分别命名为sh-aRBD-2、sh-aRBD-5、sh-aRBD-17、sh-aRBD-18。
二、一种靶向SARS-CoV-2RBD的鲨源纳米抗体的制备方法:
所述鲨源纳米抗体的制备方法按照以下步骤进行:
采用体外重组表达的SARS-CoV-2RBD蛋白作为抗原对鲨鱼进行免疫,免疫后抽取外周血,对于外周血利用质量分数为30%的蔗糖溶液采用密度梯度离心法分离获得淋巴细胞,抽提淋巴细胞溶液的总RNA,随后反转录为cDNA;
再以cDNA为模板,用特异性引物扩增出VNAR基因序列,将VNAR基因序列插入pR2噬菌粒扩增产物中,然后电转化到TG1感受态细菌中,在涂布平板上培养,刮取平板上的菌落得到噬菌体展示抗体文库,再经过多重筛选、重组表达和纯化得到SARS-CoV-2RBD鲨源纳米抗体。
这样表征纳米抗体与SARS-CoV-2RBD的亲和力,获得靶向SARS-CoV-2RBD蛋白的高亲和力鲨源纳米抗体;并利用配对的纳米抗体检测SARS-CoV-2RBD抗原。
所述的多重筛选、重组表达和纯化,具体为:对RBD噬菌体抗体文库先经过淘选和噬菌体ELISA筛选得到阳性克隆子,经测序鉴定得到抗体序列,将抗体序列构建到重组表达载体中,诱导表达并纯化获得SARS-CoV-2RBD鲨源纳米抗体。
所述对鲨鱼进行免疫的具体方案为:共进行五次免疫,其中前三次为皮下注射,后两次为尾静脉注射;皮下注射之间相间隔时间为10天,尾静脉注射之间相间隔时间为30天;第三次皮下注射免疫后30天再进行尾静脉注射;最后一次尾静脉注射后15天,通过尾静脉采集鲨鱼外周血。
所述VNAR基因序列扩增所用正向引物为SEQ ID NO.11,即5’-GCTGCA CAGCCTGCTATGGCAACTCAACGGGTTGAACAAACACCGA-3’,反向引物为SEQ ID NO.12,即5’-GAGTTTTTGTTCGGCTGCTGCTGGTTTTACAGTCAGA ATGGTGCCGC-3’。
所述pR2噬菌粒扩增产物是由pR2噬菌粒扩增获得,所用正向引物为SEQ IDNO.13,即5’-AGCAGCCGAACAAAAACTCATCTCAGAAGAG-3’;反向引物为SEQ ID NO.14,即5’-CCATAGCAGGCTGTGCAGCATAGAAAGGTACCA CTAAAGGAATTGC-3’。
三、一种双表位二聚化抗体:
用权利要求1-7任一所述SARS-CoV-2RBD鲨源纳米抗体构建双表位二聚化抗体,将两个序列不同的SARS-CoV-2RBD鲨源纳米抗体用柔性多肽链连接构成。两个SARS-CoV-2RBD鲨源纳米抗体的序列不同。
所述双表位二聚化抗体是指将分别结合于SARS-CoV-2RBD上两个独立表位的两个纳米抗体用柔性多肽链连接,从而构建的能够结合所述SARS-CoV-2RBD两个表位的抗体。
所述的柔性多肽链的核苷酸序列如SEQ ID NO.9所示,氨基酸序列如SEQ IDNO.10所示。
双表位二聚化抗体相比未用柔性多肽链连接前的SARS-CoV-2RBD鲨源纳米抗体优势性能更好。
将本发明的鲨源纳米抗体配对,采用胶体金法检测RBD抗原蛋白,其中sh-aRBD-2和sh-aRBD-18抗体配对检出限最低,为0.390μg/mL。
而后,本发明发现所制备的四种纳米抗体可识别SARS-CoV-2RBD 3个表位,其中sh-aRBD-5和sh-aRBD-17结合于SARS-CoV-2RBD同一表位,sh-aRBD-2和sh-aRBD-18分别结合另外两个不同的表位,sh-aRBD-2和sh-aRBD-18配对检出限更低,为0.390μg/mL。
因此,用它们组合构建了双表位二聚化抗体sh-aRBD-2-5、sh-aRBD-2-17、sh-aRBD-2-18、sh-aRBD-5-18、sh-aRBD-17-18。
所述鲨源纳米抗体在SARS-CoV-2RBD蛋白的免疫印迹(Western blot)、酶联免疫吸附实验(ELISA)以及COVID-19制药中的应用。
所述SARS-CoV-2RBD鲨源纳米抗体配对检测SARS-CoV-2RBD抗原蛋白,并筛选得到检出限低的配对抗体。
本发明的有益效果是:
本发明的鲨鱼纳米抗体分子量小,结构简单,稳定性好。本发明所述鲨源纳米抗体分子量仅有12kDa,是常规抗体的1/10左右,比羊驼纳米抗体小约20%,是已知脊椎动物中最小的天然单域抗体,可用原核或真核表达系统体外表达,容易制备,表达量高,且成本低。
本发明所述鲨源纳米抗体可以应用于SARS-CoV-2RBD蛋白的免疫印迹(Westernblot)、酶联免疫吸附实验(ELISA)以及COVID-19预防性、治疗性和诊断性的制药应用。
附图说明
图1是SARS-CoV-2RBD蛋白免疫鲨鱼方案。
图2是单克隆噬菌体ELISA结果。
图3是所述纳米抗体氨基酸序列比对结果。
图4是所述纳米抗体Fc融合蛋白(A)及纳米抗体(B)的SDS-PAGE凝胶电泳结果。泳道M为标准蛋白。
图5是采用ELISA表征所述纳米抗体Fc融合蛋白与SARS-CoV-2RBD之间结合的结果图。
图6是采用凝胶过滤层析表征所述纳米抗体Fc融合蛋白与SARS-CoV-2RBD之间结合的结果图。
图7是采用BLI表征所述纳米抗体Fc融合蛋白(A)及纳米抗体(B)与SARS-CoV-2RBD之间的亲和力。其中实线是实时监测的动力学曲线,虚线是软件拟合的曲线。不同抗体浓度梯度的动力学曲线从上到下与右侧标识的从上到下的浓度依次对应。
图8是所述纳米抗体配对检测RBD抗原蛋白结果。所检测RBD抗原浓度标示于试纸条上方,由50μg/mL依次二倍递减至0.097μg/mL。记录为1-2的试纸条为检测线包被sh-aRBD-2、胶体金标记sh-aRBD-18组别。其余试纸条胶体金标记均为sh-aRBD-2,检测线处包被的抗体信息显示于试纸条上方。
图9是采用BLI表征所述二聚化纳米抗体与SARS-CoV-2RBD之间的亲和力。
具体实施方式
下面结合附图及具体实施例对本发明作进一步详细说明,以下实施例是对本发明的解释而本发明并不局限于以下实施例。
实施例1SARS-CoV-2RBD蛋白免疫鲨鱼
利用重组表达的SARS-CoV-2RBD蛋白免疫鲨鱼,免疫方案如图1所示。本发明共进行五次免疫,其中前三次为皮下注射,后两次为尾静脉注射。皮下注射间隔时间为10天,尾静脉注射间隔时间为30天。第三次皮下免疫后30天,即进行尾静脉注射。尾静脉注射后15天,通过尾静脉采集鲨鱼外周血。本发明共使用3条条纹斑竹鲨,每条鲨鱼每次免疫RBD蛋白剂量均为250μg。
实施例2SARS-CoV-2RBD纳米抗体筛选
1)将抽取的鲨鱼血液缓慢加至等体积30%(m/v)蔗糖溶液上层,500g离心20min,取中间淋巴细胞层,经PBS洗涤后,离心收集细胞沉淀。利用RNA抽提试剂盒提取总RNA,并反转录为cDNA。
2)以cDNA为模板,以特异性引物扩增VNAR序列,正向引物为:5’-GCTGCACAGCCTGCTATGGCAACTCAACGGGTTGAACAAACACCGA-3’,反向引物为:5’-GAGTTTTTGTTCGGCTGCTGCTGGTTTTACAGTCAGAATG GTGCCGC-3’。扩增所用DNA聚合酶为高保真酶,扩增程序为:98℃、10s,57℃、15s,72℃、25s,循环30次。利用试剂盒回收扩增的VNAR片段。以pR2噬菌粒为模板,特异性引物扩增pR2噬菌粒,正向引物为:5’-AGCAGCCGAACAAAAACTCATCTCAGAAGAG-3’,反向引物为:5’-CCATAGCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC-3’。扩增所用DNA聚合酶为高保真酶,扩增程序为:98℃、10s,53℃、15s,68℃、4min 15s,循环30次。利用Nco I和Not I酶切扩增产物中的模板pR2噬菌粒,而后利用试剂盒回收扩增的pR2噬菌粒。利用无缝克隆连接pR2噬菌粒和VNAR片段,其摩尔比为1:4。将连接产物电转化至TG1感受态细胞中,37℃、200rpm培养1h,分别取0.2μL和0.02μL(稀释法)菌液涂布10cm固体平板,培养13h后计数菌落数目,并计算所构建抗体文库大小。将剩余的菌液经离心后涂布至150mm固体平板,37℃培养13h,而后刮取菌苔,液氮速冻后于-80℃储存,此为纳米抗体文库。
3)活化冻存的抗体库细菌,加入KM13辅助噬菌体,以辅助M13噬菌体生长和繁殖。取细菌培养上清,测量噬菌体滴度,此为扩增后的噬菌体。以0.1mg/mL浓度包被SARS-CoV-2RBD抗原至免疫板,加入1x 1011pfu以上扩增的噬菌体,室温孵育1h。利用胰酶洗脱与SARS-CoV-2RBD抗原特异性结合的噬菌体,并将其侵染TG1细菌。分别取50μL和5μL侵染的菌液涂布固体平板,记录菌落总数。
4)从以上平板中随机挑取95个单菌落,活化过夜。加入KM13辅助噬菌体,离心收集裂解后的上清,此为单克隆噬菌体。以1μg/mL浓度包被SARS-CoV-2RBD抗原至96孔免疫板,每孔分别加入以上制备的单克隆噬菌体溶液,室温孵育1h。利用HRP-anti M13抗体捕获与SARS-CoV-2RBD抗原结合的噬菌体,并用TMB底物显色5min,以1M H2SO4溶液终止反应。采用酶标仪记录OD450数值,并将其整理成如图2所示柱状图。
5)挑取OD450值大于1的孔进行测序分析,测序引物为:5’-CCCTCATAGTTAGCGTAACGA-3’。
6)比对分析所测得抗体序列,排除重复的克隆后,共得到四条不同的纳米抗体序列(图3)。SEQ ID NO.1-4所示为纳米抗体核苷酸序列,由此可得如SEQ ID NO.5-8所示的纳米抗体氨基酸序列。
实施例3表达纯化所得SARS-CoV-2RBD纳米抗体及其Fc融合蛋白
将本发明中的纳米抗体序列构建到带信号肽的哺乳动物表达载体pTT5中,使其与人IgG1 Fc融合表达,Fc片段表达在C末端,并可用TEV酶酶切获得纳米抗体。将重组质粒转染HEK 293细胞,收集培养上清,经rProtein A亲和层析柱纯化纳米抗体Fc融合蛋白。如图4A所示,我们获得了高纯度的SARS-CoV-2RBD纳米抗体Fc融合蛋白。经TEV酶切后,获得了高纯度的SARS-CoV-2RBD纳米抗体蛋白(图4B)。
实施例4表征所述SARS-CoV-2RBD纳米抗体
1)采用凝胶过滤层析表征所述纳米抗体Fc融合蛋白与SARS-CoV-2RBD的结合情况。将纳米抗体Fc融合蛋白和SARS-CoV-2RBD蛋白以摩尔比2:1混匀,冰上孵育1h,而后上样至Superdex 200凝胶过滤层析柱,记录280nm处吸光度变化情况,并绘制层析图谱。与此同时,设置纳米抗体Fc融合蛋白对照和SARS-CoV-2RBD蛋白对照。由图5可知,sh-aRBD-2-Fc、sh-aRBD-5-Fc和sh-aRBD-18-Fc均能与SARS-CoV-2RBD结合,而sh-aRBD-17-Fc与SARS-CoV-2RBD结合力较弱。
2)采用非竞争性ELISA表征所述纳米抗体Fc融合蛋白与SARS-CoV-2RBD的结合情况:将浓度为10μg/mL的SARS-CoV-2RBD蛋白包被于免疫板中,依次添加1:5梯度稀释的纳米抗体Fc融合蛋白和ACE2-Fc蛋白溶液,室温孵育1h。而后,加入HRP anti-IgG1 Fc抗体以检测结合的VNAR-Fc和ACE2-Fc。结果如图6所示,四个纳米抗体与SARS-CoV-2RBD的结合力大小依次为:sh-aRBD-5-Fc>sh-aRBD-18-Fc>sh-aRBD-2-Fc>sh-aRBD-17-Fc,其EC50值分别是0.037、2.155、145.819、510.841nM。其中sh-aRBD-5-Fc和sh-aRBD-18-Fc的亲和力高于ACE2-Fc(4.119nM)。
3)采用BLI表征纳米抗体及其Fc融合蛋白与SARS-CoV-2RBD的亲和力。为了表征纳米抗体Fc融合蛋白与RBD的亲和力,首先用生物素标记RBD,得到生物素化的RBD蛋白(biotin-RBD)。而后,将其固化在SA生物传感器上,设置不同的纳米抗体Fc融合蛋白浓度梯度,检测biotin-RBD与纳米抗体Fc融合蛋白的亲和力。结果如图7A所示,四个纳米抗体Fc融合蛋白与SARS-CoV-2RBD的结合力大小依次为:sh-aRBD-5-Fc>sh-aRBD-18-Fc>sh-aRBD-2-Fc>sh-aRBD-17-Fc,其亲和力常数KD值分别是3.88、9.20、28.3、211nM。为了表征纳米抗体与RBD的亲和力,首先将纳米抗体Fc融合蛋白固化在Protein A生物传感器上,设置不同的SARS-CoV-2RBD浓度梯度,检测RBD与纳米抗体的亲和力。结果如图7B所示,四个纳米抗体与SARS-CoV-2RBD的结合力大小依次为:sh-aRBD-5>sh-aRBD-18>sh-aRBD-2>sh-aRBD-17,其亲和力常数KD值分别是38.5、60.3、429、2720nM。
实施例5所述纳米抗体配对检测RBD抗原蛋白
将sh-aRBD-2、sh-aRBD-5、sh-aRBD-17和sh-aRBD-18两两配对,分别包被于检测线处或进行胶体金标记,制备得到RBD抗原蛋白检测试纸条。试纸条宽度为3.5mm,包被所使用的抗体浓度为1.5mg/mL,胶体金标记所使用抗体浓度为20μg/mL。所检测RBD抗原浓度由50μg/mL依次二倍递减至0.097μg/mL,RBD蛋白上样量均为40μL。结果如图8所示,sh-aRBD-2和sh-aRBD-18抗体配对检出限最低,为0.390μg/mL。
实施例6表征所述二聚化纳米抗体
本发明中所述的四种纳米抗体可识别SARS-CoV-2RBD 3个表位,其中sh-aRBD-5和sh-aRBD-17结合于SARS-CoV-2RBD同一表位,sh-aRBD-2和sh-aRBD-18分别结合另外两个不同的表位。因此,用他们组合构建了双表位二聚化抗体sh-aRBD-2-5、sh-aRBD-2-17、sh-aRBD-2-18、sh-aRBD-5-18、sh-aRBD-17-18。重组表达sh-aRBD-2-5和sh-aRBD-2-17抗体,并检测了其与SARS-CoV-2RBD的亲和力常数KD值。如图9所示,二聚化纳米抗体亲和力高于纳米抗体单体,sh-aRBD-2-5和sh-aRBD-2-17亲和力常数KD值分别为6.39和32.1nM。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
本发明涉及的序列如下:
SEQ ID NO.1:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-2的核苷酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCTTAAAAGGTTCCAGCTGTGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATGAAACAGCTGGGCCGGACTGTTCCTATAGCTGGGGATATAGCTATATTGAAGGAGGCGGCACCATTCTGACTGTAAAACCT
SEQ ID NO.2:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-5的核苷酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGATAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATAAGCCCCCTCTACAGCTGGGATCTCGGGCGTTATACCTTAGCTGGAATTGCGAGGGAGGCGGCACCATTCTGACTGTAAAACCT
SEQ ID NO.3:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-17的核苷酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGATAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGGACAGCTGAATGAGGGCTGTTATGGGAGCTGGAATCGCAACTATTATGAAGGAGGCGGCACCATTCTGACTGTAAAACCT
SEQ ID NO.4:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-18的核苷酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGTTGTACATTGACTAGCACGCACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAGGCTTATACAGCTGGATGTGAAGGGCGATACTATAACTGGGATGGAGGAGGCGGCACCATTCTGACTGTAAAACCT
SEQ ID NO.5:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-2的氨基酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
TQRVEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYETAGPDCSYSWGYSYIEGGGTILTVKP
SEQ ID NO.6:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-5的氨基酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCEAYKPPLQLGSRALYLSWNCEGGGTILTVKP
SEQ ID NO.7:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-17的氨基酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKGQLNEGCYGSWNRNYYEGGGTILTVKP
SEQ ID NO.8:
名称:SARS-CoV-2RBD鲨源纳米抗体sh-aRBD-18的氨基酸序列来源:条纹斑竹鲨(Cihiloscyllium plagiasum)
TQRVEQTPTTTTKEAGESLTINCVLRDSSCTLTSTHWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCEAYTAGCEGRYYNWDGGGGTILTVKP
SEQ ID NO.9:
名称:柔性多肽链的核苷酸序列
来源:人工序列(Artificial Sequence)
GGTGGCGGAGGGTCTGGTGGCGGAGGGTCTGGTGGCGGAGGGTCT
SEQ ID NO.10:
名称:柔性多肽链的氨基酸序列
来源:人工序列(Artificial Sequence)
GGGGSGGGGSGGGGS
SEQ ID NO.11
名称:VNAR基因序列扩增的正向引物
来源:人工序列(Artificial Sequence)
GCTGCACAGCCTGCTATGGCAACTCAACGGGTTGAACAAACACCGASEQ ID NO.12
名称:VNAR基因序列扩增的反向引物
来源:人工序列(Artificial Sequence)
GAGTTTTTGTTCGGCTGCTGCTGGTTTTACAGTCAGAATGGTGCCGC
SEQ ID NO.13
名称:pR2噬菌粒扩增的正向引物
来源:人工序列(Artificial Sequence)
AGCAGCCGAACAAAAACTCATCTCAGAAGAG
SEQ ID NO.14
名称:pR2噬菌粒扩增的反向引物
来源:人工序列(Artificial Sequence)
CCATAGCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC。
Claims (4)
1.一种靶向SARS-CoV-2RBD的鲨源纳米抗体,其特征在于:所述鲨源纳米抗体的核苷酸序列为SEQ ID NO.4。
2.一种双表位二聚化抗体,其特征在于:用权利要求1所述SARS-CoV-2RBD鲨源纳米抗体构建双表位二聚化抗体,将两个序列不同的SARS-CoV-2RBD鲨源纳米抗体用柔性多肽链连接构成。
3.根据权利要求2所述的一种双表位二聚化抗体,其特征在于:所述的柔性多肽链的核苷酸序列如SEQ ID NO.9所示,氨基酸序列如SEQ ID NO.10所示。
4.权利要求1所述靶向SARS-CoV-2RBD的鲨源纳米抗体和权利要求3所述双表位二聚化抗体的应用,其特征在于:
所述鲨源纳米抗体在SARS-CoV-2RBD蛋白的免疫印迹、酶联免疫吸附实验以及COVID-19制药中的应用。
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