CN114106164B - 抗新型冠状病毒s蛋白的单克隆抗体及其应用 - Google Patents
抗新型冠状病毒s蛋白的单克隆抗体及其应用 Download PDFInfo
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Abstract
本说明书实施例提供了一种抗新型冠状病毒S蛋白的单克隆抗体及其应用。该单克隆抗体包括重链可变区和轻链可变区。所述重链可变区包括氨基酸序列为SEQ ID NO:1所示的CDRH1、氨基酸序列为SEQ ID NO:2所示的CDRH2和氨基酸序列为SEQ IDNO:3所示的CDRH3。所述轻链可变区包括氨基酸序列为SEQ ID NO:4所示的CDRL1、SEQ ID NO:5所示的CDRL2和SEQ ID NO:6所示的CDRL3。本发明同时提供了编码该单克隆抗体的核酸分子、包括该单克隆抗体的组合物和检测新型冠状病毒的试剂盒、该单克隆抗体在预防、抑制和/或治疗抗新型冠状病毒的产品中的应用、在制备检测抗新型冠状病毒S蛋白的试剂中的应用。本发明的单克隆抗体具有较高的灵敏性和特异性,有助于设计和开发抗新冠病毒的药物并且能够应用于免疫学检测。
Description
技术领域
本说明书涉及生物医药技术领域,具体涉及一种抗新型冠状病毒S蛋白的单克隆抗体及其应用。
背景技术
新型冠状病毒导致的新型冠状病毒肺炎是当前全球关注的公共健康问题。2020年2月11日,这种新型冠状病毒被国际病毒分类委员会命名为SARS-CoV-2(重症急性呼吸系统综合症冠状病毒2,也称为2019-nCoV(2019新型冠状病毒)),同日,世界卫生组织将该病毒所感染的肺炎命名为COVID-19。SARS-CoV-2与严重急性呼吸综合征冠状病毒(SARS-CoV)、中东呼吸综合症冠状病毒(MERS-CoV)都是源自动物的人畜共患病原体。尽管冠状病毒通常与人类急性呼吸道感染有关,但其感染多种宿主物种的能力使其成为复杂的病原体。由新型冠状病毒(SARS-CoV-2)引起的COVID-19大流行在世界各地肆虐,并造成数百万住院和死亡。
因此,一方面,有必要建立快速筛选和检测新型冠状病毒的方法,以筛选出感染者或潜在者,并对他们进行隔离或治疗。目前已知的检测方法有实时荧光定量PCR法、血清抗体检测法、抗原检测法等。其中,抗原检测法为通过单克隆抗体检测SARS-CoV-2的相应抗原靶标,是一种准确、快速、简单和易于使用的诊断方法。而单克隆抗体作为抗原检测法的核心原材料,起着重要作用。另一方面,有必要研发出能够有效预防、抑制和/或治疗新型冠状病毒的药物,其中,抗体作为病毒治疗重要的手段,重要性不言而喻。
发明内容
根据本说明书的一个方面,提供了一种抗新型冠状病毒S蛋白的单克隆抗体。该单克隆抗体可以包括重链可变区和轻链可变区。所述重链可变区包括氨基酸序列为SEQ IDNO:1所示的CDRH1、氨基酸序列为SEQ ID NO:2所示的CDRH2和氨基酸序列为SEQ IDNO:3所示的CDRH3。所述轻链可变区包括氨基酸序列为SEQ ID NO:4所示的CDRL1、SEQ ID NO:5所示的CDRL2和SEQ ID NO:6所示的CDRL3。
在一些实施例中,所述重链可变区的氨基酸序列为SEQ ID NO:7所示,所述轻链可变区的氨基酸序列为SEQ ID NO:8所示。
根据本说明书的另一个方面,提供了一种核酸分子。所述核酸分子可以包括编码上述抗新型冠状病毒S蛋白的单克隆抗体的核苷酸序列。
根据本说明书的另一个方面,提供了一种组合物。所述组合物可以包括上述抗新型冠状病毒S蛋白的单克隆抗体,其中,所述组合物用于预防、抑制和/或治疗抗新型冠状病毒。
根据本说明书的另一个方面,提供了抗新型冠状病毒S蛋白的单克隆抗体在制备预防、抑制和/或治疗抗新型冠状病毒的产品中的应用。
根据本说明书的另一个方面,提供了一种检测新型冠状病毒的试剂盒。所述试剂盒包括上述抗新型冠状病毒S蛋白的单克隆抗体。
根据本说明书的再一个方面,提供了抗新型冠状病毒S蛋白的单克隆抗体在制备检测抗新型冠状病毒S蛋白的试剂中的应用。
具体实施方式
如本申请和权利要求书中所示,除非上下文明确提示例外情形,“一”、“一个”、“一种”和/或“该”等词并非特指单数,也可包括复数。一般说来,术语“包括”与“包含”仅提示包括已明确标识的步骤和元素,而这些步骤和元素不构成一个排它性的罗列,方法或者设备也可能包含其它的步骤或元素。
2019-nCoV(SARS-CoV-2)有四种主要的结构蛋白:刺突蛋白(spike protein,也称为S蛋白)、核衣壳蛋白(nucleocapsid protein,也称为N蛋白)、膜蛋白(membraneprotein,也称为M蛋白)和包膜蛋白(envelope protein,也称为E蛋白)。
S蛋白由较长的膜外区、跨膜区和膜内区组成,属于第一类病毒膜融合蛋白(ClassI viral fusion protein)。不同冠状病毒的S蛋白最显著的区别在于病毒的组装和释放过程中是否被宿主蛋白酶切割。成熟的S蛋白通常会被宿主蛋白酶(半胱氨酸蛋白酶、胰蛋白酶等)切割成两个亚基:S1和S2。S1亚基可进一步分成两个相对独立的区域(domain),分别是N-端区域(N-terminal domain,NTD)和C-端区域(C-terminal domain,CTD)。S1包含有受体结合域(receptor binding domain,RBD),大部分的冠状病毒S蛋白的RBD都位于CTD,例如SARS-CoV和MERS-CoV等。只有少部分β冠状病毒的RBD位于NTD(通常NTD结合糖类受体,CTD结合蛋白类受体)。S2亚基通过跨膜区锚定在膜上,它含有膜融合过程所需要的基本元件,包括:一个内在的膜融合肽(fusion peptide,FP),两个7肽重复序列(heptad repeat,HR)、一个跨膜临近区(juxamembrain domain,JMD)和一个跨膜结构域(transmembranedomain,TMD),以及C末端的胞浆区域(cytoplasmic domain,CD)(约40个氨基酸长度)。
新型冠状病毒的S蛋白的RBD是病毒进入宿主细胞的关键部位。当S蛋白的RBD与细胞表面受体(ACE2)的相应部位特异结合后,诱导S2的融合核心螺旋结构发生构象变化,卷曲螺旋折叠形成发夹样结构,发夹样结构使病毒与细胞膜拉在一起,最终引起融合,该步骤是病毒感染宿主细胞的关键。S蛋白是宿主中和抗体的重要作用位点。S蛋白可通过RBD的基因重组或突变可以实现不同宿主间传播,并导致较高致死率。
根据上述描述,S蛋白可选择作为针对冠状病毒疫苗设计的关键靶点。所有冠状病毒都具有保守的功能模体(motif),分别位于S1(RBD序列具有高度保守性)和S2(S2比S1区域的氨基酸序列更为保守)结构域,对RBD及S2区的研究,有助于设计冠状病毒疫苗和开发新的抗冠状病毒药物。
因此,本申请制备了抗SARS-CoV-2-RBD蛋白的抗体,以抑制病毒感染。具体地,运用HEK293F细胞表达的SARS-CoV-2-RBD蛋白免疫Balb/c小鼠,取小鼠脾细胞与骨髓瘤细胞融合,通过特异性的高通量筛选得到具有高度特异性的杂交瘤细胞,通过培养和再免疫获得大量小鼠腹水,再经多步分离及纯化获得高纯度、高灵敏度和高特异性的抗SARS-CoV-2-RBD蛋白的抗体,命名为Mouse anti-SARS-COV-2 Mab1(RBD-Ab)抗体,所获抗体经验证对SARS-CoV-2-RBD蛋白具有良好的特异性结合能力。该单克隆抗体可以通过靶向S1-RBD、S1-NTD或者S2区来阻止S2亚单位介导的S1-RBD与宿主细胞的膜融合,进而抑制病毒感染。Mouse anti-SARS-COV-2 Mab1(RBD-Ab)抗体有助于设计和开发抗新冠病毒的药物(例如,病毒疫苗、中和抗体),为开发新的抗冠状病毒药物提供了研究基础。Mouse anti-SARS-COV-2 Mab1(RBD-Ab)抗体有助于ELISA、免疫层析、免疫印迹、免疫荧光等免疫学检测,为免疫学检测试剂(例如,检测N蛋白免疫试纸条)提供了所需的原料。
根据本申请的一方面,提供了一种单克隆抗体。该单克隆抗体可以包括重链可变区和轻链可变区。所述重链可变区包括氨基酸序列为SEQ ID NO:1所示的CDRH1、氨基酸序列为SEQ ID NO:2所示的CDRH2和氨基酸序列为SEQ IDNO:3所示的CDRH3。所述轻链可变区包括氨基酸序列为SEQ ID NO:4所示的CDRL1、SEQ ID NO:5所示的CDRL2和SEQ ID NO:6所示的CDRL3。
重链可变区的氨基酸序列为SEQ ID NO:7所示,所述轻链可变区的氨基酸序列为SEQ ID NO:8所示。
本申请的抗新型冠状病毒S蛋白的单克隆抗体具有高效价、高纯度、高灵敏度和高特异性的优点。本申请的抗新型冠状病毒S蛋白的单克隆抗体可以应用于检测抗新型冠状病毒S蛋白的试剂,例如,检测S蛋白免疫试纸条。在一些实施例中,抗新型冠状病毒S蛋白的单克隆抗体可以用于ELISA、免疫层析、免疫印迹、免疫荧光等免疫学检测,为免疫学检测试剂(例如,检测N蛋白免疫试纸条)提供了所需的原料。以该抗体为原料开发的检测试剂盒具备很好的应用价值。本申请的抗新型冠状病毒S蛋白的单克隆抗体可以应用于预防、抑制和/或治疗新型冠状病毒的产品(例如,疫苗,药物组合物)中,为治疗(例如,中和新型冠状病毒)、预防和/或抑制新型冠状病毒SARS-CoV-2的产品(例如,疫苗、药物组合物)的开发具有广泛的应用前景。
根据本申请的另一方面,还提供了一种核酸分子。该核酸分子可以包括编码上述抗新型冠状病毒S蛋白的单克隆抗体的核苷酸序列。在一些实施例中,编码重链可变区的CDRH1、CDRH2和CDRH3的核苷酸序列分别为SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示。编码所述轻链可变区的CDRL1、CDRL2和CDRL3的核苷酸序列分别为SEQ ID NO:12、SEQID NO:13和SEQ ID NO:14所示。在一些实施例中,编码所述重链可变区的核苷酸序列为SEQID NO:15所示,以及编码所述重链可变区的核苷酸序列为SEQ ID NO:16所示。
根据本申请的另一方面,还提供了一种组合物。所述药物组合物可以包括上述抗新型冠状病毒S蛋白的单克隆抗体。所述药物组合物用于预防、抑制和/或治疗抗新型冠状病毒。这里的药物组合物指的是能够预防、抑制和/或治疗抗新型冠状病毒的各种组合物的形式,包括但不限于,抗体组合、抗体对、药物组合物、抗体药物偶联物、疫苗等。在一些实施例中,该组合物可以包括抗体组合或抗体对,其包括上述抗新型冠状病毒S蛋白的单克隆抗体以及靶向新型冠状病毒其他靶点(例如,N蛋白)或靶向其他病毒(例如,其他冠状病毒、风疹病毒等)的抗体。在一些实施例中,该组合物可以包括药物组合物。该药物组合物可以包括上述抗新型冠状病毒S蛋白的单克隆抗体和药物上可接受的载体等。药物上可接受的载体可以为本领域常规的药物辅料,包括药学上可接受的赋形剂、填充剂或稀释剂等,本申请并不对此进行限制。该药物组合物可以以片剂、胶囊、颗粒、粉末、液体、悬浮液、乳霜、泡沫、凝胶、洗剂、膏状或软膏等形式制成。该药物组合物可以以口服给药、注射给药、局部给药等方式施用于受试者。在一些实施例中,该组合物可以包括抗体药物偶联物。该抗体药物偶联物可以包括上述抗新型冠状病毒S蛋白的单克隆抗体和细胞毒性药物,例如,微管相关抑制剂(MMAE、MMAF)等。在一些实施例中,该组合物可以包括疫苗。
根据本申请的另一方面,还提供了一种检测新型冠状病毒的试剂盒。该试剂盒可以包括上述抗新型冠状病毒S蛋白的单克隆抗体。以该抗新型冠状病毒S蛋白的单克隆抗体为原料开发的检测试剂盒具备很好的应用价值,具有高的灵敏性和特异性,并且可以早期检测出患有新型冠状病毒的患者。
根据本申请的另一方面,还提供了上述抗新型冠状病毒S蛋白的单克隆抗体在制备预防、抑制和/或治疗抗新型冠状病毒的产品中的应用以及在制备检测抗新型冠状病毒S蛋白的试剂中的应用。
实施例
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂公司购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
构建重组质粒
1、重组载体的构建
取灭活的SARS-CoV-2毒株(来源于浙江省疾病预防控制中心),提取病毒的RNA,反转录为cDNA模板,根据SARS-CoV-2的RBD蛋白的序列(Genebank登录号:43740568),利用带有BamH I和EcoR I的限制性内切酶位点的引物(正向引物为:GAATTCATGAGAGTCCAACCAACAGAATC(SEQ ID NO:17);反向引物为:GGATCCGAAATTGACACATTTGTTTT(SEQ ID NO:18))从cDNA模板中PCR扩增669bp的RBD片段。PCR条件为:94℃预变性5分钟,94℃30秒,60℃30秒,72℃1分钟,35次循环,72℃延伸10分钟。
获得PCR产物后,将PCR产物连接到pMD18T载体(购自TAKARA),得到pMD18T-RBD,然后通过热击法将连接产物转化DH5a感受态细胞,测序挑选RBD核苷酸序列完全正确的克隆。酶切验证后,测序正确,将pMD18T-RBD进行EcoR I和BamH I双酶切,之后连接到经过同样酶切的pcDNA3.1/HIS A真核表达载体中,得到重组载体。
2、转染HEK293F细胞
将重组载体转化大肠杆菌,按常规方法进行质粒扩增,之后用大提质粒试剂盒(购自天根生物)提取质粒,去除内毒素;按照Lipofectin试剂盒手册配制DNA-脂质体混合物,加入DMEM培养基培养的HEK293F细胞中,37℃温育2小时;换液成含10%BSF的DMEM培养基,继续培养48小时。
3、NEO抗性克隆筛选
把转染后的细胞从培养瓶中分离,按1×105细胞/孔加到96孔板中,以含500微克/ml NEO的DMEM培养基(加10%BSF)继续培养转染后的细胞,经7天后,选取形成克隆的细胞,扩增培养到6孔板。
4、表达RBD克隆分析
NEO抗性克隆经培养后,以1.5×105/ml细胞密度接种到T25培养瓶,在含5%的CO2培养箱中37℃培养72小时,取上清进行RBD蛋白含量分析。
制备SARS-CoV-2-RBD蛋白
参照GE公司Glutathione Ni Sepharose 6Fast Flow4B使用指南过柱收集目标蛋白。
具体操作如下:
(1)样品的澄清过滤:使用50ml注射器以及0.22μm滤膜将HEK293F细胞表达的细胞悬液上清进行澄清;
(2)采用蛋白层析柱,在AKTA上进行捕获、纯化;
(3)进行系统冲洗,再用平衡液冲洗AKTA的A1泵,洗脱液冲洗B1泵;
(4)设置好系统流速0.1ml/min,选择相应的柱位1号位连接蛋白层析柱,用平衡液对AKTA及柱子进行平衡,平衡结束后紫外调零;
(5)开始上样,将A1泵转移至上样离心管内上样;
(6)上样结束后,将A1泵转移至平衡缓冲液中,冲洗平衡液至检测波长稳定后分布洗脱,收集洗脱液;
(7)再用平衡液A冲洗,最后冲20%的乙醇,保存。
结果,用洗脱缓冲液从柱上洗脱重组6X His标记的RBD蛋白,用SDS-PAGE分析纯化蛋白,用考马斯亮蓝染色法观察,从而获得高纯度的SARS-CoV-2-RBD蛋白。
单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)的制备
选用6-8周龄且健康的雌性Balb/c小鼠,按照预先指定的免疫方案进行免疫注射。上述步骤中获得的SARS-CoV-2-RBD蛋白作为免疫原,免疫Balb/c小鼠,提取免疫成功的小鼠脾脏淋巴细胞。通过细胞融合技术将淋巴细胞与小鼠骨髓瘤细胞SP2/0进行融合。经过二轮亚克隆筛选后获得稳定分泌抗SARS-CoV-2-RBD的单克隆抗体的杂交瘤细胞株,从而获得抗SARS-CoV-2-RBD单克隆抗体。
利用前述表达的SARS-CoV-2-RBD蛋白,进行动物免疫实验。
动物免疫实验具体步骤包括:
1、将体重、周龄均值一致的Balb/c小鼠随机分为3组(根据上述步骤中获得的免疫抗原RBD蛋白的剂量分了3组,分别为:A组小鼠中含有的免疫抗原的计量为50ug/只,B组小鼠中含有的免疫抗原的计量为100ug/只,C组小鼠中含有的免疫抗原的计量为150ug/只,每一组小鼠都有对照组)。
2、实验开始前,各收集小鼠免疫前血清(第五天通过眼球取血,适量取血,保证小鼠正常状态)作为阴性对照,收集好的血清存于-80℃。
3、加铝佐剂(氢氧化铝佐剂)组配制方式:免疫前,每一抗原在75μL PBS中分别稀释至对应剂量(50ug/只,100ug/只,150ug/只小鼠),并与明矾佐剂(即氢氧化铝佐剂)(1mg/只小鼠)混合,按照体积抗原:佐剂=3:1(即75ul免疫原稀释液中加入25ul佐剂);佐剂使用前摇匀,将注射佐剂(25ul)缓慢滴加至免疫原溶液中;佐剂与免疫原稀释液充分混合后,两者充分混合30分钟。使佐剂有效吸附抗原;后续根据免疫动物实验操作进行。
4、不加铝佐剂组:抗原在100μL的PBS中分别稀释至对应剂量,即50ug/只,100ug/只,150ug/只小鼠,免疫原为100μL稀释后的抗原,后续根据免疫动物实验操作进行。
5、以2周的间隔皮下注射:实验设计为3次免疫方式,但分别在每一次免疫注射7天后眼球取血,离心法获得部分小鼠上清,先进行血清抗体滴度检测,最后一次免疫结束7天后,心脏取血取最大血量,离心获得上清,存于-80℃。3次免疫后的部分检测数据如下表1所示,其中,A1、A2、B1、B2、C1分别表示小鼠的编号,A1和A2属于A组,B1和B2属于B组,C1属于C组。
表1 3次免疫后血清抗体效价检测数据
免疫脾细胞与骨髓瘤细胞系SP2/0细胞融合,通过HAT选择培养基(HAT选择培养基含有次黄嘌呤、氨基喋呤和胸腺嘧啶)筛选融合细胞,并对融合细胞进行ELISA阳性筛选及亚克隆;筛选出的阳性单克隆,取腹水,用Protein A/G抗体纯化柱纯化杂交瘤细胞产生的腹水抗体,纯化后的腹水抗体ELISA效价>1:128,000,纯度>90%,此数据通过下步进行确定的。
酶联反应ELISA检测识别RBD蛋白的结合活性
1、IgG抗体滴度检测方式
(1)底板包被:将所用抗原用包被稀释液稀释到3ug/ml,每孔各加入配好的包被液100μl,置4℃冰箱,放置24h。
(2)24h后,从冰箱取出后置于37℃平衡30min,之后弃去孔中液体;用洗涤液满孔洗涤3遍,每遍3min。
(3)封闭酶标反应孔:每孔加入200ul 5%小牛血清后置37℃封闭90min,封闭结束后用洗涤液满孔洗涤3遍,每遍3min。
(4)加入待检测样品:将样本按照需要的比例进行稀释,将稀释好的样品加入酶标反应孔中,每孔100μl,置于37℃,90min;用洗涤液满孔洗涤3遍,每遍3min。
(5)加入酶标抗体:按照说明书加入适宜浓度的二抗;37℃,90min之间,每孔加100μl洗涤同前。
(6)加入底物液:底物加入量每孔100μl,置37℃避光放置15~30min。
(7)终止反应:每孔加入终止液50μl终止反应,于20min内测定实验结果。
检测单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)识别RBD蛋白的结合活性
(1)、细胞融合和克隆筛选数据
共进行5轮融合后,小鼠编号依次为A1、A2、B1、B2、C1;A2小鼠融合筛选共挑出20个阳性克隆进行亚克隆,最终完成了14个细胞株;经融合筛选,共挑选出140个OD450值>2.2以上的阳性克隆进行倍比稀释检测效价,再进行第二次亚克隆筛选。得到4个细胞株,分别命名为A2-1到A2-5。B1小鼠融合筛选共挑出56个阳性孔进行亚克隆,经融合筛选,共挑选出110个OD450值>2.1以上的阳性孔进行倍比稀释检测效价,再进行第二次和第三次亚克隆筛选,最终完全了5个细胞株,分别命名为B1-1到B1-5。其余小鼠融合未筛选到阳性孔。
(2)、腹水制备和检测数据
A2小鼠总共5个杂交瘤细胞株,B1小鼠总共5个杂交瘤细胞株,每个完全细胞株分别打3只F1小鼠,共制备了10个腹水,所有腹水检测效价数据如下表2:
表2腹水抗体检测效价数据
(3)、抗体纯化条件摸索和检测数据
稀释倍数 | A2-1 | A2-2 | A2-3 | A2-4 | A2-5 | B1-1 | B1-2 | B1-3 | B1-4 | B1-5 |
100 | 1.967 | 1.529 | 1.87 | 1.394 | 1.874 | 1.425 | 1.85 | 1.587 | 1.927 | 2.119 |
500 | 1.566 | 1.491 | 1.778 | 1.359 | 0.897 | 1.306 | 1.608 | 1.327 | 1.548 | 1.802 |
2500 | 1.273 | 1.46 | 1.603 | 1.427 | 0.38 | 1.398 | 1.003 | 1.132 | 0.589 | 0.231 |
12500 | 0.773 | 1.061 | 0.715 | 0.84 | 0.176 | 1.256 | 0.297 | 0.647 | 0.211 | 0.105 |
62500 | 0.519 | 0.508 | 0.373 | 0.373 | 0.092 | 0.545 | 0.118 | 0.255 | 0.099 | 0.08 |
312500 | 0.158 | 0.155 | 0.147 | 0.121 | 0.071 | 0.19 | 0.091 | 0.111 | 0.07 | 0.075 |
1562500 | 0.139 | 0.077 | 0.114 | 0.072 | 0.067 | 0.084 | 0.081 | 0.088 | 0.073 | 0.08 |
PBS | 0.124 | 0.095 | 0.093 | 0.086 | 0.089 | 0.084 | 0.078 | 0.171 | 0.083 | 0.144 |
以上腹水经3.3%正辛酸-硫胺沉淀法纯化,共得到10个抗体,所有抗体的效价检测数据见下表3:
表3所有抗体的效价检测数据
稀释浓度 | A2-1 | A2-2 | A2-3 | A2-4 | A2-5 | B1-1 | B1-2 | B1-3 | B1-4 | B1-5 |
20ug/ml | 1.848 | 1.721 | 2.196 | 1.954 | 2.093 | 1.352 | 2.044 | 2.015 | 2.126 | 2.315 |
4ug/ml | 1.729 | 1.616 | 2.048 | 1.335 | 1.588 | 1.192 | 1.487 | 1.323 | 1.787 | 1.931 |
800ng/ml | 1.489 | 1.268 | 1.609 | 0.584 | 0.752 | 0.929 | 0.647 | 0.408 | 1.262 | 1.406 |
160ng/ml | 1.149 | 0.689 | 1.009 | 0.279 | 0.345 | 0.38 | 0.26 | 0.254 | 0.615 | 0.851 |
32ng/ml | 0.587 | 0.447 | 0.423 | 0.149 | 0.241 | 0.154 | 0.127 | 0.122 | 0.274 | 0.522 |
6.4ng/ml | 0.241 | 0.229 | 0.157 | 0.126 | 0.114 | 0.088 | 0.085 | 0.077 | 0.185 | 0.317 |
1.28ng/ml | 0.154 | 0.115 | 0.11 | 0.106 | 0.118 | 0.072 | 0.073 | 0.08 | 0.073 | 0.18 |
PBS | 0.127 | 0.109 | 0.101 | 0.109 | 0.08 | 0.081 | 0.112 | 0.095 | 0.052 | 0.095 |
根据上表3,将B1-5号抗体命名Mouse anti-SARS-COV-2 Mab1(RBD-Ab),其具有高效价、高纯度、高灵敏度和高特异性。单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)可用于ELISA、免疫层析、免疫印迹、免疫荧光等免疫学检测以及新冠药物,为新冠药物的研究和开发提供了基础。
为检测Mouse anti-SARS-COV-2 Mab1(RBD-Ab)抗体是否特异结合新冠病毒2019-nCoV(SARS-CoV-2)的S蛋白的RBD蛋白,从北京义翘神州公司购买了SARS病毒的RBD蛋白(SARS-CoV Spike/RBD Protein(RBD,His Tag),货号40150-V08B2)和MERS病毒的RBD蛋白(MERS-CoV Spike/RBD Protein fragment(RBD,aa 367-606,His Tag),货号40071-V08B1)。通过酶联反应ELISA检测Mouse anti-SARS-COV-2 Mab1(RBD-Ab)抗体是否结合SARS-CoV-RBD和MERS-CoV-RBD,交叉实验结果数据见下表4:
表4交叉实验结果数据
以上数据显示单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)只对SARS-CoV-2-RBD抗原(即新型冠状病毒的RBD蛋白)具有良好的特异性结合能力,而不结合SARS-CoV-RBD和MERS-CoV-RBD抗原,对检测新冠病毒具有特异性。
对单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)的重链可变区及轻链可变区进行序列分析
设计扩增单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)的重链可变区及轻链可变区基因的引物。
取单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)对数生长期的杂交瘤细胞株(约107个细胞),按照Trizol RNA提取试剂盒的说明书抽提细胞的总RNA,以总RNA为模板反转录合成cDNA第一链,以上述扩增产物为模板PCR扩增抗体的重链可变区和轻链可变区基因。
回收单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)的重链可变区和轻链可变区基因片段,进行测序。测序结果如下:
编码单克隆抗体Mouse anti-SARS-COV-2 Mab1(RBD-Ab)的重链可变区的核苷酸序列(339bp)如下:
GTGCAGCTGCAGGAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTAACTACAATGAGAAGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCGGTTTATTTCTGTGCAAGAGGCCGGGCGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:15)
编码重链可变区的CDRH1的序列为:GGATACGCCTTCACTAATTACTTG(SEQ ID NO:9)。
编码重链可变区的CDRH2的序列为:ATTAATCCTGGAAGTGGTGGTACT(SEQ ID NO:10)。
编码重链可变区的CDRH3的序列为:GCAAGAGGCCGGGCGGACTAC(SEQ ID NO:11)。
重链可变区的氨基酸序列(113aa)如下所示:
VKLQESGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARGRADYWGQGTSVTVSS(SEQ ID NO:7)。
重链可变区的CDRH1的氨基酸序列为:GYAFTNYL(SEQ ID NO:1)。
重链可变区的CDRH2的氨基酸序列为:INPGSGGT(SEQ ID NO:2)。
重链可变区的CDRH3的氨基酸序列为:ARGRADY(SEQ IDNO:3)。
编码单克隆抗体MOUSEANTI-SARS-COV-2 MAB1(RBD-AB)的轻链可变区的核苷酸序列(321bp)如下所示:
GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCTTTAGCAGTAATATAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCAGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO:16)。
编码轻链可变区的CDRL1的序列为:CAGGGCTTTAGCAGTAAT(SEQ ID NO:12)。
编码轻链可变区的CDRL2的序列为:CATGGAACC(SEQ ID NO:13)。
编码轻链可变区的CDRL3的序列为:GTACAGTATGCTCAGTTTCCGTACACG(SEQ ID NO:14)。
轻链可变区的氨基酸序列(107aa)如下所示:
DILMTQSPSSMSVSLGDTVSITCHASQGFSSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPYTFGGGTKLEIK(SEQ ID NO:8)
轻链可变区的CDRL1的氨基酸序列为:QGFSSN(SEQ ID NO:4)。
轻链可变区的CDRL2的氨基酸序列为:HGT(SEQ ID NO:5)。
轻链可变区的CDRL3的氨基酸序列为:VQYAQFPYT(SEQ ID NO:6)。
本申请所披露的一种,带来的有益效果包括但不限于:(1)本申请得到的抗新型冠状病毒S蛋白的单克隆抗体具有高纯度、高灵敏度和高特异性;(2)本申请的抗新型冠状病毒S蛋白的单克隆抗体可用于ELISA、免疫层析、免疫印迹、免疫荧光等免疫学检测,为免疫学检测试剂(例如,检测S蛋白免疫试纸条)提供了所需的原料;(3)本申请的抗新型冠状病毒S蛋白的单克隆抗体对于预防、抑制和/或治疗新型冠状病毒的药物(例如,疫苗)开发具有广泛的应用前景。需要说明的是,不同实施例可能产生的有益效果不同,在不同的实施例里,可能产生的有益效果可以是以上任意一种或几种的组合,也可以是其他任何可能获得的有益效果。
本领域的技术人员应当理解,以上实施例仅为说明本发明,而不对本发明构成限制。凡在本发明的精神和原则内所作的任何修改、等同替换和变动等,均应包含在本发明的保护范围之内。
序列表
<110> 杭州旭科生物技术有限公司
<120> 抗新型冠状病毒S蛋白的单克隆抗体及其应用
<160> 18
<170> SIPOSequenceListing 1.0
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<211> 8
<212> PRT
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Gly Tyr Ala Phe Thr Asn Tyr Leu
1 5
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Ile Asn Pro Gly Ser Gly Gly Thr
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Ala Arg Gly Arg Ala Asp Tyr
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Gln Gly Phe Ser Ser Asn
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<213> 人工序列(Artificial Sequence)
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His Gly Thr
1
<210> 6
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<213> 人工序列(Artificial Sequence)
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Val Gln Tyr Ala Gln Phe Pro Tyr Thr
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<210> 7
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
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Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser
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Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Leu
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Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
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Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys
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Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
65 70 75 80
Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys Ala
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Arg Gly Arg Ala Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
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Ser
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<213> 人工序列(Artificial Sequence)
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Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly
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Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Phe Ser Ser Asn
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ggatacgcct tcactaatta cttg 24
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<212> DNA
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attaatcctg gaagtggtgg tact 24
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<212> DNA
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gcaagaggcc gggcggacta c 21
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<400> 12
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catggaacc 9
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<212> DNA
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gtacagtatg ctcagtttcc gtacacg 27
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gtgcagctgc aggagtctgg agctgagctg gtaaggcctg ggacttcagt gaaggtgtcc 60
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ggacagggcc ttgagtggat tggagtgatt aatcctggaa gtggtggtac taactacaat 180
gagaagttca agggcaaggc aacactgact gcagacaaat cctccagcac tgcctacatg 240
cagctcagca gcctgacatc tgatgactct gcggtttatt tctgtgcaag aggccgggcg 300
gactactggg gtcaaggaac ctcagtcacc gtctcctca 339
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<211> 321
<212> DNA
<213> 人工序列(Artificial Sequence)
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gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc 60
atcacttgcc atgcaagtca gggctttagc agtaatatag ggtggttgca gcagaaacca 120
gggaaatcat ttaagggcct gatctatcat ggaaccaact tggaagatgg agttccatca 180
aggttcagtg gcagtggatc tggagcagat tattctctca ccatcagcag cctggaatct 240
gaagattttg cagactatta ctgtgtacag tatgctcagt ttccgtacac gttcggaggg 300
gggaccaagc tggaaataaa a 321
<210> 17
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
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gaattcatga gagtccaacc aacagaatc 29
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ggatccgaaa ttgacacatt tgtttt 26
Claims (10)
1.一种抗新型冠状病毒S蛋白的单克隆抗体,其特征在于,包括重链可变区和轻链可变区,其中,
所述重链可变区包括氨基酸序列为SEQ ID NO:1所示的CDRH1、氨基酸序列为SEQ IDNO:2所示的CDRH2和氨基酸序列为SEQ ID NO:3所示的CDRH3;以及
所述轻链可变区包括氨基酸序列为SEQ ID NO:4所示的CDRL1、SEQ ID NO:5所示的CDRL2和SEQ ID NO:6所示的CDRL3。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述重链可变区的氨基酸序列为SEQ ID NO:7所示,所述轻链可变区的氨基酸序列为SEQ ID NO:8所示。
3.一种核酸分子,其特征在于,所述核酸分子包括编码权利要求1或2所述的抗新型冠状病毒S蛋白的单克隆抗体的核苷酸序列。
4.根据权利要求3所述的核酸分子,其特征在于,编码所述重链可变区的CDRH1、CDRH2和CDRH3的核苷酸序列分别为SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示,编码所述轻链可变区的CDRL1、CDRL2和CDRL3的核苷酸序列分别为SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示。
5.根据权利要求4所述的核酸分子,其特征在于,编码所述重链可变区的核苷酸序列为SEQ ID NO:15所示,编码所述轻链可变区的核苷酸序列为SEQ ID NO:16所示。
6.一种组合物,其特征在于,所述组合物包括权利要求1或2所述的抗新型冠状病毒S蛋白的单克隆抗体,其中,所述组合物用于预防、抑制和/或治疗抗新型冠状病毒。
7.权利要求1或2所述的抗新型冠状病毒S蛋白的单克隆抗体在制备预防、抑制和/或治疗抗新型冠状病毒的产品中的应用。
8.一种检测新型冠状病毒的试剂盒,其特征在于,所述试剂盒包括权利要求1或2所述的抗新型冠状病毒S蛋白的单克隆抗体。
9.权利要求1或2所述的抗新型冠状病毒S蛋白的单克隆抗体在制备检测抗新型冠状病毒S蛋白的试剂中的应用。
10.根据权利要求9所述的应用,其特征在于,所述抗新型冠状病毒S蛋白的单克隆抗体用于新型冠状病毒的免疫学检测。
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