CN114213531B - 抗新型冠状病毒中和抗体、其抗原结合片段及其应用 - Google Patents
抗新型冠状病毒中和抗体、其抗原结合片段及其应用 Download PDFInfo
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Abstract
本发明公开了抗新型冠状病毒中和抗体、其抗原结合片段及其应用。具体地公开了特异性结合新型冠状病毒S蛋白受体结合域(RBD)的抗体或其抗原结合片段。本发明通过抗体库的筛选获得了能够特异性结合RBD的鼠源单链抗体A‑1F,在此基础上开发了人鼠嵌合抗体mhA‑1F和人源化抗体HSA‑1F。本发明的抗体mhA‑1F和HSA‑1F具有较高的人源化程度,不易引发免疫原性反应,亲和力高,能高效地中和新型冠状病毒,可制成临床上用于预防和治疗新型冠状病毒肺炎的特异性抗体药物,或制成SARS‑CoV‑2的诊断试剂或试剂盒等,在药物应用和临床诊断等领域具有十分广阔的前景和重要的意义。
Description
技术领域
本发明属于生物医药领域,具体涉及抗新型冠状病毒中和抗体、其抗原结合片段及其应用。
背景技术
新型冠状病毒(SARS-CoV-2)与SARS-CoV基因组的同源性约为80%,主要引起新型冠状病毒肺炎。SARS-CoV-2隶属冠状病毒的β亚属,为单股正链的RNA病毒,全长约30kb,由蛋白质和RNA组成,其膜表面主要由3种结构蛋白组成:刺突蛋白(Spike Protein,S蛋白)、包膜蛋白(Envelop Protein,E蛋白)和膜蛋白(Membrane Protein, M蛋白)。病毒里面是负责病毒繁殖的核酸物质RNA,它由核衣壳蛋白(Nucleoprotein, N蛋白)包裹并保护着。在这四种蛋白中,最重要的就是刺突蛋白(S蛋白),S蛋白是形成病毒“冠状”形态的主要蛋白之一,介导SARS-CoV-2进入细胞,其分为S1区与S2区,起主要感染作用的受体结合域(receptor binding domain,RBD)就位于 S1区。病毒感染人体时,其RBD会与哺乳动物细胞表面受体血管紧张素转换酶2 (angiotensin converting enzyme 2,ACE2)结合,引起自身构象变化,使疏水性的融合肽与细胞膜接近并融合介导病毒进入细胞内,从而侵染受体细胞,因此, SARS-CoV-2病毒的RBD结构决定了其与潜在受体ACE2结合的效率以及感染的种属特异性,是重要的中和抗体识别与开发靶点。
人体感染SARS-CoV-2病毒后的主要症状为发烧或轻微咳嗽,部分患者会发展为肺炎,甚至死亡。目前尚没有针对SARS-CoV-2的特异性治疗药物用于临床,临床上以对症支持治疗为主,仍缺乏特异、高效的抗病毒药物。
抗体(antibody,Ab)是B细胞被B细胞抗原表位特异性刺激后增殖分化为浆细胞所产生的效应免疫分子,介导体液免疫。当抗体与病原体表面,或者细菌毒素关键表位相结合,则封闭了病原体或毒素的毒力结构,使病毒丧失感染能力,并使毒素丧失毒力,称为中和作用(neutralization)。大部分抗体是通过向T-淋巴细胞发出锁定抗原的信号,激发细胞免疫反应,杀死病毒,而中和抗体是B淋巴细胞产生的能够与病原微生物表面的抗原结合的一类抗体,是特异针对病毒中和表位产生的抗体,可直接靶定到病毒中和表位,使病毒失去结合受体的能力。
作为天然适应性免疫系统的一部分,中和抗体(neutralizing antibody)在人体抵抗病毒感染的过程中起到了不可或缺的作用。中和抗体具备阻断病毒感染目的细胞的潜力。研究表明,对患有新型冠状病毒并同时伴有急性呼吸窘迫综合征(ARDS)的危重症患者使用含有中和抗体的康复期血浆治疗后,病人体内的病毒载量迅速降低,患者的临床症状得到有效改善。这些研究表明了体液免疫在SARS-CoV-2中的重要性,显示出中和抗体在SARS-CoV-2治疗方面的潜力。研究和开发靶向SARS-CoV-2的中和抗体药物,作为增强SARS-CoV-2治疗和预防的手段具有重要的意义。
发明内容
本发明所要解决的技术问题是提供一种能够有效抑制新型冠状病毒SARS-CoV-2的中和抗体(抗新型冠状病毒中和抗体)及其人源化抗体、抗原结合片段,和应用。所要解决的技术问题不限于如所描述的技术主题,本领域技术人员通过以下描述可以清楚地理解本文未提及的其它技术主题。
为解决上述技术问题,本发明首先提供了特异性结合新型冠状病毒S蛋白受体结合域的抗体或其抗原结合片段,所述抗体包含重链可变区和轻链可变区,所述重链可变区包含氨基酸序列分别是SEQ ID No.1、SEQ ID No.2和SEQ ID No.3的HCDR1、HCDR2 和HCDR3;所述轻链可变区包含氨基酸序列分别是SEQ ID No.4、SEQ ID No.5和SEQ ID No.6的LCDR1、LCDR2和LCDR3。
所述抗体为特异性结合新型冠状病毒(SARS-CoV-2)S蛋白(刺突蛋白,SpikeProtein)受体结合域(receptor binding domain,RBD)的中和抗体,又称抗新型冠状病毒中和抗体,所述抗体可为鼠源抗体、嵌合抗体、人源化抗体或人源抗体。
其中,HCDR1、HCDR2和HCDR3为重链可变区中的三个互补决定区(CDR),LCDR1、LCDR2和LHCDR3为轻链可变区中的三个互补决定区(CDR)。互补决定区的序列根据 Kabat编号系统定义。
所述抗体可为全长抗体,所述抗原结合片段可为Fab片段、Fv片段、Fab′片段、 F(ab′)2片段、单链抗体(ScFv)、纳米抗体(单域抗体)、双特异性抗体或最小识别单位(MRU)。
进一步地,上述抗体或其抗原结合片段中,所述重链可变区的氨基酸序列可为SEQID No.7的第1-118位或与SEQ ID No.7的第1-118位具有至少90%的同一性;所述轻链可变区的氨基酸序列可为SEQ ID No.8的第1-107位或与SEQ ID No.8的第1-107 位具有至少90%的同一性。其中,氨基酸序列的不一致处可在框架区(FR)。
在本发明的一个实施例中,上述抗体为人鼠嵌合抗体,名称为mhA-1F。
上述抗体或其抗原结合片段中,所述重链可变区的氨基酸序列可为SEQ ID No.9的第1-118位或与SEQ ID No.9的第1-118位具有至少90%的同一性;所述轻链可变区的氨基酸序列可为SEQ ID No.10的第1-107位或与SEQ ID No.10的第1-107位具有至少90%的同一性。其中,氨基酸序列的不一致处可在框架区(FR)。
在本发明的一个实施例中,上述抗体可为人源化抗体,名称为HSA-1F。
所述人源化抗体可包含来源于人抗体的框架区或框架区变体。
在本发明保护范围内,所述抗体还包括选自IgG1或IgG4的重链恒定区和包含选自kappa或Lambda亚型的轻链恒定区。
上述抗体或其抗原结合片段中,所述抗体的重链的氨基酸序列可为SEQ ID No.7或与SEQ ID No.7具有至少80%的同一性;所述抗体的轻链的氨基酸序列可为SEQ ID No.8或与SEQ ID No.8具有至少80%的同一性。其中,氨基酸序列的不一致处可在框架区(FR)。
在本发明的一个实施例中,上述抗体为人鼠嵌合抗体mhA-1F。
上述抗体或其抗原结合片段中,所述抗体的重链的氨基酸序列可为SEQ ID No.9或与SEQ ID No.9具有至少80%的同一性;所述抗体的轻链的氨基酸序列可为SEQ IDNo.10或与SEQ ID No.10具有至少80%的同一性。其中,氨基酸序列的不一致处可在框架区(FR)。
在本发明的一个实施例中,上述抗体为人源化抗体HSA-1F。
在本发明的一个实施方案中,所述抗原结合片段为单链抗体(ScFv),名称为A-1F。所述单链抗体A-1F也在本发明的保护范围内。所述单链抗体A-1F的重链可变区的氨基酸序列为SEQ ID No.7的第1-118位;轻链可变区的氨基酸序列为SEQ ID No.8的第1-107位;所述重链可变区包含氨基酸序列分别是SEQ ID No.1、SEQ ID No.2、和 SEQ ID No.3的HCDR1、HCDR2和HCDR3;所述轻链可变区包含氨基酸序列分别是SEQ ID No.4、SEQ ID No.5、和SEQ ID No.6的LCDR1、LCDR2和LCDR3。
上述至少80%或至少90%的同一性,可为至少80%、85%或95%的同一性。
本文中,同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter 设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambdaratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
本文中,所述至少80%的同一性可为至少80%、81%、82%、83%、84%、85%、86%、 87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同一性。
本文中,所述至少90%的同一性可为至少90%、91%、92%、93%、94%、95%、96%、 97%、98%或99%的同一性。
具有改善的亲和力和/或效价的本发明所述抗体的变体可通过采用在本领域已知的方法来获得,并包括在本发明的范围内。例如,氨基酸置换可用于获得具有进一步改善的亲合力的抗体。或者,核苷酸序列的密码子优化也可用来改善用于产生抗体的表达系统中的翻译效率。此外,包含通过对本发明的任何核酸序列应用定向进化法而优化抗体特异性或中和活性的序列的多核苷酸也属于本发明的范围内。
本发明还提供了生物材料,所述生物材料可为下述B1)至B7)中的任一种:
B1)编码所述抗体或其抗原结合片段的核酸分子;
B2)编码所述抗体或其抗原结合片段的重链和/或轻链的核酸分子;
B3)编码所述抗体或其抗原结合片段的重链可变区和/或轻链可变区的核酸分子;
B4)含有B1)-B3)任一所述核酸分子的表达盒;
B5)含有B1)-B3)任一所述核酸分子的重组载体、或含有B4)所述表达盒的重组载体;
B6)含有B1)-B3)任一所述核酸分子的重组微生物、或含有B4)所述表达盒的重组微生物、或含有B5)所述重组载体的重组微生物;
B7)含有B1)-B3)任一所述核酸分子的细胞系、或含有B4)所述表达盒的细胞系、或含有B5)所述重组载体的细胞系。
其中,B6)所述的重组微生物和B7)所述的细胞系可表达所述抗体或其抗原结合片段。
上述生物材料中,所述核酸分子可为下述任一种:
C1)编码序列是SEQ ID No.11或SEQ ID No.13的重链DNA分子;
C2)编码序列是SEQ ID No.12或SEQ ID No.14的轻链DNA分子;
C3)编码序列是SEQ ID No.11的第1-354位或SEQ ID No.13的第1-354位的重链可变区DNA分子;
C4)编码序列是SEQ ID No.12的第1-321位或SEQ ID No.14的第1-321位的轻链可变区DNA分子。
在本发明的一个实施例中,所述人鼠嵌合抗体mhA-1F的重链核苷酸序列为SEQ IDNo.11;轻链核苷酸序列为SEQ ID No.12;重链可变区的核苷酸序列为SEQ ID No.11 的第1-354位;轻链可变区的核苷酸序列为SEQ ID No.12的第1-321位;其中:
重链可变区HCDR1的核苷酸序列是SEQ ID No.11的第76-99位;
重链可变区HCDR2的核苷酸序列是SEQ ID No.11的第151-174位;
重链可变区HCDR3的核苷酸序列是SEQ ID No.11的第289-321位;
轻链可变区LCDR1的核苷酸序列是SEQ ID No.12的第79-96位;
轻链可变区LCDR2的核苷酸序列是SEQ ID No.12的第148-156位;
轻链可变区LCDR3的核苷酸序列是SEQ ID No.12的第265-291位。
在本发明的一个实施例中,所述人源化抗体HSA-1F的重链核苷酸序列为SEQ IDNo.13;轻链核苷酸序列为SEQ ID No.14;重链可变区的核苷酸序列为SEQ ID No.13 的第1-354位;轻链可变区的核苷酸序列为SEQ ID No.14的第1-321位;其中:
重链可变区HCDR1的核苷酸序列是SEQ ID No.13的第76-99位;
重链可变区HCDR2的核苷酸序列是SEQ ID No.13的第151-174位;
重链可变区HCDR3的核苷酸序列是SEQ ID No.13的第289-321位;
轻链可变区LCDR1的核苷酸序列是SEQ ID No.14的第79-96位;
轻链可变区LCDR2的核苷酸序列是SEQ ID No.14的第148-156位;
轻链可变区LCDR3的核苷酸序列是SEQ ID No.14的第265-291位。
在本发明的一个实施例中,所述单链抗体A-1F的重链可变区的核苷酸序列为SEQID No.11的第1-354位;轻链可变区的核苷酸序列为SEQ ID No.12的第1-321位;其中:
重链可变区HCDR1的核苷酸序列是SEQ ID No.11的第76-99位;
重链可变区HCDR2的核苷酸序列是SEQ ID No.11的第151-174位;
重链可变区HCDR3的核苷酸序列是SEQ ID No.11的第289-321位;
轻链可变区LCDR1的核苷酸序列是SEQ ID No.12的第79-96位;
轻链可变区LCDR2的核苷酸序列是SEQ ID No.12的第148-156位;
轻链可变区LCDR3的核苷酸序列是SEQ ID No.12的第265-291位。
本文所述载体是本领域技术人员公知的,包括但不限于:质粒、噬菌体(如λ噬菌体或M13丝状噬菌体等)、黏粒(即柯斯质粒)、病毒载体(如杆状病毒载体、逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒或疱疹病毒(如单纯疱疹病毒)等)。在本发明的一个实施例中,载体具体可为pADSCFV-S载体或pcDNA3.1(+)载体。
本文所述微生物可为酵母、细菌或真菌。其中,细菌可来自埃希氏菌属(Escherichia),欧文氏菌(Erwinia),根癌农杆菌属(Agrobacterium)、黄杆菌属(Flavobacterium),产碱菌属(Alcaligenes),假单胞菌属(Pseudomonas),芽胞杆菌属(Bacillus)等;酵母可为毕赤酵母(P.pastoris)。
所述细胞系(宿主细胞)是指可用于导入载体的细胞,其包括但不限于:真核细胞(如酵母细胞、曲霉菌)、动物细胞(如哺乳动物细胞、昆虫细胞)或原核细胞。在本发明的一个实施例中,所述细胞系具体可为HEK293-F细胞。
术语“细胞”和“细胞系”可互换使用,并且所有这类名称都包括其后代。
在本发明的一个实施例中,所述重组载体具体可为pcDNA3.1-mhA-1FH和/或pcDNA3.1-mhA-1FK。
所述重组载体pcDNA3.1-mhA-1FH是用鼠源单链抗体A-1F重链可变区(VH)基因(核苷酸序列是SEQ ID No.11的第1-354位)与人IgG1恒定区重链基因(核苷酸序列是GenBank Accession No.BC016381.1(Update Date 24-MAR-2009)的第510-1502 位核苷酸)连接得到的融合基因,替换pcDNA3.1(+)载体的HindIII识别位点和BamH I识别位点之间的片段,得到的表达嵌合抗体mhA-1F重链的重组表达载体。
所述重组载体pcDNA3.1-mhA-1FK是用鼠源单链抗体A-1F轻链可变区(VK,VL) 基因(核苷酸序列是SEQ ID No.12的第1-321位)与人Kappa恒定区轻链基因(核苷酸序列是GenBank Accession No.AM408494.1(Update Date 12-JAN-2007)的第565-888 位核苷酸)连接得到的融合基因,替换pcDNA3.1(+)载体的Hind III识别位点和BamH I识别位点之间的片段,得到的表达嵌合抗体mhA-1F轻链的重组表达载体。
所述重组细胞具体可为将所述重组载体pcDNA3.1-mhA-1FH和pcDNA3.1-mhA-1FK导入宿主细胞(如HEK293-F细胞)得到的表达嵌合抗体mhA-1F的重组细胞。
本发明还提供了制备所述抗体或其抗原结合片段的方法,所述方法包括,在允许所述抗体或其抗原结合片段表达的条件下,培养所述重组细胞,和从培养的重组细胞培养物中回收所述抗体或其抗原结合片段。
本发明还提供了药物组合物,所述药物组合物包含所述抗体或其抗原结合片段,以及一种或多种药学上可接受的载体。
所述药学上可接受的载体可为稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂但不限于此。
其中,所述药物组合物具有抑制或中和SARS-CoV-2活性的中和抗病毒作用。所述药物组合物用于改善、预防或治疗SARS-CoV-2感染引起的疾病和/或用于抑制 SARS-CoV-2感染。
所述抑制或中和SARS-CoV-2活性包括特异性结合SARS-CoV-2刺突蛋白(S蛋白)RBD区域,从而使SARS-CoV-2失去结合受体ACE2的能力。
进一步地,本发明的药物组合物可包含第一抗体和第二抗体或其抗原结合片段,其中第一抗体为本发明的中和抗体,而第二抗体为抑制SARS-CoV-2病毒感染的其他任何抗体。
本发明还提供了缀合物(conjugate),所述缀合物包含本发明所述的抗体或其抗原结合片段,以及与所述抗体或其抗原结合片段连接的可检测的标记;具体地,所述可检测的标记可选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
本发明还提供了下述任一项所述的应用:
A1)所述生物材料在制备所述抗体或所述药物组合物中的应用;
A2)所述抗体,和/或,所述生物材料,和/或,所述药物组合物在制备用于预防和/或治疗SARS-CoV-2感染引起的疾病的产品中的应用;
A3)所述抗体,和/或,所述生物材料,和/或,所述药物组合物在制备用于抑制SARS-CoV-2感染的产品中的应用;
A4)所述抗体,和/或,所述生物材料,和/或,所述药物组合物在制备用于抑制或中和SARS-CoV-2的产品中的应用;
A5)所述抗体,和/或,所述生物材料,和/或,所述药物组合物在制备用于结合SARS-CoV-2的RBD蛋白的产品中的应用;
A6)所述抗体,和/或,所述生物材料在制备用于检测SARS-CoV-2和/或SARS-CoV-2的RBD蛋白的产品中的应用;
A7)所述抗体,和/或,所述生物材料在制备用于诊断或辅助诊断SARS-CoV-2感染引起的疾病的产品中的应用。
本发明的抗体通过检测抗SARS-CoV-2疫苗的抗原是否含有具有正确构象的特异性表位来监测所述疫苗的质量的用途也设想处于本发明的范围内。
所述用于检测SARS-CoV-2和/或SARS-CoV-2的RBD蛋白的产品包括利用酶联免疫吸附法、免疫荧光检测法、放射免疫法、发光免疫测定法、胶体金免疫层析法、凝集法或免疫比浊法等检测抗原抗体结合的产品。
上述应用中,所述产品可为药物、试剂或试剂盒。
所述药物、试剂或试剂盒含有本文任一所述抗体或其抗原结合片段或其组合。所述试剂盒可为化学发光免疫试剂盒、酶联免疫检测试剂盒、胶体金免疫试剂盒、或荧光免疫试剂盒,但不限于此。
上述应用中,所述SARS-CoV-2感染引起的疾病可为呼吸系统感染和/或消化系统感染。
所述呼吸系统感染可为呼吸道感染和/或肺部感染,所述呼吸道感染可为鼻咽炎、鼻炎、咽喉炎、气管炎和/或支气管炎,所述肺部感染可为肺炎,如为新型冠状病毒肺炎(简称新冠肺炎)。所述消化系统感染可为腹泻。
本文中,术语“中和抗体”是指一种能中和,即防止、抑制、减小、阻碍或干扰病原体引发和/或保持宿主内感染的能力的抗体。如本文所述,这些抗体可单独地或以组合形式,在经适当配制后用作预防剂或治疗剂,与活性疫苗接种联合使用,用作诊断工具或用作生产工具。
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物,其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。所述抗原结合片段保留母体抗体的至少某些结合特异性。通常,当基于摩尔来表示活性时,所述抗原结合片段保留至少10%的母体结合活性。具体地,所述抗原结合片段保留至少20%、50%、70%、80%、90%、95%或100%或更多的母体抗体对靶标的结合亲和力。
术语“Fab片段”是由重链Fd和完整轻链通过二硫键结合而成的异二聚体,仅含一个抗原结合位点。将重链Fd和完整轻链的编码基因连接,融合细菌蛋白信号肽基因后可在大肠杆菌内分泌表达Fab抗体(Fab片段),并有完整的立体折叠和链内、链间二硫键。所述重链Fd指Fab中约1/2的H链部分(约含225个氨基酸残基,包括VH、 CH1和部分铰链区)。
术语“Fv片段”是指可分别构建含有VH和VL基因的载体,共转染细胞,使之分别表达,再组装成功能性Fv抗体;也可在载体中的VH和VL之间设置终止密码,分别表达两个小分子蛋白片段,再通过非共价键结合而形成Fv抗体(Fv片段)。
术语“Fab′片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2 结构域之间区域的一条重链的部分,由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′)2分子。
术语“F(ab′)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链,由此在两条重链间形成链间二硫键。因此,F(ab′)2片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。
术语“单链抗体(ScFv)”是指用适当的寡核苷酸接头(linker)连接轻链和重链可变区基因,使之表达单一的多肽链,称为单链抗体(ScFv)。多肽链能自发折叠成天然构象,保持Fv的特异性和亲和力。
术语“纳米抗体(单域抗体)”是指将抗体重链V区通过基因工程方法表达,获得仅含VH片段的抗体。单域抗体与抗原结合的能力及其稳定性,与完全抗体基本一致。
术语“双特异性抗体”是指将两套轻链、重链基因导入骨髓瘤细胞中,选择合适的抗体恒定区及Ig类型,可获得产量大、均一性和纯度高的双特异性抗体。另外,以化学交联技术或杂交-杂交瘤技术也可获得双特异性抗体。
术语“最小识别单位(MRU)”是指仅含可变区中单一CDR结构,分子质量仅为完整抗体的1%左右,可结合相应抗原。
本发明的抗体可以本领域已知的各种方法来制备,例如通过基因工程重组技术来获得。例如,通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA 分子。将所得DNA分子插入表达载体内,然后转染宿主细胞,在特定条件下培养转染后的宿主细胞,并表达本发明的抗体。
本领域技术人员熟知,所述抗原结合片段可以可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。
本申请的发明人经过广泛而深入的研究,通过抗体库的筛选获得了能够特异性结合新型冠状病毒(SARS-CoV-2)S蛋白(刺突蛋白,Spike Protein)受体结合域(receptorbinding domain,RBD)的鼠源单链抗体(scFv)A-1F,在此基础上,发明人又付出了大量的创造性劳动,对鼠源单链抗体(scFv)A-1F进行了深入的研究和改造,开发了人鼠嵌合抗体mhA-1F和人源化抗体HSA-1F。实验表明,本发明制备的人鼠嵌合抗体 mhA-1F和人源化抗体HSA-1F具有较高的人源化程度,不易引发免疫原性反应,亲和力高,能通过特异性结合SARS-CoV-2刺突蛋白(S蛋白)RBD区域来抑制或中和 SARS-CoV-2活性,使SARS-CoV-2失去结合受体ACE2的能力,高效地中和新型冠状病毒(SARS-CoV-2)。本发明的抗新型冠状病毒中和抗体可以在原核细胞、酵母细胞、真核细胞及任何重组系统中表达和生产,获得具有中和SARS-CoV-2感染的抗体产物,可制成临床上用于预防和治疗新型冠状病毒肺炎的特异性抗体药物用于新型冠状病毒感染的防治,或制成SARS-CoV-2的诊断试剂或试剂盒等,在药物应用和临床诊断等领域具有十分广阔的前景和重要的生物学及医学意义。
附图说明
图1为纯化后的mhA-1F抗体SDS-PAGE电泳检测结果。其中M为Marker,1为mhA-1F(非还原);2为mhA-1F(还原)。
图2为抗体mhA-1F与RBD蛋白的亲和力检测。
图3为SARS-CoV-2假病毒检测抗体mhA-1F的中和能力。
图4为利用在线软件Abysis(www.abysis.org)对鼠源抗体A-1F重、轻链可变区氨基酸分布及特性进行分析的结果。图4中A为鼠源抗体A-1F重链可变区氨基酸序列性质分析。图4中B为鼠源抗体A-1F轻链可变区氨基酸序列性质分析。图4中C 和D分别为图4中A和B中的相关图注说明。
图5为利用在线软件Abysis(www.abysis.org)对鼠源抗体A-1F重、轻链可变区人源化程度进行分析。图5中A为鼠源抗体A-1F重链可变区人源化程度。图5中B 为鼠源抗体A-1F轻链可变区人源化程度。
图6为利用在线软件Abysis(www.abysis.org)对鼠源抗体A-1F重、轻链可变区氨基酸序列人源化情况进行分析。图6中A为鼠源抗体A-1F重链可变区氨基酸序列人源化情况分析。图6中B为鼠源抗体A-1F轻链可变区氨基酸序列人源化情况分析。
图7为人源化抗体HSA-1F的表达与纯化及SDS-PAGE电泳检测图。其中M为 Marker,1为HSA-1F(非还原),2为HSA-1F(还原)。
图8为人源化抗体HSA-1F与RBD的亲和力检测。
图9为人源化抗体HSA-1F中和SARS-CoV-2假病毒的活性检测。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、抗体的发现
一、小鼠免疫及免疫抗体库的制备
选取6-8周龄雌性Balb/C小鼠,小鼠尾静脉采血留本底血清。将新型冠状病毒S 蛋白RBD结构域重组蛋白(北京义翘神州科技有限公司,货号:40592-V08H5,简称重组RBD蛋白)用弗氏完全佐剂乳化,每只小鼠腹腔注射100μg重组RBD蛋白,注射之前先对小鼠取本底血清作为对照。首次免疫后1周进行加强免疫,用费氏不完全佐剂乳化重组RBD蛋白,每只小鼠腹腔注射100μg重组RBD蛋白,注射前断尾采血,共进行两轮加强免疫。三轮免疫后改用冲击免疫,每只小鼠腹腔注射100ug不加佐剂的SARS-CoV-2RBD重组蛋白(商品化RBD,义翘神州,40592-V08H5),冲击免疫后3 天处死小鼠,收集脾细胞。
利用细胞总RNA提取试剂盒(天根,DP430),提取小鼠脾细胞的总RNA。以提取的总RNA为模板,利用第一链cDNA合成试剂盒(Thermo scientific,K1621)分别合成抗体重链可变区(VH)和轻链可变区(VL),用基因特异性引物进行反转录,合成第一链cDNA,引物分别于抗体重链恒定区和抗体轻链恒定区配对,具体序列分别为 PmCGR:5’-TGCATTTGAACTCCTTGCC-3’和PmCKR:5’-CCATCAATCTTCCACTTGAC-3’。将合成好的cDNA立即存放-70℃保存。然后用反转录的cDNA为模板,根据参考文献 (Journal of ImmunologicalMethods,201(1997),35–55)设计合成引物,利用 PCR分别扩增鼠源抗体重链可变区(VH)和轻链可变区(VL)基因,然后利用重叠延伸PCR技术将VH和VL连接,组装构建单链抗体(scFv)。最后将制备的小鼠单链抗体基因克隆至载体pADSCFV-S载体(构建方法记载于如下专利中:抗人IL-17单克隆抗体,专利号为201510097117.0、公开号为CN105315371B的第0057-0059段)中,转化宿主菌,构建小鼠scFv库。此抗体库的库容达到8×108,正确率为60%。
二、抗新型冠状病毒RBD小鼠单链抗体(scFv)库的筛选
以SARS-CoV-2RBD重组蛋白(商品化RBD,义翘神州,40592-V08H5)为抗原,利用固相筛选策略(实验方案参考噬菌体展示:通用实验指南/(美)克拉克森 (Clackson,T),(美)洛曼(Lowman,H.B.)编;马岚等译。化学工业出版社,2008.5)对上述构建的小鼠单链抗体噬菌体库总共进行三轮筛选,最终获得能特异性结合人新冠病毒S蛋白RBD结构域的亲和力较高的单链抗体克隆A-1F。
测序分析结果表明,所述单链抗体A-1F的重链可变区的氨基酸序列为SEQ IDNo.7 的第1-118位;轻链可变区的氨基酸序列为SEQ ID No.8的第1-107位;所述重链可变区包含氨基酸序列分别是SEQ ID No.1、SEQ ID No.2、和SEQ ID No.3的HCDR1、 HCDR2和HCDR3;所述轻链可变区包含氨基酸序列分别是SEQ ID No.4、SEQ ID No.5、和SEQ ID No.6的LCDR1、LCDR2和LCDR3。
所述单链抗体A-1F的重链可变区的核苷酸序列为SEQ ID No.11的第1-354位;轻链可变区的核苷酸序列为SEQ ID No.12的第1-321位;其中:
重链可变区HCDR1的核苷酸序列是SEQ ID No.11的第76-99位;
重链可变区HCDR2的核苷酸序列是SEQ ID No.11的第151-174位;
重链可变区HCDR3的核苷酸序列是SEQ ID No.11的第289-321位;
轻链可变区LCDR1的核苷酸序列是SEQ ID No.12的第79-96位;
轻链可变区LCDR2的核苷酸序列是SEQ ID No.12的第148-156位;
轻链可变区LCDR3的核苷酸序列是SEQ ID No.12的第265-291位。
实施例2、抗新冠病毒嵌合抗体的制备
嵌合抗体是将鼠源抗体的可变区(V区)和人抗体的恒定区(C区)组装成嵌合抗体,这两部分在空间结构上相对独立,所以其特异性和亲和力保持得很好。本实施例基于实施例1获得的鼠源单链抗体A-1F制备人鼠嵌合的全抗体,命名为mhA-1F(重链恒定区为IgG1,轻链为Kappa)。制备过程如下:
一、重组质粒的构建
为了表达嵌合抗体mhA-1F,分别制备重链表达载体和轻链表达载体:
1、将鼠源单链抗体A-1F重链可变区(VH)基因(SEQ ID No.11的第1-354位) 与人IgG1恒定区重链基因(GenBank Accession No.BC016381.1(Update Date 24-MAR-2009)的第510-1502位核苷酸的基因)连接得到融合基因(该融合基因的核苷酸序列是重链的核苷酸序列SEQ ID No.11),用该融合基因替换pcDNA3.1(+) (Invitrogen,V79020)载体的HindIII识别位点和BamH I识别位点之间的片段,保持pcDNA3.1(+)的其它核苷酸序列不变,得到嵌合抗体mhA-1F的重链表达载体,命名为pcDNA3.1-mhA-1FH。
2、将鼠源单链抗体A-1F轻链可变区(VK,VL)基因(SEQ ID No.12的第1-321 位)与人Kappa恒定区轻链基因(GenBank Accession No.AM408494.1(Update Date 12-JAN-2007)的第565-888位核苷酸的基因)连接得到融合基因(该融合基因的核苷酸序列是轻链的核苷酸序列SEQ ID No.12),用该融合基因替换pcDNA3.1(+) (Invitrogen,V79020)载体的Hind III识别位点和BamH I识别位点之间的片段,保持pcDNA3.1(+)的其它核苷酸序列不变,得到嵌合抗体mhA-1F的轻链表达载体,命名为pcDNA3.1-mhA-1FK。
二、抗体的制备
1、抗体的表达
利用转染试剂FectoPRO DNA Transfection Reagent(Polyplus,116-001)将构建好的重链表达载体pcDNA3.1-mhA-1FH和轻链表达载体pcDNA3.1-mhA-1FK共转染FreeStyleTM HEK293-F细胞(Invitrogen,R79007)。具体步骤如下:转染前一天将密度在2-3×106/mL左右、活度90%以上的FreeStyleTM HEK293-F细胞1000rpm离心3 分钟,弃上清后用FreeStyle 293培养基(Gibco,12338-018)重悬细胞沉淀,并将细胞密度调整为1.0×106/mL,按30mL细胞悬液/瓶进行分装,细胞摇床中125rpm, 5%CO2,37℃震荡培养;第二天,进行转染复合物的制备:将轻重链质粒 (pcDNA3.1-mhA-1FK和pcDNA3.1-mhA-1FH)各取12ug,并将其稀释于3mL FreeStyle 293培养基中,轻轻混匀后添加24ul的FectoPRO转染试剂,混匀后室温放置15min;然后将混合溶液添加至前一天准备好的FreeStyleTM HEK293-F细胞中,轻轻混匀,放回细胞摇床中;转染后48小时开始监测细胞活度,在细胞活度降到80-85%时8,000rpm 离心10min,收集抗体表达上清液进行纯化。
2、抗体的纯化
用0.45μm滤膜抽滤抗体表达上清液以去除杂质,用10×PBS配置1×PBS结合缓冲液;将HiTrapTMMabSelect SuRe预装柱安装到AKTA系统,先用1×PBS结合缓冲液冲洗5-10个柱体积,待基线平稳,开始上样;上样完毕后,用结合缓冲液冲洗掉杂蛋白,继续用结合缓冲液冲洗5-10个柱体积,待基线水平后观察UV280是否接近零,如果不接近可手动调零;用0.1M柠檬酸盐缓冲液(pH 3.0)冲洗预装柱以洗脱抗体, UV280升到50时开始收集,UV280降到50时结束收集。
用HitrapTMDedalting层析柱将HiTrapTMMabSelect SuRe亲和层析纯化样品进行脱盐处理,流动相为0.01M的柠檬酸盐缓冲液(pH 6.0)。首先用0.01M的柠檬酸盐缓冲液(pH6.0)冲洗整个体系,待基线平稳后再冲洗3-5个柱体积,然后进行上样, (上样体积不大于层析柱总体积的五分之一)观察UV280值是否接近零,如不接近需要手动调零,进行缓冲液置换,流速均为2.5mL/min,UV280升到50时开始收集,UV280 降到50时结束收集,若盐峰与蛋白峰值重叠,则结束置换。获得的洗脱峰即为纯化的 mhA-1F抗体,取部分样品进行SDS-PAGE检测和浓度检测,其余的分装冻存于-80℃。
图1为纯化后的mhA-1F抗体SDS-PAGE检测结果。由图1可见,纯化后的mhA-1F 抗体还原性样品有两条清晰的条带,分子量较大的条带为重链,分子量较小的条带为轻链。
3、抗体的定量
将纯化并置换后的抗体溶液用滤膜过滤除菌,取样用NanoDrop紫外分光光度计(Thermo Scientific)测定蛋白浓度(300μg/mL)。
人鼠嵌合抗体mhA-1F(mhA-1F抗体)的重链的氨基酸序列为SEQ ID No.7;轻链的氨基酸序列为SEQ ID No.8;SEQ ID No.7的第1-118位为重链可变区(VH),其中: SEQ IDNo.7的第26-33位为HCDR1(SEQ ID No.1);SEQ ID No.7的第51-58位为HCDR2 (SEQ IDNo.2);SEQ ID No.7的第97-107位为HCDR3(SEQ ID No.3);SEQ ID No.7 的第119-216位为重链恒定区CH1,SEQ ID No.7的第217-231位为重链铰链区Hinge, SEQ ID No.7的第232-341位为重链恒定区CH2,SEQ ID No.7的第342-448位为重链恒定区CH3。
SEQ ID No.8的第1-107位为轻链可变区(VL),其中:SEQ ID No.8的第27-32 位为LCDR1(SEQ ID No.4);SEQ ID No.8的第50-52位为LCDR2(SEQ ID No.5);SEQ ID No.8的第89-97位为LCDR3(SEQ ID No.6);SEQ ID No.8的第108-214位为轻链恒定区CL。
人鼠嵌合抗体mhA-1F(mhA-1F抗体)的重链的核苷酸序列为SEQ ID No.11,编码氨基酸序列是SEQ ID No.7的重链;轻链的核苷酸序列为SEQ ID No.12,编码氨基酸序列是SEQ ID No.8的轻链;
SEQ ID No.11的第1-354位为重链可变区(VH),其中:SEQ ID No.11的第76-99 位为HCDR1;SEQ ID No.11的第151-174位为HCDR2;SEQ ID No.11的第289-321位为HCDR3;SEQID No.11的第355-648位为重链恒定区CH1,SEQ ID No.11的第649-693 位为重链铰链区Hinge;SEQ ID No.11的第694-1023位为重链恒定区CH2;SEQ ID No.11 的第1024-1344位为重链恒定区CH3;SEQ ID No.11的第1345-1347位为终止密码子。
SEQ ID No.12的第1-321位为轻链可变区(VL),其中:SEQ ID No.12的第79-96 位为LCDR1;SEQ ID No.12的第148-156位为LCDR2;SEQ ID No.12的第265-291位为LCDR3;SEQID No.12的第322-642位为轻链恒定区CL;SEQ ID No.12的第643-645 位为终止密码子。其中,互补决定区的序列根据Kabat编号系统定义。抗体mhA-1F 为IgG1,轻链类型为kappa(κ)型。
实施例3、mhA-1F抗体的特异性结合能力检测
1、用碳酸盐包被缓冲液(pH 9.6)稀释SARS-CoV-2RBD重组蛋白(商品化RBD (义翘神州,40592-V08H5))至2ng/ul,按照每孔100μL的体积加入到酶标板 (Corning,9018)中,每个实验孔设置3个复孔,4℃包被过夜。
2、第二天,用PBST对过夜包被的酶标板洗板6次,加入含有2%(质量百分含量) 脱脂奶粉的PBS封闭液,37℃孵育封闭2h。
3、封闭完成后,弃去封闭液,每孔加入100μL按照2倍倍比稀释的mhA-1F抗体溶液(起始浓度1.2nM),一共设置11个梯度,37℃孵育90min,然后PBST洗板 6次。
4、完成上述步骤后,取所述酶标板,每孔加入100μL的1:4000倍稀释HRP 标记的抗人IgG抗体(中杉金桥,ZB-2304),37℃孵育1h,PBST洗板6次。
5、向清洗好的酶标板每孔加入50μL OPD底物显色液,室温下避光孵育10min。
6、显色完成后,每孔加入50μL 1M硫酸溶液终止酶联显色反应。
7、将上述酶标板用酶标仪492nm/630nm双波长测定光密度(OD)值。
结果如图2所示。图2中,横坐标为蛋白摩尔浓度的对数值,纵坐标为光密度值。结果分析显示mhA-1F抗体与RBD重组蛋白的结合能力为EC50=0.073nM。
实施例4、mhA-1F抗体中和新冠病毒假病毒活性检测
一、假病毒的制备
1、S蛋白表达质粒的构建
根据GenBank公布的SARS-CoV-2基因序列(GenBank号:NC_045512.2),委托上海生工生物工程有限公司合成SARS-CoV-2病毒S刺突蛋白基因(S基因)全长基因序列(去除跨膜区末端的19个氨基酸,序列如SEQ ID No.15所示),利用常规的酶切连接法将合成的S蛋白基因片段(SEQ ID No.15)替换载体pcDNA3.1(+)(Invitrogen, V79020)的Kpn I和Xho I识别位点间的片段(小片段),保持pcDNA3.1(+)载体的其他序列不变,得到的重组表达载体即为SARS-CoV-2S基因表达质粒,命名为 pcDNA3.1-ST19。
2、假病毒包装
2.1假病毒包装前一天调整悬浮细胞FreeStyleTM HEK293-F(Invitrogen公司,R79007)浓度至1×106个/mL,接种70mL至250mL细胞瓶,37℃,150rpm过夜培养;
2.2第二天,将42μg pNL4.3-Luc-E-R-(NTCC,3767994)和14μg pcDNA3.1-ST19 混匀,反复冻融3次,用3mL opti-MEM培养基稀释质粒后再加入80uL FectoPRO 转染试剂,上下颠倒混匀后室温放置15min,后将溶液全部移至步骤2.1的细胞中, 37℃培养72h;
2.3 72h后,用50ml离心管将细胞悬液4℃下3000rpm离心30min,收获含有SARS-CoV-2假型病毒的培养上清液,将上清液分装到1.5mL EP管,每支1.2mL,存放于-80℃冰箱保存。
3、粗测假病毒滴度
3.1向96孔板中第二行至第八行加入50μL DMEM,第1行加入50μL假病毒样品(每个样品做2-3个复孔),第二行加入25μL假病毒样品,然后将第二行混匀,取25μL加入第三行,依次向下稀释,直至第八行混匀后吸弃25μL。
3.2将96孔板放入37℃孵箱,孵育1h。
3.3 1h孵育结束后,将浓度4×105/mL的Huh7细胞(NTCC,SCSP-526),添加到 96孔板中,每孔100ul。
3.4将96孔板放入37℃孵箱培养48h。
3.5 48h后,弃96孔板中上清液,在干净纸巾上拍干残液,每孔加入40μL 1×Passive Lysis Buffer(Promega,E194A,用前用水稀释至1×),摇床上避光摇晃20 min;裂解完成后轻拍96孔板使每孔细胞脱落至裂解液中,将孔中裂解物转移到检测 Luciferase的检测白板中。
3.6向检测白板中每孔加入40μL Luciferase Assay Substrate(Promega,E1501),用MD公司的Spectra Max L检测单荧光素酶,根据Reed-Muench法计算假病毒滴度。经计算假病毒滴度为:1×105TCID50/mL。
4、利用假病毒检测抗体的中和活性
4.1稀释抗体:96孔板中第二行至第八行加入140μL DMEM,第1行加入210μL mhA-1F抗体(此为3个复孔的量),然后从第一行取70μL加入第二行,依次向下稀释,直至第八行,转移至另一块96孔板,共做15个稀释度,最后一个稀释度混匀后吸弃70μL。
4.2每孔加入35μL假病毒,混匀后将每孔中的样品分至另一96孔板,1列分3 列,每孔50μL,放入37℃孵箱,孵育1h。
4.3孵育至0.5h时,可开始消化Huh7细胞(NTCC,SCSP-526),单细胞悬液密度调整至4×105个/mL。
4.4 96孔板中每孔加入100μL Huh7细胞,37℃孵箱培养48h。
4.5 48h后,弃96孔板中上清液,在干净纸巾上拍干残液,每孔加入40μL 1×Passive Lysis Buffer(Promega,E194A,用前用水稀释至1×),摇床上避光摇晃20 min;裂解完成后轻拍96孔板使每孔细胞脱落至裂解液中,将每孔中的裂解物转移到检测Luciferase的检测白板中。
4.6用MD公司的Spectra Max L检测单荧光素酶,每孔加入40μL LuciferaseAssay Substrate(Promega,E1501);利用软件GraphPad Prism 8作图,并计算EC50。
结果显示,mhA-1F抗体对SARS-CoV-2假型病毒具有较强的抑制活性和中和活性,表明mhA-1F抗体可以有效中和新冠S蛋白假病毒,具有抑制SARS-CoV-2假病毒感染的能力,EC50为2.27nM(图3)。
实施例5、鼠源单链抗体A-1F抗体的人源化改造及亲和力、中和活性检测一、鼠源抗体A-1F可变区氨基酸序列分析及空间结构模拟
为了降低抗体分子在体内的免疫原性,进一步减少鼠源成分,对A-1F抗体进行了人源化设计:利用在线软件Abysis(www.abysis.org)对实施例1获得的鼠源抗体A-1F 重、轻链可变区氨基酸序列进行特性分析(图4),借助Z-score打分模式对鼠源抗体 A-1F的人源化程度进行了预测(图5),并分析其具有鼠源特征的潜在位点(图6)。最终设计得到的人源化抗体命名为HSA-1F。
二、利用常规分子生物学方法制备重组人源化HSA-1F的全抗体
人源化抗体HSA-1F的重链的氨基酸序列为SEQ ID No.9;轻链的氨基酸序列为SEQID No.10。
SEQ ID No.9的第1-118位为重链可变区(VH),其中:SEQ ID No.9的第26-33 位为HCDR1(SEQ ID No.1);SEQ ID No.9的第51-58位为HCDR2(SEQ ID No.2);SEQ ID No.9的第97-107位为HCDR3(SEQ ID No.3);SEQ ID No.9的第119-216位为重链恒定区CH1,SEQ IDNo.9的第217-231位为重链铰链区Hinge,SEQ ID No.9的第 232-341位为重链恒定区CH2,SEQ ID No.9的第342-448位为重链恒定区CH3。
SEQ ID No.10的第1-107位为轻链可变区(VL),其中:SEQ ID No.10的第27-32 位为LCDR1(SEQ ID No.4);SEQ ID No.10的第50-52位为LCDR2(SEQ ID No.5); SEQ IDNo.10的第89-97位为LCDR3(SEQ ID No.6);SEQ ID No.10的第108-214位为轻链恒定区CL。
人源化抗体HSA-1F的重链的核苷酸序列为SEQ ID No.13,编码氨基酸序列是SEQID No.9的重链;人源化抗体HSA-1F的轻链的核苷酸序列为SEQ ID No.14,编码氨基酸序列是SEQ ID No.10的轻链;
SEQ ID No.13的第1-354位为重链可变区(VH),其中:SEQ ID No.13的第76-99 位为HCDR1;SEQ ID No.13的第151-174位为HCDR2;SEQ ID No.13的第289-321位为HCDR3;SEQID No.13的第355-648位为重链恒定区CH1,SEQ ID No.13的第649-693 位为重链铰链区Hinge;SEQ ID No.13的第694-1023位为重链恒定区CH2;SEQ ID No.13 的第1024-1344位为重链恒定区CH3;SEQ ID No.13的第1345-1347位为终止密码子。
SEQ ID No.14的第1-321位为轻链可变区(VL),其中:SEQ ID No.14的第79-96 位为LCDR1;SEQ ID No.14的第148-156位为LCDR2;SEQ ID No.14的第265-291位为LCDR3;SEQID No.14的第322-642位为轻链恒定区CL;SEQ ID No.14的第643-645 位为终止密码子。
其中,互补决定区的序列根据Kabat编号系统定义。抗体HSA-1F为IgG1,轻链类型为kappa(κ)型。
HSA-1F抗体的具体制备方法参见实施例2,除将实施例2中的pcDNA3.1-mhA-1FH替换为pcDNA3.1-HSA-1FH,将实施例2中的pcDNA3.1-mhA-1FK替换为 pcDNA3.1-HSA-1FK外,其它方法相同。
pcDNA3.1-HSA-1FH是用核苷酸序列是SEQ ID No.13的人源化抗体HSA-1F的重链基因替换pcDNA3.1(+)(Invitrogen,V79020)载体的HindIII识别位点和BamH I 识别位点之间的片段,保持pcDNA3.1(+)的其它核苷酸序列不变,得到人源化抗体 HSA-1F的重链表达载体。
pcDNA3.1-HSA-1FK是用核苷酸序列是SEQ ID No.14的人源化抗体HSA-1F的轻链基因替换pcDNA3.1(+)(Invitrogen,V79020)载体的HindIII识别位点和BamH I 识别位点之间的片段,保持pcDNA3.1(+)的其它核苷酸序列不变,得到人源化抗体 HSA-1F的轻链表达载体。
HSA-1F抗体的表达纯化及SDA-PAGE电泳检测见图7。
三、HSA-1F抗体与RBD的亲和力检测方法同实施例3,结果显示抗体HSA-1F结合RBD的EC50为0.052nM(图8)。
四、HSA-1F抗体中和新冠病毒S蛋白假病毒的活性检测的方法同实施例4,结果显示HSA-1F抗体较亲本抗体mhA-1F中和活性下降,但仍可以有效中和新冠肺炎假病毒,EC50为9.638nM(图9)。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
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Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 8
<211> 214
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 8
Asp Val Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 9
<211> 448
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 9
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Met Tyr Trp Val Lys Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Gly Val Asn Pro Tyr Thr Val Asp Thr Lys Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Arg Asp Arg Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 10
<211> 214
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 10
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Val Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser
65 70 75 80
Glu Asp Val Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 11
<211> 1347
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 11
gaggttcagc tgcaacagtc tggaactgag ctggtgaagc ctggggcttc agtgaagata 60
tcctgcaaga cttctggata cacattcact gaatacacca tgtactgggt gaagcagagc 120
catggaaaga gccttgagtg gattggaggt gttaatcctt acactgttga tactaagtac 180
aaccagaggt tcaagggcaa ggccacattg actgtagaca agtcctctag tacagcctac 240
atggagctcc gcagcctgac atcggaggat tctgcagtct attactgtgt aagagatagg 300
tacgactatg ctatggacta ctggggtcaa ggaacctcag tcaccgtgtc ctcagcctcc 360
accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420
gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480
tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540
tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600
tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga gcccaaatct 660
tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900
taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
agcctctccc tgtccccggg taaatga 1347
<210> 12
<211> 645
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 12
gatgttgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60
gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120
gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240
gaagacttgg cagagtattt ctgtcagcaa tataacagct atcccctcac gttcggaggg 300
gggaccaagc tggaaataaa aagaactgtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645
<210> 13
<211> 1347
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 13
gaggtgcagc tgcagcagag cggccccgag ctggtgaagc ccggcgccag cgtgaagatc 60
agctgcaaga ccagcggcta caccttcacc gagtacacca tgtactgggt gaagcagagc 120
cccggcaagg gcctggagtg gatcggcggc gtgaacccct acaccgtgga caccaagtac 180
aaccagagat tcaagggcaa ggccaccatc accgtggaca agagcaccag caccgcctac 240
atggagctga gaagcctgac cagcgaggac accgccgtgt actactgcgt gagagacagg 300
tacgactacg ccatggacta ctggggccag ggcaccaccg tgaccgtgtc ctcagcctcc 360
accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420
gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480
tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540
tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600
tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga gcccaaatct 660
tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900
taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
agcctctccc tgtccccggg taaatga 1347
<210> 14
<211> 645
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 14
gacatcgtga tgacccagag ccccagcagc gtgagcgtga gcgtgggcga cagagtgagc 60
atcacctgca aggccagcca gaacgtgggc accaacgtgg cctggtacca gcagaagccc 120
ggccagagcc ccaaggccct gatctacagc gccagctaca gatacagcgg cgtgcccgac 180
agattcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cgtgcagagc 240
gaggacgtgg ccgactactt ctgccagcag tacaacagct accccctgac cttcggcggc 300
ggcaccaagc tggagatcaa gagaactgtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645
<210> 15
<211> 3820
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 15
atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60
agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120
aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180
aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240
aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300
ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360
aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420
ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480
tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540
ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600
tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660
tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720
ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780
ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840
gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900
tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960
caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020
gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080
tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140
ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200
gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260
tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320
cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380
ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440
aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500
aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560
ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620
ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680
cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740
acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800
ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860
cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920
aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980
gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040
cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100
gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160
agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220
tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280
acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340
gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400
aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460
ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520
cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580
ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640
acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700
caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760
aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820
acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880
acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940
ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000
cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060
tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120
gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180
gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240
atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300
cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360
tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420
ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480
tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540
aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600
caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660
atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720
tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780
tctgagccag tgctcaaagg agtcaaatta cattacacat 3820
Claims (9)
1.特异性结合新型冠状病毒S蛋白受体结合域的抗体或其抗原结合片段,其特征在于,所述抗体包含重链可变区和轻链可变区,所述重链可变区包含氨基酸序列分别是SEQ IDNo.1、SEQ ID No.2和SEQ ID No.3的HCDR1、HCDR2和HCDR3;所述轻链可变区包含氨基酸序列分别是SEQ ID No.4、SEQ ID No.5和SEQ ID No.6的LCDR1、LCDR2和LCDR3。
2.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述重链可变区的氨基酸序列为SEQ ID No.7的第1-118位或与SEQ ID No.7的第1-118位具有至少90%的同一性;所述轻链可变区的氨基酸序列为SEQ ID No.8的第1-107位或与SEQ ID No.8的第1-107位具有至少90%的同一性。
3.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述重链可变区的氨基酸序列为SEQ ID No.9的第1-118位或与SEQ ID No.9的第1-118位具有至少90%的同一性;所述轻链可变区的氨基酸序列为SEQ ID No.10的第1-107位或与SEQ ID No.10的第1-107位具有至少90%的同一性。
4.根据权利要求1或2所述的抗体或其抗原结合片段,其特征在于,所述抗体的重链的氨基酸序列为SEQ ID No.7或与SEQ ID No.7具有至少80%的同一性;所述抗体的轻链的氨基酸序列为SEQ ID No.8或与SEQ ID No.8具有至少80%的同一性。
5.根据权利要求1或3所述的抗体或其抗原结合片段,其特征在于,所述抗体的重链的氨基酸序列为SEQ ID No.9或与SEQ ID No.9具有至少80%的同一性;所述抗体的轻链的氨基酸序列为SEQ ID No.10或与SEQ ID No.10具有至少80%的同一性。
6.生物材料,其特征在于,所述生物材料为下述B1)至B5)中的任一种:
B1)编码权利要求1-5中任一所述抗体或其抗原结合片段的核酸分子;
B2)含有B1)所述核酸分子的表达盒;
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体;
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物;
B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系、或含有B3)所述重组载体的细胞系。
7.药物组合物,其特征在于,所述药物组合物包含权利要求1-5中任一所述抗体或其抗原结合片段,以及一种或多种药学上可接受的载体。
8.下述任一项所述的应用:
A1)权利要求6所述的生物材料在制备权利要求1-5中任一所述抗体或权利要求7所述的药物组合物中的应用;
A2)权利要求1-5中任一所述抗体,和/或,权利要求6所述的生物材料,和/或,权利要求7所述的药物组合物在制备用于治疗SARS-CoV-2感染引起的疾病的产品中的应用;
A3)权利要求1-5中任一所述抗体,和/或,权利要求6所述的生物材料,和/或,权利要求7所述的药物组合物在制备用于抑制或中和SARS-CoV-2的产品中的应用;
A4)权利要求1-5中任一所述抗体,和/或,权利要求6所述的生物材料,和/或,权利要求7所述的药物组合物在制备用于结合SARS-CoV-2的RBD蛋白的产品中的应用;
A5)权利要求1-5中任一所述抗体,和/或,权利要求6所述的生物材料在制备用于检测SARS-CoV-2和/或SARS-CoV-2的RBD蛋白的产品中的应用;
A6)权利要求1-5中任一所述抗体,和/或,权利要求6所述的生物材料在制备用于诊断或辅助诊断SARS-CoV-2感染引起的疾病的产品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述SARS-CoV-2感染引起的疾病为呼吸系统感染和/或消化系统感染。
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