CN114790240B - SARS-CoV-2中和性单克隆抗体及应用 - Google Patents
SARS-CoV-2中和性单克隆抗体及应用 Download PDFInfo
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Abstract
本发明属于单克隆抗体领域,具体涉及SARS‑CoV‑2中和性单克隆抗体及应用。该SARS‑CoV‑2中和性单克隆抗体为单克隆抗体8H9或单克隆抗体9D4。本发明的SARS‑CoV‑2中和性单克隆抗体,对SARS‑CoV‑2野生株和突变株如B.1.617.2株和B.1.1.529株假病毒具有较强的中和活性,在应对SARS‑CoV‑2野生株及其突变株引起的疾病的特异性预防、治疗和诊断中具有重要的研究和应用价值。
Description
技术领域
本发明属于单克隆抗体领域,具体涉及SARS-CoV-2中和性单克隆抗体及应用。
背景技术
人感染新冠病毒后呈现多种临床表现,主要常见症状有发热、呼吸道症状、咳嗽、气促等,感染的持续迁延可发展为重症肺炎,引起呼吸衰竭、休克和器官衰竭等严重并发症,部分轻症或出院患者在恢复后会出现病情反复。疫苗能有效预防传染病,且目前已有部分新冠疫苗被批准使用,但其主要是针对健康人群。针对SARS-CoV-2的中和性抗体能有效阻断病毒与细胞的结合,且在临床试验上显示出积极效果。
目前,有部分SARS-CoV-2的单克隆抗体已被批准紧急使用,且有大量处于不同的开发阶段。然而,现有的大部分抗体主要是针对未突变的SARS-CoV-2野生型。新出现的突变株如B.1.617.2株和B.1.1.529株,在刺突蛋白氨基酸序列上的多个点突变不仅会影响突变株的传播速度、致病性,还会对单克隆抗体的中和活性产生较大的影响,可能导致病毒免疫逃逸的发生。因此亟需开发针对SARS-CoV-2野生型和流行突变株具有广谱中和能力的单克隆抗体,这在针对包括SARS-CoV-2野生型和流行突变株的检测及治疗方面具有较大的应用前景。
发明内容
本发明的目的是提供SARS-CoV-2中和性单克隆抗体,其能够特异性地与SARS-CoV-2刺突蛋白结合,并能中和SARS-CoV-2野生株和流行突变株具有广谱中和能力。
本发明的第二个目的是提供编码上述SARS-CoV-2中和性单克隆抗体的核酸分子。
本发明的第三个目的是提供上述SARS-CoV-2中和性单克隆抗体的应用。
为了实现以上目的,本发明所采用的技术方案是:
SARS-CoV-2中和性单克隆抗体,其为单克隆抗体8H9或单克隆抗体9D4;所述单克隆抗体8H9包含氨基酸序列如SEQ ID NO:1-3所示的VHCDR1、VHCDR2、VHCDR3,和氨基酸序列如SEQ ID NO:4-6所示的VLCDR1、VLCDR2、VLCDR3;所述单克隆抗体9D4包含氨基酸序列如SEQ ID NO:11-13所示的VHCDR1、VHCDR2、VHCDR3,和氨基酸序列如SEQ ID NO:14-16所示的VLCDR1、VLCDR2、VLCDR3。
本发明的SARS-CoV-2中和性单克隆抗体,对SARS-CoV-2野生株和突变株如B.1.617.2株和B.1.1.529株假病毒具有较强的中和活性,在应对SARS-CoV-2野生株及其突变株引起的疾病的特异性预防、治疗和诊断中具有重要的研究和应用价值。
优选地,所述单克隆抗体8H9重链可变区的氨基酸序列如SEQ ID NO.7所示,所述单克隆抗体8H9轻链可变区的氨基酸序列如SEQ ID NO.8所示。进一步优选地,所述单克隆抗体8H9的轻链型为Kappa,亚型为IgG 2a。
优选地,所述单克隆抗体9D4重链可变区的氨基酸序列如SEQ ID NO.17所示,所述单克隆抗体9D4轻链可变区的氨基酸序列如SEQ ID NO.18所示。进一步优选地,所述单克隆抗体9D4的轻链型为Kappa,亚型为IgG 2a。
在本发明提供的单克隆抗体的重链和轻链可变区氨基酸序列基础上,可通过常规蛋白质工程方法进行一个或多个氨基酸的添加、删除、替换等修饰,获得保守型变异体或其片段,仍能够中和SARS-CoV-2野生株和两种流行突变株的假病毒,包括B.1.617.2株和B.1.1.529株。
编码上述SARS-CoV-2中和性单克隆抗体的核酸分子。
以上述核酸分子编码得到的单克隆抗体能够中和SARS-CoV-2野生株和两种流行突变株的假病毒,包括B.1.617.2株和B.1.1.529株。
抗体核酸分子可以利用基因工程重组技术或化学合成方法获得。在本发明提供的上述核酸分子经一个或多个核苷酸添加、删除、替换、修饰等突变后得到的重链可变区核苷酸序列和/或轻链可变区核苷酸序列的变异序列,其所编码的氨基酸序列组成的单链抗体或嵌合单克隆抗体或改型单克隆抗体或其他形式的单克隆抗体或抗体片段,仍能够中和SARS-CoV-2野生株和两种流行突变株的假病毒,包括B.1.617.2株和B.1.1.529株。
优选地,编码所述单克隆抗体8H9重链可变区的基因核苷酸序列如SEQ ID NO:9所示,编码所述单克隆抗体8H9轻链可变区的基因核苷酸序列如SEQ ID NO:10所示。
优选地,编码所述单克隆抗体9D4重链可变区的基因核苷酸序列如SEQ ID NO:19所示,编码所述单克隆抗体9D4轻链可变区的基因核苷酸序列如SEQ ID NO:20所示。
上述SARS-CoV-2中和性单克隆抗体及其人源化改造抗体在制备检测SARS-CoV-2的试剂、抑制SARS-CoV-2的试剂、预防和/或治疗因SARS-CoV-2感染导致的疾病的药物中的应用。
优选地,所述SARS-CoV-2包括野生株、B.1.617.2株和B.1.1.529株。
目前已开发的疫苗和部分被批准紧急使用的抗体主要是针对未突变的SARS-CoV-2野生株。SARS-CoV-2突变株如B.1.617.2株和B.1.1.529株显示出对部分在不同开发阶段的抗体、康复者血浆和疫苗免疫后血浆具有不同程度的耐受性。本发明所涉及的8H9和9D4单克隆抗体的实验结果表明,该抗体8H9和9D4对SARS-CoV-2野生株和突变株如B.1.617.2株和B.1.1.529株假病毒具有较强的中和活性。
附图说明
图1为本发明中稳定分泌表达重组RBD蛋白的CHO细胞株的鉴定;其中(A)为亚克隆后细胞荧光/白光图;(B)细胞表达上清的Western blot鉴定:M:Marker;1:CHO/RBD细胞培养上清;2:CHO细胞培养上清;
图2为本发明中RBD蛋白纯化的鉴定M:Marker;1:细胞培养上清;2:孵育后流穿液;3:杂蛋白洗脱;4:目的蛋白洗脱;
图3为本发明中免疫后的小鼠血清效价检测结果图;
图4为本发明中单克隆抗体8H9和9D4纯化后的SDS-PAGE鉴定结果图;
图5为本发明中纯化后的单克隆抗体8H9和9D4与RBD结合活性检测结果图;
图6为本发明中单克隆抗体8H9和9D4对SARS-CoV-2野生株假病毒、SARS-CoV-2突变株B.1.617.2株和B.1.1.529株的假病毒的中和活性检测结果图。
具体实施方式
下面结合具体实施例对本发明的具体实施过程进行详细说明。若未特别说明,以下实施例中所用的技术手段均为本领域技术人员所熟知的常规手段,所有试剂耗材均为市售商品。
实施例1SARS-CoV-2中和性单克隆抗体
本实施例的SARS-CoV-2中和性单克隆抗体,为单克隆抗体8H9或单克隆抗体9D4。
单克隆抗体8H9包含氨基酸序列如SEQ ID NO:1-3所示的VHCDR1、VHCDR2、VHCDR3,和氨基酸序列如SEQ ID NO:4-6所示的VLCDR1、VLCDR2、VLCDR3;8H9抗体的重链可变区的氨基酸序列如SEQ ID NO.7所示,轻链可变区的氨基酸序列如SEQ ID NO.8所示。8H9抗体的轻链型为Kappa,亚型为IgG 2a。
单克隆抗体9D4包含氨基酸序列如SEQ ID NO:11-13所示的VHCDR1、VHCDR2、VHCDR3,和氨基酸序列如SEQ ID NO:14-16所示的VLCDR1、VLCDR2、VLCDR3;9D4抗体的重链可变区的氨基酸序列如SEQ ID NO.17所示,轻链可变区的氨基酸序列如SEQ ID NO.18所示。9D4抗体的轻链型为Kappa,亚型为IgG 2a。
本实施例的SARS-CoV-2中和性单克隆抗体的制备过程如下:
(1)重组RBD蛋白的制备、鉴定与纯化
利用慢病毒系统将RBD基因构建至慢病毒表达载体中pLVX-RBD-IRES-ZsGreen1,选用HEK293T细胞包装慢病毒。RBD基因的核苷酸序列如SEQ ID NO:21所示,RBD的氨基酸序列如SEQ ID NO:22所示。
具体过程如下:
a)HEK293T细胞在T25细胞培养瓶中生长到70~90%汇合度时,小心地弃掉培养基。然后,在细胞培养瓶中加入3mL 37℃预热的PBS洗涤液。之后,弃去洗涤液,在细胞培养瓶中加入1mL 0.25%胰蛋白酶以覆盖细胞。室温静置1min后除去胰蛋白酶,将细胞转移到细胞培养箱中继续消化5min。之后,加入3mL DMEM完全培养基并反复吹吸6~8次,确保细胞分散成单个细胞悬液。细胞计数,用DMEM完全培养基将细胞密度调整为8×105个细胞/mL。
b)将2mL/孔细胞悬液转移到多聚赖氨酸处理过的6孔细胞培养板,放回细胞培养箱中过夜培养。转染前检查细胞密度,细胞应达到70%汇合度。小心弃掉6孔板上清培养基,每孔加入2mL Opti-MEM完全培养基,放回细胞培养箱中。开始制备转染复合物,将200μLbuffer加入2mL离心管,然后以1:2:2的摩尔比分别加入pLVX-RBD-IRES-ZsGreen1、pMD2.G和psPAX2质粒共2μg,用涡旋仪涡旋10s以充分混匀。再向管中加入4μLreagent,涡旋1s,在室温下孵育10min。孵育结束后将转染复合物逐滴滴加至6孔板中,轻轻晃动板子混合均匀。转染后6h,小心吸取上清弃掉,补加2mL Opti-MEM完全培养基,放回细胞培养箱培养。培养24h后观察荧光强度以确认转染效率,继续培养24h后观察细胞生长状态和荧光强度,当镜检观察到荧光强度表达趋于稳定且50%细胞破裂脱落时吸取上清,12000rpm离心10min收集上清。
c)用流式细胞仪将转导后的细胞以1个细胞/孔将荧光强度高的单细胞克隆分选到96孔细胞培养板中。分选的细胞在分裂时有时会出现荧光消失的情况,所以待细胞在96孔板中汇合度达到60%左右,挑选细胞状态好,荧光强度高的细胞进行亚克隆,直至筛选得到荧光强度高且稳定的单克隆细胞(图1)。将能稳定分泌表达重组RBD蛋白的CHO细胞株,命名为CHO/RBD。将CHO/RBD细胞大量培养并收集表达的上清液,用Ni-NTA亲和纯化,经SDS-PAGE验证后获得大小为26kDa的高纯度RBD蛋白(图2)。
(2)分泌SARS-CoV-2RBD蛋白抗体的杂交瘤细胞株的制备
2.1、小鼠免疫
经过纯化的重组RBD蛋白以完全弗氏佐剂乳化,采用皮下多点注射方法免疫6-8周龄BALB/c小鼠(购自郑州大学实验动物中心,2只),免疫剂量为50μg/只,间隔两周后进行加强免疫,以不完全弗氏佐剂乳化,免疫剂量为50μg/只。第二次加强免疫后尾部采血,以间接ELISA法测定血清效价(图3),两只小鼠效价达到1:1.28×105。
2.2、杂交瘤细胞融合和筛选
采用聚乙二醇的方法,将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0按细胞数量为8:1的比例进行细胞融合,融合后的细胞用HAT选择培养基进行筛选。10天后,开始使用下文所述的ELISA检测方法来检测针对RBD蛋白的抗体的存在情况。
ELISA检测方法:
间接ELISA用于评估上清液中抗体对于RBD蛋白的结合能力。ELISA板用4μg/mL的重组RBD蛋白在4℃下包被过夜。用PBST(0.05%吐温)洗涤板,并将其用150μL/孔的含1%BSA的PBST在37℃封闭1h。随后弃去封闭液,向每个板加100μL杂交瘤细胞培养上清液,然后在37℃孵育1h。将板用PBST洗涤三次,并用100μL/孔的1:10000稀释的羊抗鼠IgG-HRP二抗37℃孵育1h。将板用PBST洗涤五次然后加入TMB显色液并在室温下在避光显色10min。用2M浓硫酸终止液终止反应。使用酶标仪在450nm下读板。
2.3、杂交瘤细胞亚克隆
对阳性孔细胞进行有限稀释法亚克隆,每次亚克隆后7-8天测定间接ELISA值,挑取OD450值高于1.5的单克隆进行有限稀释法进行亚克隆3-4次直至获得稳定分泌抗SARS-CoV-2RBD蛋白抗体的杂交瘤细胞株。
(3)单克隆抗体8H9和9D4的制备及纯化
3.1、制备:在提前7天注射弗氏不完全佐剂后,将中和性单克隆抗体8H9和9D4的单克隆杂交瘤细胞株(0.5mL,1×106cells/mL)注射至小鼠腹腔,继续培养7-10天。
3.2、纯化:收获腹水,37℃静置2h后,5000rpm离心30min,收集中层上清液并过滤,随后用Protein A亲和层析柱进行纯化。纯化步骤如下:
用pH为7.4的0.01M PBS缓冲液平衡柱子;
上样后,用pH为7.4的0.01M PBS缓冲液淋洗;
用pH为3.0的0.1M Gly缓冲液进行洗脱,并用pH为8.0的Tris中和洗脱液。
收集洗脱液于0.01M PBS缓冲液中透析。取纯化后的抗体进行SDS-PAGE分析。
3.3、结果分析:SDS-PAGE结果见图4,在还原条件下呈现分子量约为50kDa和25kDa的两条带,分别对应抗体的重链和轻链。纯化后的单克隆抗体纯度达到95%以上。
(4)中和性单克隆抗体8H9和9D4与抗原的结合活性检测
采用间接ELISA法测定8H9和9D4抗体与SARS-CoV-2RBD蛋白结合能力。步骤如下:
ELISA板用4μg/mL的重组RBD蛋白在4℃下包被过夜。用PBST(0.05%吐温)洗涤板,并将其用150μL/孔的含1%BSA的PBST在37℃封闭1h。随后弃去封闭液,向每个板加100μL系列稀释的抗体,然后在37℃孵育1h。将板用PBST洗涤三次,并用100μL/孔的1:10000稀释的羊抗鼠IgG-HRP二抗37℃孵育1h。将板用PBST洗涤五次然后加入TMB显色液并在室温下在避光显色10min。用2M浓硫酸终止液终止反应。使用酶标仪在450nm下读板。
8H9和9D4抗体与抗原结合活性检测结果见图5,结果表明:
单克隆抗体8H9针对RBD蛋白的结合活性为:EC50=25.15ng/mL。
单克隆抗体9D4针对RBD蛋白的结合活性为:EC50=24.18ng/mL。
实施例2核酸分子
编码上述SARS-CoV-2中和性单克隆抗体的核酸分子,其中,8H9抗体的重链可变区编码基因的核苷酸序列如SEQ ID NO:9所示,轻链可变区编码基因的核苷酸序列如SEQ IDNO:10所示。9D4抗体的重链可变区编码基因的核苷酸序列如SEQ ID NO:19所示,轻链可变区编码基因的核苷酸序列如SEQ ID NO:20所示。
实施例3单克隆抗体的应用
本实施例进行上述单克隆抗体8H9和9D4对野生株和(突变型)假病毒的中和试验。
采用假病毒检测体系检测8H9和9D4对SARS-CoV-2野生株及其突变株的中和活性,假病毒由本实验室保存。步骤说明如下:将系列稀释的抗体8H9和9D4与假病毒混合后37℃孵育1h,细胞对照组为不加抗体和病毒,病毒对照组不加抗体。然后以2×104cells/100μL加入预先准备好的293T-hACE2单层细胞培养24h,随后将100μL培养上清替换为等体积荧光底物,室温孵育2min后将150μL裂解液转移到不透明96孔板测定荧光值,根据中和抑制率的结果,利用Reed-Muench法计算待测抗体的IC50。
抗体8H9和9D4对假病毒中和实验检测结果见图6。
结果表明,抗体8H9和9D4对SARS-CoV-2野生株和突变株B.1.6.7.2株和B.1.1.529株假病毒中和活性IC50分别为,8H9:32.96ng/mL、56.57ng/mL和92.91ng/mL;9D4:112.25ng/mL、154.68ng/mL和187.74ng/mL
因此,单克隆抗体8H9和9D4对SARS-CoV-2野生株和突变株都具有中和活性。
实施例4单克隆抗体可变区基因扩增与序列测定
将前期筛选出的8H9和9D4单克隆杂交瘤细胞进行扩大培养,Trizol法提取总RNA;利用RT-PCR获得鼠源抗体可变区编码片段并进行测序。
测序结果如下:
本发明的单克隆抗体8H9重链可变区和轻链可变区的核苷酸序列分别为SEQ IDNO:9和SEQ ID NO:10所示。进一步分析得到单克隆抗体8H9重链可变区氨基酸序列如SEQID NO:7所示;单克隆抗体8H9的轻链可变区的氨基酸序列如SEQ ID NO:8所示。
本发明的单克隆抗体9D4重链可变区和轻链可变区的核苷酸序列分别为SEQ IDNO:19和SEQ ID NO:20所示。进一步分析得到单克隆抗体9D4重链可变区氨基酸序列如SEQID NO:17所示;单克隆抗体9D4的轻链可变区的氨基酸序列如SEQ ID NO:18所示。
具体的,8H9抗体的六个CDR区的序列结构为:
重链CDR1(VHCDR1),SEQ ID NO.1:GYTFTTYG
重链CDR2(VHCDR2),SEQ ID NO.2:INTYSGVP
重链CDR3(VHCDR3),SEQ ID NO.3:ARYDPTAPDYAMDY
轻链CDR1(VLCDR1),SEQ ID NO.4:ENVGTY
轻链CDR2(VLCDR2),SEQ ID NO.5:GAS
轻链CDR3(VLCDR3),SEQ ID NO.6:GQSYIYPLT
8H9抗体的重链可变区和轻链可变区的氨基酸序列分别为:SEQ ID NO.7和SEQ IDNO.8;
SEQ ID NO.7:
QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQTPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTASLQINNLKNEDTATYFCARYDPTAPDYAMDYWGQGTSVTVSS
SEQ ID NO.8:
NIVMTQSPKSMSMSVGERVTLSCKASENVGTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYIYPLTFGAGTNLELK
8H9抗体的重链可变区和轻链可变区的核苷酸序列分别为:SEQ ID NO.9和SEQ IDNO.10;
SEQ ID NO.9:
CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAACCTATGGAATGAGCTGGGTGAAACAGACTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTCTTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGATATGATCCTACGGCTCCAGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA
SEQ ID NO.10:
AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGGTACTTATGTATCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGAGCTACATCTACCCGCTCACGTTCGGTGCTGGGACCAACCTGGAGCTGAAA
9D4抗体的六个CDR区的序列结构为:
重链CDR1(VHCDR1),SEQ ID NO.11:GYALTDYY
重链CDR2(VHCDR2),SEQ ID NO.12:IFPGNDYT
重链CDR3(VHCDR3),SEQ ID NO.13:ARYGDGNYVYFDY
轻链CDR1(VLCDR1),SEQ ID NO.14:SSISSSN
轻链CDR2(VLCDR2),SEQ ID NO.15:GTS
轻链CDR3(VLCDR3),SEQ ID NO.16:QQWSSYPLT
9D4抗体的重链可变区和轻链可变区的氨基酸序列分别为:SEQ ID NO.17和SEQID NO.18;
SEQ ID NO.17:
QVHLQQSGPELVKPGASVKISCKSSGYALTDYYINWVKQRPGQGLEWIGWIFPGNDYTYFNEKFKDKATLTLDKSSRTAYMLLSSLTTEDSAVYFCARYGDGNYVYFDYWGQGTSLTVSS
SEQ ID NO.18:
ETVLIQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGNSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGAGTKLELK
9D4抗体的重链可变区和轻链可变区的核苷酸序列分别为:SEQ ID NO.19和SEQID NO.20;
SEQ ID NO.19:
CAGGTCCACCTACAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGTCTTCTGGCTACGCCCTCACTGACTACTATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAAATGATTATACTTACTTCAATGAGAAGTTCAAGGACAAGGCCACACTTACTTTAGACAAATCCTCCAGGACAGCCTACATGTTGCTCAGCAGCCTGACCACTGAGGACTCTGCGGTCTATTTCTGTGCAAGATACGGGGATGGTAATTACGTCTACTTTGACTACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCA
SEQ ID NO.20:
GAAACTGTGCTCATTCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTACACTGGTACCAGCAGAAGTCAGGAAACTCCCCCAAACCCTGGATTTATGGCACATCCAATCTTGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA
<110> 郑州大学 河南农业大学
<120> SARS-CoV-2中和性单克隆抗体及应用
<160> 22
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> 人工序列
<221> 8H9-VHCDR1
<400> 1
Gly Tyr Thr Phe Thr Thr Tyr Gly
1 5
<210> 2
<211> 8
<212> PRT
<213> 人工序列
<221> 8H9-VHCDR2
<400> 2
Ile Asn Thr Tyr Ser Gly Val Pro
1 5
<210> 3
<211> 14
<212> PRT
<213> 人工序列
<221> 8H9-VHCDR3
<400> 3
Ala Arg Tyr Asp Pro Thr Ala Pro Asp Tyr Ala Met Asp Tyr
1 5 10
<210> 4
<211> 6
<212> PRT
<213> 人工序列
<221> 8H9-VLCDR1
<400> 4
Glu Asn Val Gly Thr Tyr
1 5
<210> 5
<211> 3
<212> PRT
<213> 人工序列
<221> 8H9-VLCDR2
<400> 5
Gly Ala Ser
1
<210> 6
<211> 9
<212> PRT
<213> 人工序列
<221> 8H9-VLCDR3
<400> 6
Gly Gln Ser Tyr Ile Tyr Pro Leu Thr
1 5
<210> 7
<211> 121
<212> PRT
<213> 人工序列
<221> 8H9-重链可变区
<400> 7
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Gly Met Ser Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Ser
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Tyr Asp Pro Thr Ala Pro Asp Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 8
<211> 107
<212> PRT
<213> 人工序列
<221> 8H9-轻链可变区
<400> 8
Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly
1 5 10 15
Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Ile Tyr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys
100 105
<210> 9
<211> 363
<212> DNA
<213> 人工序列
<221> 8H9-重链可变区编码基因
<400> 9
cagatccagt tggtacagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgcaagg cttctgggta taccttcaca acctatggaa tgagctgggt gaaacagact 120
ccaggaaagg gtttaaagtg gatgggctgg ataaacacct actctggagt gccaacatat 180
gctgatgact tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctct 240
ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagatatgat 300
cctacggctc cagactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360
tca 363
<210> 10
<211> 321
<212> DNA
<213> 人工序列
<221> 8H9-轻链可变区编码基因
<400> 10
aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 60
ttgagctgca aggccagtga gaatgtgggt acttatgtat cctggtatca acagaaacca 120
gagcagtctc ctaaactgct gatatacggg gcatccaacc ggtacactgg ggtccccgat 180
cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcaggct 240
gaagaccttg cagattatca ctgtggacag agctacatct acccgctcac gttcggtgct 300
gggaccaacc tggagctgaa a 321
<210> 11
<211> 8
<212> PRT
<213> 人工序列
<221> 9D4-VHCDR1
<400> 11
Gly Tyr Ala Leu Thr Asp Tyr Tyr
1 5
<210> 12
<211> 8
<212> PRT
<213> 人工序列
<221> 9D4-VHCDR2
<400> 12
Ile Phe Pro Gly Asn Asp Tyr Thr
1 5
<210> 13
<211> 13
<212> PRT
<213> 人工序列
<221> 9D4-VHCDR3
<400> 13
Ala Arg Tyr Gly Asp Gly Asn Tyr Val Tyr Phe Asp Tyr
1 5 10
<210> 14
<211> 7
<212> PRT
<213> 人工序列
<221> 9D4-VLCDR1
<400> 14
Ser Ser Ile Ser Ser Ser Asn
1 5
<210> 15
<211> 3
<212> PRT
<213> 人工序列
<221> 9D4-VLCDR2
<400> 15
Gly Thr Ser
1
<210> 16
<211> 9
<212> PRT
<213> 人工序列
<221> 9D4-VLCDR3
<400> 16
Gln Gln Trp Ser Ser Tyr Pro Leu Thr
1 5
<210> 17
<211> 120
<212> PRT
<213> 人工序列
<221> 9D4-重链可变区
<400> 17
Gln Val His Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ser Ser Gly Tyr Ala Leu Thr Asp Tyr
20 25 30
Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Phe Pro Gly Asn Asp Tyr Thr Tyr Phe Asn Glu Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Arg Thr Ala Tyr
65 70 75 80
Met Leu Leu Ser Ser Leu Thr Thr Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Tyr Gly Asp Gly Asn Tyr Val Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Leu Thr Val Ser Ser
115 120
<210> 18
<211> 108
<212> PRT
<213> 人工序列
<221> 9D4-轻链可变区
<400> 18
Glu Thr Val Leu Ile Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser
20 25 30
Asn Leu His Trp Tyr Gln Gln Lys Ser Gly Asn Ser Pro Lys Pro Trp
35 40 45
Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
85 90 95
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 19
<211> 360
<212> DNA
<213> 人工序列
<221> 9D4-重链可变区编码基因
<400> 19
caggtccacc tacagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60
tcctgcaagt cttctggcta cgccctcact gactactata taaactgggt gaagcagagg 120
cctggacagg gacttgagtg gattggatgg atttttcctg gaaatgatta tacttacttc 180
aatgagaagt tcaaggacaa ggccacactt actttagaca aatcctccag gacagcctac 240
atgttgctca gcagcctgac cactgaggac tctgcggtct atttctgtgc aagatacggg 300
gatggtaatt acgtctactt tgactactgg ggccaaggca cctctctcac agtctcctca 360
<210> 20
<211> 324
<212> DNA
<213> 人工序列
<221> 9D4-轻链可变区编码基因
<400> 20
gaaactgtgc tcattcagtc tccagcactc atggctgcat ctccagggga gaaggtcacc 60
atcacctgca gtgtcagctc aagtataagt tccagcaact tacactggta ccagcagaag 120
tcaggaaact cccccaaacc ctggatttat ggcacatcca atcttgcttc tggagtccct 180
gttcgcttca gtggcagtgg atctgggacc tcttattctc tcacaatcag cagcatggag 240
gctgaagatg ctgccactta ttactgtcaa cagtggagta gttacccact cacgttcggt 300
gctgggacca agctggagct gaaa 324
<210> 21
<211> 606
<212> DNA
<221> RBD基因
<400> 21
atgaacatca ccaacctgtg ccccttcggc gaggtgttca acgccacccg cttcgccagc 60
gtgtacgcct ggaaccgcaa gcgcatcagc aactgcgtgg ccgactacag cgtgctgtac 120
aacagcgcca gcttcagcac cttcaagtgc tacggcgtga gccccaccaa gctgaacgac 180
ctgtgcttca ccaacgtgta cgccgacagc ttcgtgatcc gcggcgacga ggtgcgccag 240
atcgcccccg gccagaccgg caagatcgcc gactacaact acaagctgcc cgacgacttc 300
accggctgcg tgatcgcctg gaacagcaac aacctggaca gcaaggtggg cggcaactac 360
aactacctgt accgcctgtt ccgcaagagc aacctgaagc ccttcgagcg cgacatcagc 420
accgagatct accaggccgg cagcaccccc tgcaacggcg tggagggctt caactgctac 480
ttccccctgc agagctacgg cttccagccc accaacggcg tgggctacca gccctaccgc 540
gtggtggtgc tgagcttcga gctgctgcac gcccccgcca ccgtgcacca ccaccaccac 600
cactaa 606
<210> 22
<211> 200
<212> PRT
<221> RBD
<400> 22
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
1 5 10 15
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
20 25 30
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
35 40 45
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
50 55 60
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
65 70 75 80
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
85 90 95
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
100 105 110
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
115 120 125
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
130 135 140
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
145 150 155 160
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
165 170 175
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
180 185 190
Thr Val His His His His His His
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Claims (10)
1.SARS-CoV-2中和性单克隆抗体,其特征在于,其为单克隆抗体8H9或单克隆抗体9D4;所述单克隆抗体8H9包含氨基酸序列如SEQ ID NO:1-3所示的VHCDR1、VHCDR2、VHCDR3,和氨基酸序列如SEQ ID NO:4-6所示的VLCDR1、VLCDR2、VLCDR3;所述单克隆抗体9D4包含氨基酸序列如SEQ ID NO:11-13所示的VHCDR1、VHCDR2、VHCDR3,和氨基酸序列如SEQ ID NO:14-16所示的VLCDR1、VLCDR2、VLCDR3。
2.如权利要求1所述的SARS-CoV-2中和性单克隆抗体,其特征在于,所述单克隆抗体8H9重链可变区的氨基酸序列如SEQ ID NO.7所示,所述单克隆抗体8H9轻链可变区的氨基酸序列如SEQ ID NO.8所示。
3.如权利要求2所述的SARS-CoV-2中和性单克隆抗体,其特征在于,所述单克隆抗体8H9的轻链型为Kappa,亚型为IgG 2a。
4.如权利要求1所述的SARS-CoV-2中和性单克隆抗体,其特征在于,所述单克隆抗体9D4重链可变区的氨基酸序列如SEQ ID NO.17所示,所述单克隆抗体9D4轻链可变区的氨基酸序列如SEQ ID NO.18所示。
5.如权利要求4所述的SARS-CoV-2中和性单克隆抗体,其特征在于,所述单克隆抗体9D4的轻链型为Kappa,亚型为IgG 2a。
6.编码如权利要求1~5中任一项所述的SARS-CoV-2中和性单克隆抗体的核酸分子。
7.如权利要求6所述的核酸分子,其特征在于,编码所述单克隆抗体8H9重链可变区的基因核苷酸序列如SEQ ID NO:9所示,编码所述单克隆抗体8H9轻链可变区的基因核苷酸序列如SEQ ID NO:10所示。
8.如权利要求6所述的核酸分子,其特征在于,编码所述单克隆抗体9D4重链可变区的基因核苷酸序列如SEQ ID NO:19所示,编码所述单克隆抗体9D4轻链可变区的基因核苷酸序列如SEQ ID NO:20所示。
9.如权利要求1~5中任一项所述的SARS-CoV-2中和性单克隆抗体在制备检测SARS-CoV-2的试剂、预防和/或治疗因SARS-CoV-2感染导致的疾病的药物中的应用。
10.如权利要求9所述的应用,其特征在于,所述SARS-CoV-2包括野生株、B.1.617.2株和B.1.1.529株。
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