CN114349850A - 抗体及其制备方法和编码核酸以及工程细胞 - Google Patents
抗体及其制备方法和编码核酸以及工程细胞 Download PDFInfo
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- CN114349850A CN114349850A CN202011506854.9A CN202011506854A CN114349850A CN 114349850 A CN114349850 A CN 114349850A CN 202011506854 A CN202011506854 A CN 202011506854A CN 114349850 A CN114349850 A CN 114349850A
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Abstract
本公开涉及一种制备抗体的方法、抗体、编码该抗体的核酸和一种工程细胞。本公开提供的抗体能够抗严重急性呼吸综合征冠状病毒2(SARS‑CoV‑2),其特异性好、敏感性高,病原中和效果优异。
Description
技术领域
本公开涉及生物技术领域,具体地,涉及一种制备抗体的方法、抗体、编码该抗体的核酸和一种工程细胞。
背景技术
中和抗体是当病原微生物侵入机体时会产生相应的抗体。病原微生物入侵细胞时需要依赖病原体自身表达的特定分子与细胞上的受体结合,才能感染细胞,并进一步扩增。中和抗体是B淋巴细胞产生的某些抗体,能够与病原微生物表面的抗原结合,从而阻止该病原微生物黏附靶细胞受体,防止侵入细胞。
严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起的新冠肺炎COVID-19已作为严重的疾病在全球传播大流行(Callaway et al.,2020)。目前,尚无针对病毒与靶标相互作用的经过验证的疗法可用于新冠肺炎。而中和抗体由人体免疫系统产生,可以有效阻止病毒感染细胞。但运用康复患者血浆进行治疗虽成效显著,但由于血浆来源有限,不能广泛使用。因此亟需对筛选出有效的中和抗体并实现量产以供大批量治疗需求。
发明内容
本公开的目的是提供一种针对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的中和抗体。
本发明的发明人对抗体的筛选方法进行了改进,较快地获得了一株性能优异的抗体的核酸编码序列和氨基酸序列,由此得到了本发明。
第一方面,本公开提供一种制备抗体的方法,其特征在于,该方法包括如下步骤:
S1、获得抗原免疫后的人体或动物体的血液样本,并将所述血液样本分为第一样本和第二样本;
S2、从所述第一样本中提取mRNA并进行B细胞受体测序,得到第一测序结果;
S3、从所述第二样本中提取mRNA并进行B细胞的单细胞VDJ测序,得到第二测序结果;
S4、将所述第一测序结果和所述第二测序结果的重链部分混合进行抗原特异性聚类,得到B细胞受体重链簇;
S5、在所述B细胞受体重链簇中根据表达量筛选来源于单细胞VDJ测序的候选中和抗体mRNA序列重链;
S6、对筛选得到的候选中和抗体序列重链查找其配对的轻链,对于多条轻链根据表达量进行筛选进行配对,得到轻-重链对;
S7、从所述轻-重链对中根据表达量、种型、CDR1/CDR2突变率、匹配注释得分、拼接质量得分、可产生性、被测序细胞完整性、全长完整度筛选出候选轻-重链对;
S8、提取候选轻-重链对的可变区域部分的序列,根据参考基因组序列添加V基因头部不变序列部分,并且添加恒定区片段形成完整的中和抗体序列;
S9、将所述中和抗体序列进行表达并通过ELISA试验进行筛选,得到目标抗体。
第二方面,本公开提供了一种抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体,该抗体具有轻链和重链,所述轻链的可变区的氨基酸序列如SEQ ID NO.1所示,所述重链的可变区的氨基酸序列如SEQ ID NO.2所示。
可选地,所述轻链的氨基酸序列如SEQ ID NO.3所示,所述重链的氨基酸序列如SEQ ID NO.4所示。
第三方面,本公开还提供了一种核酸组,所述核酸组包括编码所述的抗抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体的轻链的第一核酸和编码所述的抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体的重链的第二核酸,优选地,所述核酸的核苷酸序列如SEQ ID NO.5及SEQ ID NO.6所示。
第四方面,本公开还提供了一种工程细胞,该工程细胞表达如上所述的抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体。
通过上述技术方案,本公开提供的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体特异性好、敏感性高,病原中和效果优异。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本公开的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本公开,但并不构成对本公开的限制。在附图中:
图1是ELISA竞争性实验结果图。
图2是假病毒中和实验结果图。
图3是噬斑减少中和测试结果图。
具体实施方式
以下结合附图对本公开的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本公开,并不用于限制本公开。
本公开提供了一种制备抗体的方法,该方法包括如下步骤:
S1、获得抗原免疫后的人体或动物体的血液样本,并将所述血液样本分为第一样本和第二样本;
S2、从所述第一样本中提取mRNA并进行B细胞受体测序,得到第一测序结果;
S3、从所述第二样本中提取mRNA并进行B细胞的单细胞VDJ测序,得到第二测序结果;
S4、将所述第一测序结果和所述第二测序结果的重链部分进行抗原特异性聚类,得到B细胞受体重链簇;
S5、在所述B细胞受体重链簇中根据表达量筛选来源于单细胞VDJ测序的候选中和抗体序列重链;
S6、对筛选得到的候选中和抗体序列重链查找其配对的轻链,对于多条轻链根据表达量进行筛选进行配对,得到轻-重链对;
S7、从所述轻-重链对中根据表达量、种型、CDR1/CDR2突变率、匹配注释得分、拼接质量得分、可产生性、被测序细胞完整性、全长完整度筛选出候选轻-重链对;
S8、提取候选轻-重链对的可变区域部分的序列,根据参考基因组序列添加V基因头部不变序列部分,并且添加恒定区片段形成完整的中和抗体序列;
S9、将所述中和抗体序列进行表达并通过ELISA试验进行筛选,得到目标抗体。
可选地,其中,所述抗原包括病毒、细菌、小分子化合物半抗原;所述动物体包括哺乳动物、鸟类,优选包括牛、羊、兔、鼠、犬。
可选地,其中,所述单细胞VDJ测序采用10x GenomicsImmune Profiling或SMART-SEQ/SMART-SEQ2测序协议。
可选地,其中,抗原特异性聚类方法采用iSMART方法(Zhang H,Liu L,Zhang J,etal.Investigation of Antigen-Specific T-Cell Receptor Clusters in HumanCancers.Clin Cancer Res.2020;26(6):1359-1371.)或GliPh2方法(Huang,H.,Wang,C.,Rubelt,F.et al.Analyzing the Mycobacterium tuberculosis immune response by T-cell receptor clustering with GLIPH2 and genome-wide antigen screening.NatBiotechnol(2020).)。
可选地,其中,所述抗原特异性聚类的筛选条件包括:(1)簇内包含来自单细胞VDJ测序的重链序列;(2)簇内所有重链CDR3区域氨基酸序列不全相同;(3)簇内重链序列种型包含至少两种,且符合类型转换事件顺序。
可选地,其中,所述V基因头部不变序列部分的重链为SEQ ID NO.7所示,轻链为SEQ ID NO.8所示;所述恒定区片段的序列的重链为SEQ ID NO.9所示,轻链为SEQ IDNO.10所示。
可选地,其中,所述抗原免疫后的人体为传染病人,特别是严重急性呼吸综合征冠状病毒2(SARS-CoV-2)感染者。
本公开还提供了一种严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体,该抗体具有轻链和重链,所述轻链的可变区的氨基酸序列如SEQ ID NO.1所示,所述重链的可变区的氨基酸序列如SEQ ID NO.2所示。
优选地,所述轻链的氨基酸序列如SEQ ID NO.3所示,所述重链的氨基酸序列如SEQ ID NO.4所示。
本公开还提供了一种核酸组,所述核酸组包括编码如上所述的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体的轻链的第一核酸和编码如上所述的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体的重链的第二核酸。优选地,所述核酸的核苷酸序列如SEQ ID NO.5及SEQ ID NO.6所示。
本公开还提供了一种工程细胞,该工程细胞表达如上所述的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体。
下面通过实施例来进一步说明本公开,但是本公开并不因此而受到任何限制。
实施例1
本实施例用于说明本公开的抗体筛选方法的过程。
S1、获得抗原免疫后的人体或动物体的血液样本,并将所述血液样本分为第一样本和第二样本;
S2、将所述第一样本进行B细胞受体测序,得到第一测序结果;
S3、将所述第二样本进行B细胞的单细胞VDJ测序,得到第二测序结果;所述单细胞VDJ测序的条件为使用10x Genomics Immune Profiling测序方案。
S4、将所述第一测序结果和所述第二mRNA测序结果进行抗原特异性聚类,得到B细胞受体重链mRNA簇;所述抗原特异性聚类的筛选条件包括:(1)簇内包含来自单细胞VDJ测序的重链序列;(2)簇内所有重链CDR3区域氨基酸序列不全相同;3)簇内重链序列种型包含至少两种,且符合类型转换事件顺序;
S5、在所述B细胞受体重链mRNA簇中根据表达量筛选来源于单细胞VDJ测序的候选中和抗体mRNA序列重链;
S6、对筛选得到的候选中和抗体序列重链查找其配对的轻链,对于多条轻链根据表达量进行筛选进行配对,得到轻-重链对;
S7、从所述轻-重链对中根据表达量、种型、CDR1/CDR2突变率、匹配注释得分、拼接质量得分、可产生性、被测序细胞完整性、全长完整度筛选出候选轻-重链对;
S8、提取候选轻-重链对的可变区域部分的序列,根据参考基因组补完V基因头部不变序列部分,再加上恒定区片段形成完整的中和抗体序列;所述V基因头部不变序列部分重链如SED NO.7所示,具体为MGWSLILLFLVAVATRVLS,轻链如SEQ ID NO.8所示,具体为MGWSCIILFLVATATGVHS;所述恒定区片段的序列重链如SEQ ID NO.9所示,具体为ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,轻链如SEQ ID NO.10所示,具体为RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
S9、将所述中和抗体序列进行表达并通过ELISA试验、假病毒中和实验以及噬斑减少中和测试进行筛选,得到目标抗体。
目标抗体命名为GD1-69,其氨基酸序列如SEQ ID NO.3及SEQ IDNO.4所示,目标抗体的编码核酸的核苷酸序列如SEQ ID NO.5及SEQ IDNO.6所示。
基于COVID-19患者的PBMC的单细胞转录组和免疫组库抗体筛选
出院时从总共16名COVID-19患者中采集新鲜血液样本。PBMC分为三个等分试样,以分别生成数据:1)通过10x Genomics 5'V(D)J系统进行单细胞RNA-seq文库(n=12);2)使用mRNA进行深层TCR和BCR谱表测序(n=15)。总共,从490万个TCR克隆和690万个BCR克隆中的88974个免疫细胞和免疫受体高变区获得了scRNA-seq数据。
使用SHM和CSR作为选择标准,选择了374个BCR组具有最高的抗原特异性。为了选择潜在的高亲和力抗体,去除了IgM/D/E同种型,并去除了低表达水平(UMI≤5,000)的克隆。其余的174种IgG或IgA抗体在患者血液库中高度扩增,并具有抗原选择的证据。因此,选择了前100名(按UMI计数排名)进行体外验证实验。对于每种抗体,合成包含成对的Ig重链和轻链的DNA质粒,并将其转染到HEK-293T细胞系中进行蛋白质生产。纯化后,使用酶联免疫吸附测定(ELISA)测试抗体分别与1)整个Spike蛋白和2)RBD区的结合。发现了12种对S蛋白具有强结合但对RBD区没有强结合的抗体,可用于COVID-19诊断。鉴定了3种抗体GD1-68,GD1-69和GD1-75与RBD区结合,它们是可能具有临床实用性的推定中和抗体
其中,发现GD1-69具有最高的结合亲和力,在与ACE2的竞争性ELISA实验中,测得其半数抑制浓度为IC50=0.26μg/ml(图1)。GD1-68和GD1-75都没有达到GD1-69的高中和活性。通过基于假病毒的中和试验进一步验证GD1-69的中和活性,测得IC50值为1.77μg/ml(图2)。为了评估其对真病毒的中和潜力,我们使用GD1-69和从COVID-19患者中分离的活SARS-CoV-2病毒在Vero E6细胞中进行了噬斑减少中和测试试验。GD1-69表现出病毒抑制作用,IC50为0.44μg/ml(图3)。鉴于以上结果,GD1-69是针对SARS-Cov-2的有效中和抗体。
测试实施例1
按照与SARS-COV-2病毒S蛋白与S蛋白RBD区域分别按0.01ug/ml及5ug/ml浓度结合的方法,对GD1-69抗体进行间接ELISA实验,结果如表1和表2所示。
按照加入ACE2蛋白,使GD1-69与ACE2竞争性结合SARS-COV-2病毒S蛋白与S蛋白RBD区域的方法,对GD1-69抗体进行竞争性ELISA实验,结果如图1所示。
按照将GD1-69连续稀释孵育1h的假病毒(2×104RLU)与每孔2×104个HEK293T细胞(293T-ACE2细胞)进行混合的方法,对GD1-69抗体进行假病毒实验,结果如图2所示。
按照将GD1-69进行连续稀释并在多孔板中加入预镀的Vro E6细胞单层的方法,对GD1-69抗体进行斑块减少中和检测,结果如图3所示。
将GD1-69抗体与其他抗体相比,结果如表3所示。
表1 SARS-Cov-2 Spike蛋白结合
表2 SARS-Cov-2 Spike RBD区域蛋白结合
表3已于知名医学期刊公开的SARS-COV-2抗体情况
根据表1、表2、表3、图1、图2及图3的数据可以看出,本公开的方法得到的GD1-69抗体能够与SARS-CoV-2Spike蛋白结合,从而能够中和严重急性呼吸综合征冠状病毒2(SARS-CoV-2),并且具有较高的特异性和稳定性。
以上结合附图详细描述了本公开的优选实施方式,但是,本公开并不限于上述实施方式中的具体细节,在本公开的技术构思范围内,可以对本公开的技术方案进行多种简单变型,这些简单变型均属于本公开的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本公开对各种可能的组合方式不再另行说明。
此外,本公开的各种不同的实施方式之间也可以进行任意组合,只要其不违背本公开的思想,其同样应当视为本公开所公开的内容。
序列表
<110> 蒋庆华,王平平,步志高,赵东明
<120> 抗体及其制备方法和编码核酸以及工程细胞
<130> 17156-K-JQH
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Claims (10)
1.一种制备抗体的方法,其特征在于,该方法包括如下步骤:
S1、获得抗原免疫后的人体或动物体的血液样本,并将所述血液样本分为第一样本和第二样本;
S2、对所述第一样本进行B细胞mRNA受体测序,得到第一测序结果;
S3、对所述第二样本进行B细胞的单细胞mRNA VDJ测序,得到第二测序结果;
S4、将所述第一测序结果和所述第二测序结果进行抗原特异性聚类,得到B细胞受体重链簇;
S5、在所述B细胞受体重链簇中根据表达量筛选来源于单细胞VDJ测序的候选中和抗体序列重链;
S6、对筛选得到的候选中和抗体序列重链查找其配对的轻链,对于多条轻链根据表达量进行筛选进行配对,得到轻-重链对;
S7、从所述轻-重链对中根据表达量、种型、CDR1/CDR2突变率、匹配注释得分、拼接质量得分、可产生性、被测序细胞完整性、全长完整度筛选出候选轻-重链对;
S8、提取候选轻-重链对的可变区域部分的序列,根据参考基因组序列添加V基因头部不变序列部分,并且添加恒定区片段形成完整的中和抗体序列;
S9、将所述中和抗体序列进行表达并通过ELISA试验进行筛选,得到目标抗体。
2.根据权利要求1所述的方法,其中,所述抗原包括病毒、细菌、小分子化合物半抗原;所述动物体包括哺乳动物、鸟类,优选包括猴、牛、羊、兔、鼠、犬。
3.根据权利要求1所述的方法,其中,所述单细胞VDJ测序采用10x Genomics ImmuneProfiling或Smart-SEQ/Smart-SEQ2协议;所述抗原特异性聚类方法采用iSMART方法或Gliph2方法。
4.根据权利要求1所述的方法,其中,所述抗原特异性聚类的筛选条件包括:(1)簇内包含来自单细胞VDJ测序的重链序列;(2)簇内所有重链CDR3区域氨基酸序列不全相同;3)簇内重链序列种型包含至少两种,且符合类型转换事件顺序。
5.根据权利要求1所述的方法,其中,所述V基因头部不变序列部分的重链为SEQ IDNO.7所示,轻链为SEQ ID NO.8所示;所述恒定区片段的序列的重链为SEQ ID NO.9所示,轻链为SEQ ID NO.10所示。
6.根据权利要求1所述的方法,其中,所述抗原免疫后的人体为传染病人,特别是严重急性呼吸综合征冠状病毒2(SARS-CoV-2)感染者。
7.一种抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体,该抗体具有轻链和重链,其特征在于,所述轻链的可变区的氨基酸序列如SEQ ID NO.1所示,所述重链的可变区的氨基酸序列如SEQ ID NO.2所示。
8.根据权利要求7所述的抗体,其特征在于,所述轻链的氨基酸序列如SEQ ID NO.3所示,所述重链的氨基酸序列如SEQ ID NO.4所示。
9.一种核酸组,其特征在于,所述核酸组包括编码权利要求8所述的抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体的轻链的第一核酸和编码权利要求8所述的抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体的重链的第二核酸,优选地,所述核酸的核苷酸序列如SEQ ID NO.5及SEQ ID NO.6所示。
10.一种工程细胞,其特征在于,该工程细胞表达权利要求8或9所述的抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的抗体。
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