CN111303280B - 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用 - Google Patents
高中和活性抗SARS-CoV-2全人源单克隆抗体及应用 Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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Abstract
本发明公开了一种抗SARS‑CoV‑2的全人源单克隆抗体，所述抗体通过流式分选‑单细胞PCR技术筛选获得，具有独特的CDR分区，本发明还公开了所述抗体在制备2019‑冠状病毒病治疗药物中的应用。本发明公开的单克隆抗体具有高效、特异的抗SARS‑COV‑2病毒活性，还具有高表达、全人源、稳定性好的特点，适合产业化生产。
Description
技术领域
本发明公开了一种抗体，属于微生物学和免疫学领域。
背景技术
COVID-19的病原体是SARS-CoV-2，也被称为2019-冠状病毒。SARS-CoV-2属冠状病毒科(Coro-naviridae)冠状病毒属(Coronavirus)，是一类有包膜的单股正链RNA病毒。基因组全长为30kb，依次由5＇-端非编码区、非结构蛋白开放阅读框(ORF)1a/b编码区、刺突糖蛋白S编码区、包膜蛋白E编码区、膜蛋白M蛋白编码区、核衣壳蛋白N蛋白编码区及3＇-端非编码区组成。其中，非结构蛋白ORF1a/b区编码的聚蛋白可被切割，形成RNA依赖的RNA聚合酶(RdRp)等，指导病毒基因组复制、转录和翻译。结构蛋白S可特异性地与宿主细胞的受体结合，是病毒入侵宿主易感细胞的关键蛋白。M蛋白和E蛋白参与病毒包膜的形成，而N蛋白则参与病毒的装配。
病毒侵染宿主细胞的过程需要宿主细胞膜表面受体的参与。不同冠状病毒可利用不同的细胞受体来完成入侵，如SARS-CoV的受体是血管紧张素转化酶2(ACE2)，而MERS-CoV的受体是氨基肽酶4(DPP4，也称CD26)。研究证明，氨肽酶N不是SARS-CoV-2的受体，而ACE2可作为其受体。
目前已知引起人致病的冠状病毒包括7种，其中HCoV-229E、HCoV-OC43、HCoV-NL63和HCoV-HKU1致病性较低，一般引起轻微的呼吸道症状。基因测序结果显示，SARS-CoV-2与SARS-CoV的同源性约79％,与MERS-CoV的同源性约50％。文献报道，新型冠状病毒S蛋白ACE2亲和力较SARS-COV强，提示具有更强的传染性。到目前为止，还没有专门用于预防的疫苗和治疗冠状病毒的特效药物。一般采用非特异性治疗，预防严重的并发症，降低重症发病率和死亡率，提高治愈率。研发新型冠状病毒病的疫苗和特异治疗药物成为全球应急科研攻关的重要任务。
SARS-CoV-2感染恢复者体内存在抗病毒多克隆抗体，可中和病毒并阻止新一轮的感染，血浆可用于重症治疗。不足之处：献浆来源有限，难以大规模制备，批间差异等；是否存在抗体依赖的增强作用(ADE效应)还有争议。相比血浆，单克隆抗体(mAb)为单一B淋巴细胞分泌，成分单一明确，可规模化制备，副作用小，且已成功用于治疗多种传染性疾病。中和mAb可以通过杂交瘤技术，人源化转基因小鼠，噬菌体文库筛选以及单细胞技术制备。单细胞-PCR技术特点：全人源，天然稳定性好；需要获取恢复者外周血、特殊分选设备、后续克隆和筛选技术要求高，需要高通量。单细胞PCR技术的原理是新型冠状病毒感染恢复者体内存在对抗病毒的保护性单克隆抗体，编码抗体的基因位于人体外周血单个淋巴细胞内，通过流式细胞仪分选和单细胞-PCR技术可以“钓取”此基因。然后通过基因工程手段，体外规模化制备此分子。在细胞感染模型、动物感染模型上验证单克隆抗体的有效性，在非人灵长类动物体内评价其安全性。
本发明拟采用单细胞PCR技术从SARS-CoV-2感染者恢复后的外周血中获得具有优异中和活性的单抗，目的是提供针对COVID-19具有良好保护效果的全人源单克隆治疗性抗体。
发明内容
基于上述发明目的，本发明通过流式分选-单细胞PCR技术筛选到了一种抗SARS-CoV-2的单克隆抗体，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-117位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第27-37、55-57、94-102位氨基酸序列所示。所述单克隆抗体在本申请中被命名为“4A8”。
在一个优选的实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:5所示。
在一个更为优选的实施方案中，所述抗体的重链恒定区的氨基酸序列如SEQ IDNO:3所示，所述轻链恒定区的氨基酸序列如SEQ ID NO:7或SEQ ID NO:9所示。
第二，本发明还提供了一种编码上述单克隆抗体重链和轻链的多核苷酸，编码所述抗体的重链可变区的多核苷酸序列由SEQ ID NO:2所示，编码所述抗体的轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
在一个优选的实施方案中，编码所述抗体的重链恒定区的多核苷酸序列由SEQ IDNO:4所示，编码所述抗体的轻链恒定区的多核苷酸序列由SEQ ID NO:8或者SEQ ID NO:10所示。
第三，本发明还提供了一种表达上述编码单克隆抗体重链和/或轻链的多核苷酸的功能元件，这种功能元件可以是传统的表达载体。
在一个优选的实施方案中，所述功能元件为线性表达框。
在另一个优选的实施方案中，所述功能元件为哺乳动物表达载体。
第四，本发明还提供了一种含有上述线性表达框的宿主细胞。
在一个优选的实施方案中，所述细胞为Expi 293F细胞。
在另一个优选的实施方案中，所述细胞为CHO-S细胞，本发明可以使用CHO-S细胞构建稳转工程细胞株，实现产业化生产。
最后，本发明还提供了上述单克隆抗体在制备COVID-19治疗药物中的应用。
本发明提供的单克隆抗体显示出对SARS-COV-2感染细胞良好的中和保护效果。本发明的结果显示，所述抗体在制备COVID-19治疗药物中具有广泛应用的前景。本发明公开的单克隆抗体还具有以下的技术优势:(1)全人源，在临床应用上，不需要进行人源化改造以降低人抗鼠抗体反应(HAMA反应)，即低免疫原性。(2)高亲和活性和高中和活性，单抗与S抗原的亲和常数KD是1.0nM,在SARS-COV-2感染的细胞模型上，半数有效浓度EC50是4nM。(3)靶向非受体结合区域：单抗能够与S蛋白和S1区特异结合，但不与RBD特异结合，提示单抗的识别区域为S1非RBD区，可以与靶向RBD或者S2的人源单抗联合应用，制备“鸡尾酒”疗法，避免耐药毒株的产生和提高疗效。(4)稳定性好因为单抗基因来自人体的同一个细胞，是天然配对的，已知人体内IgG1抗体的半衰期是21～28天，理论上公开的单抗具有一致的人体内半衰期。
附图说明
图1.流式细胞仪单细胞分选图；
图2.单克隆抗体可变区基因扩增和T载体上的酶切结果鉴定图谱；
图3.单克隆抗体重链和轻链线性表达框的核酸电泳鉴定图谱；
图4.单克隆抗体可变区序列的检索结果输出图；
图5.单克隆抗体重链和轻链表达载体的核酸电泳检测酶切鉴定图谱；
图6.亲和层析纯化后的单抗SDS-PAGE检测图谱；
图7.ELISA检测不同单抗与S蛋白的结合活性随浓度变化的曲线图；
图8.ELISA检测纯化单抗与S蛋白、S1蛋白、S2蛋白、RBD蛋白的结合活性随浓度变化的曲线图；
图9.单抗在细胞模型上的IC50测定曲线图；
图10.Fortibio测定与S蛋白的亲和力结果输出图。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1人源抗SARS-CoV-2单克隆抗体的筛选和制备
1.1流式细胞仪分选单细胞
将采集的血样利用Ficoll密度梯度离心法分离PBMC，过程如下：
1.1.1取新鲜抗凝全血(EDTA抗凝)用等体积PBS稀释全血。
1.1.2在离心管中加入一定体积的分离液，将稀释后的血样平铺到分离液液面上方，保持两液面界面清晰。分离液、抗凝未经稀释全血、PBS(或生理盐水)体积为1:1:1。
1.1.3配平，室温，水平转子700-800g(2000-2500rpm)，加速度3～4acc，离心20-30min。
1.1.4离心结束后，管底是红细胞，中间层是分离液，最上层是血浆/组织匀浆层，血浆层与分离液层之间是一层薄且较致密的白膜，即：单个核细胞(包括淋巴细胞和单核细胞)层。小心吸取白膜层到另一离心管中。
1.1.5用PBS/1640稀释到一定体积，颠倒混匀。室温，水平转子250-400g(1000-1500rpm)，离心5-10min，弃上清。重复洗涤1-2次。
1.1.6用PBS或合适的培养基将淋巴细胞重悬备用。
将PBMC使用流式抗体染色：anti-CD3-PerCP,anti-CD20-PerCP,anti-CD19-APCAlexa Fluor700,anti-CD27-PE-Cy7，anti-CD38-FITC，每5×106个PBMC细胞各加入5μL上述抗体，室温孵育30分钟/4℃孵育1小时后，使用含2％FBS的PBS重复洗涤2-3次。利用MoFloXDP流式分选细胞仪，选择浆细胞特异的细胞表面标记物(CD3neg/CD20low/CD19high/CD27high/CD38high)分选浆细胞，直接将单个浆细胞分选至96孔板中，每孔中预先加入含有20U RNA酶抑制剂及19.8μL去RNA酶的水，-80℃冻存备用。流式分选的结果见图1，图1-A显示圈定人血单个核细胞群；图1-B显示从1-A圈定的细胞中选择CD3-CD20-CD19+的细胞；图1-C显示从1-B中圈定的细胞中进一步选择CD27++CD38++的细胞。
1.2利用单细胞-PCR技术扩增全人源单抗可变区基因
1.2.1反转录PCR
参考说明书(QIAGEN，210212)，程序简单介绍如下：
通过流式细胞仪分选了1840(共20块96孔PCR反应板，每块板94个细胞，2个空白对照孔)个单细胞。向每个反应体系中同时加入以下全部的针对重链(heavy chain，H)、Kappa轻链(kappa chain，κ)、Lamda轻链(Lamda chain，λ)各亚型的特异引物(引物序列见表1)。
引物：
H：5′L-VH 1、L-VH 3、L-VH 4/6，5′L-VH 5、Hu IgG-const-anti、3′CμCH1
κ：5′L Vκ1/2、5′L Vκ3、5′L Vκ4、3′Cκ543–566
λ：5′L Vλ1、5′L Vλ2、5′L Vλ3、5′L Vλ4/5、5′L Vλ6、5′L Vλ7、5′LVλ8、3′Cλ
表1.反转录PCR引物
PCR反应体系中包含：5×缓冲液6μL、dNTP 1.2μL、反转录酶(全式金生物技术有限公司，AT311)1.2μL、引物如上、模板为单细胞，水补齐至30μL。
PCR反应条件为：50℃反转录30min；接着，95℃预变性15min，95℃40s，55℃30s，72℃1min，40个循环，最后72℃延伸10min。
1.2.2巢式PCR
取反转录产物1ul为模板，进行PCR反应扩增H、κ、λ的可变区：扩增重链可变区、kappa轻链可变区和λ轻链可变区的引物如下表2所示
表2.巢式PCR引物
※单独划线部分用于与上游片段融合，划线黑体部分用于与下游片段的融合。
PCR反应体系中包含：10×缓冲液2.5μL、10mM dNTP 0.5μL、DNA聚合酶(全式金生物技术有限公司，AP141)0.25μL、引物如上、模板为反转录产物1μL、水补齐至25μL。
PCR反应条件为：94℃预变性4min，接着，94℃30s，57℃30s，72℃45min，40个循环，最后72℃延伸10min。
1.2.3琼脂糖凝胶电泳
一个单细胞中重链和轻链基因均扩增成功的克隆，被认为是配对成功的克隆。取5μL巢式PCR扩增产物经1％琼脂糖凝胶电泳后，取配对的阳性克隆连接到T载体上进行测序，测序获得的抗体可变区序列用Vector NTI软件及登录IMGT网站进行分析。
1.2.4 PCR扩增：结果如图2所示，其中泳道1为重链可变区VH的PCR产物，扩增片段约400bp；泳道2是重链可变区VH连接到T载体上的酶切产物(T-H-1,EcoRI/HinDIII)结果，预期空载体片段2700bp,VH片段约400bp；泳道M为Marker，分子量从大到小分别为2000、1000、750、500、250、100bp；泳道3为轻链可变区VL的PCR产物，预期400bp；泳道4为轻链可变区VL连接到T载体上的酶切产物(T-L-14，EcoRI/HinDIII)，预期空载体片段2700bp，VL片段约400bp。
1.2.5序列分析经PCR鉴定阳性的克隆进行DNA序列测定和分析，登录IMGT网站(http://www.imgt.org/IMGT_vquest/analysis)进行可变区检索，为典型的抗体序列，符合预期。检索结果如图4所示，图4-A显示重链可变区的检索结果，V区同源性最高为97.92％，J区同源性最高为93.55％，D区使用读框2。图4-B显示轻链的检索结果，V区同源性最高为98.30％，J区同源性最高为91.89％。
1.3线性表达框的构建
相比传统的表达载体构建方法，构建线性表达框更加快速。设计的线性表达框含有单抗在哺乳细胞内表达的所有原件，线性表达框从5’端依次含有CMV启动子序列(Genbank登记号：X03922.1)、抗体可变区(从单细胞中扩增获得)、抗体恒定区(生工生物合成，重链恒定区序列由SEQ ID NO:3所示，DNA编码序列由SEQ ID NO:4所示，Kappa型轻链恒定区序列由SEQ ID NO:7所示，DNA编码序列由SEQ ID NO:8所示，Lamda型轻链恒定区序列由SEQ ID NO:9所示，DNA编码序列由SEQ ID NO:10所示)、多聚A尾(Genbank登记号：X03896.1)连接起来，将该线性形式的DNA转染入细胞中进行抗体表达。
具体过程是通过体外重叠延伸PCR技术将各个PCR片段连接构建：
1.3.1以pcDNA3.4(ThermoFisher Scientific，A14697)为模板，扩增启动子-前导序列片段、多聚A尾片段。扩增启动子-前导序列片段的PCR反应体系中包括：模板质粒pcDNA3.4 1ng，10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-CMV-forward(与CMV启动子上游序列匹配)(5'-CGATGTACGGGCCAGATATACGCGTTG-3')、引物3'-leader-sequence(5'-ACACTGGACACCTTTTAAAATTAG-3',用于重链的融合,信号肽序列的核苷酸序列5'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGT C-3')，编码的氨基酸序列为MNFGLSLIFLVLILKGV；用于轻链的融合引物序列为5'-GTCACCAGTGGAACCTGGAACCCA-3'，全长信号肽序列核苷酸序列为5-ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGG，氨基酸序列为MDSQAQVLMLLLLWVSGTCG，信号肽序列来源鼠源单抗可变区)、水补齐至50μL。
扩增多聚A尾片段的PCR反应体系中包括：模板质粒pSecTag2(Invitrogen，V90020)1ng，10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-BGH POLY-(A)(5'-GCCTCGACTGTGCCTTCTAG-TTGC-3')、引物3'-BGH-POLY(A)(5'-TCCCCAGCATGCCTGCTATTGTCT-3')、水补齐至50μL。扩增片段长度215bp。
PCR反应条件：94℃预变性4min，接着94℃30s，60℃30s，72℃1min，30个循环，最后72℃延伸10min。
1.3.2扩增抗体恒定区。
H链恒定区PCR体系中包含：重链恒定区模板10ng、10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-CH1(5'-ACCAAGGGCCCATCGGTCTTCCCC-3')、引物3′-CH3
水补齐至50μL。
κ链恒定区PCR体系中包含：κ链恒定区模板10ng、10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-Cκ(5'-ACTGTGGCTGCACCATCTGTCTTC-3')、引物3′Cκ
水补齐至50μL。
λ链恒定区PCR体系中包含：λ链恒定区模板10ng、10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'Cλ(GAGGAGCTTCAAG CCAACAAGGCCACA)、引物3′Cλ
水补齐至50μL。
其中黑体字序列部分
是与polyA互补序列，用于融合扩增。
PCR反应条件为：94℃预变性4min，接着94℃30s，60℃60s，72℃3min，30个循环，最后72℃延伸10min。
1.3.3扩增抗体可变区。
见巢式PCR部分。
1.3.4分别扩增重链和轻链的线性表达框。
PCR反应体系中包括：
模板：纯化后的启动子-前导序列片段10ng、重链/轻链可变区片段10ng、重链/轻链恒定区片段10ng、多聚A尾片段10ng，10×缓冲液2.5μL、10mM dNTP 0.5μL、DNA聚合酶(全式金生物技术有限公司，AP151-13)0.25μL、引物5'-CMV-FORWARD(5'-CGATGTACGGGCCAGATATACGC GTTG-3')和3'-POLY(A)(5'-TCCCCAGCATGCCTGCTATTGTCT-3'、水补齐至25μL。
PCR反应条件为：94℃预变性4min，接着94℃30s，60℃30s，72℃3min，30个循环，最后72℃延伸10min。
1.3.5 PCR产物回收纯化和定量：
PCR反应产物直接用OMEGA公司回收试剂盒回收。DNA定量：用Nano(GEHealthcare)对PCR回收产物进行定量。
1.3.6细胞接种：将293T细胞以2×105/mL接种于24孔细胞培养板中，在含有5％CO2的细胞温箱中，37℃培养过夜。
1.3.7细胞共转染：次日，向200μL无血清的MEM培养基中，加入构建成功的重链和轻链线性表达框PCR产物各1μg，混匀后加入4μL转染试剂Turbofect(Thermo Scientific,R0531)，共同孵育15-20min后逐滴加至过夜培养的293T细胞培养孔中。在含有5％CO2的细胞温箱中，37℃培养48h后收细胞培养上清备用。
线性表达框的PCR融合扩增核酸电泳检测结果见图3，其中泳道1，2，3，分别是重链表达框构建中使用的上游片段、可变区和下游片段，泳道4是构建成功的重链表达框，扩增片段3700左右，符合预期；泳道M是分子量Marker，从大到小分别为5000、3000、2000、1500、1000、750、500、250、100bp；泳道5，6，7分别是轻链表达框构建中使用的上游片段、可变区和下游片段，泳道8为成功构建的轻链表达框，扩增片段3000左右，符合预期。
1.4表达载体的构建和酶切鉴定
以线性表达框为模板，扩增重链，切胶回收约1.4kb大小的重链片段，表达载体pCDNA3.4(ThermoFisher Scientific，A14697)使用Eco RI/BamHI酶切后回收，将重链和载体片段通过同源重组(NEBuilder HIfi DNA Assembly Master Mix,E2621L)方法进行连接，转化TOP10挑取克隆进行PCR检测、双酶切鉴定和序列测定，构建成功重链的表达载体pCDNA3.4-4A8-H-24。以轻链表达框为模板，扩增轻链，胶回收约0.7kb大小的轻链片段，将轻链和载体片段通过同源重组方法进行连接，TOP10挑取克隆进行PCR检测、双酶切鉴定和序列测定，构建成功轻链的表达载体pCDNA3.4-4A8-L-1。重链和轻链表达载体的酶切鉴定核酸电泳检测见图5，泳道1是重链的PCR产物，预期1400bp，泳道2是重链表达载体的双酶切鉴定结果(H-24EcoRI/BamHI酶切)，泳道M是分子量Marker，从大到小分别为5000、3000、2000、1500、1000、750、500、250、100bp、泳道3是轻链的PCR结果，预期700bp，符合预期，泳道4是L表达载体的双酶切鉴定结果(L-1EcoRI/BamHI酶切)，符合预期。
1.5单抗的瞬时表达和亲和层析纯化
使用Expi293表达系统，取15ug重链和15ug轻链混合后转染Expi 293F细胞，按照说明书进行操作(ThermoFisher Scientific，A14635)，5-6天后收获培养液，离心后上清约30ml，使用体积为5ml的预装Protein A亲和层析柱，上样前使用20mM PBS平衡，待电导显示到基线后进样，上样结束后，使用20mMPBS洗涤色谱柱至基线平稳，使用0.1M pH3.0的甘氨酸缓冲液洗脱目的蛋白，待OD280近基线后，停止收集，使用至少3个柱体积的20mM的PBS洗涤色谱柱，至基线平稳后，用20％的乙醇洗涤色谱柱。亲和层析纯化后的单抗SDS-PAGE检测结果见图6(泳道1为单抗4A8的还原电泳结果，预期重链和轻链的分子量分别为50kDa和25kDa，泳道M为分子量标记，泳道2为单抗4A8的非还原电泳，预期150kDa，符合预期)。泳道3，4是筛选的L12单抗的非还原和还原电泳检测结果；泳道5，6是筛选的L16单抗的非还原和还原电泳检测结果。
实施例2人源抗SARS-CoV-2单克隆抗体与S蛋白的结合活性筛选及抗体4A8识别表位分析
2.1包被：取重组的S抗原、S1抗原、RBD抗原和S2抗原用包被液稀释至浓度2μg/mL，包被酶标板，每孔100μL，4℃包被过夜。
2.2封闭：每孔加入300μL PBST洗液，洗涤3次×3min/次；拍净孔内液体，加入2％BSA，200μL/孔，37℃封闭1h。
2.3样品孵育：每孔加入300μL PBST洗液，洗涤3次×3min/次；拍净孔内液体，加入PBS稀释后的纯化单抗，首孔9ug/ml,3倍系列稀释100μL/孔，37℃孵育1h。
2.4二抗孵育：洗涤，同上；加入HRP羊抗人FC二抗(1:20000稀释)，100μL/孔，37℃孵育1h。
2.5显色：洗涤，同上；每孔加入100μL TMB单组分显色液，37℃显色5min后每孔加50μL终止液终止反应，使用酶标仪检测450nm处的吸光值。使用Logistic四参数拟合绘制标准曲线，并根据标准曲线及稀释倍数计算单抗的EC50浓度。
2.6结果：检测不同单抗与S蛋白的结合活性，具体见图7，显示29种单抗与S蛋白的结合活性，最左边的红色曲线即图示中的4A8结合活性随单抗浓度的变化曲线，显示了最高的结合活性(EC50＝0.0046μg/ml)，其余曲线显示28株单抗随单抗浓度的变化曲线，呈现不同的结合活性，但都低于本申请提供的单抗4A8。
2.7选取单克隆抗体4A8进行识别表位分析，方法同上，结果如图8所示，
圆标曲线表示与S蛋白的检测结果，显示特异结合呈现剂量反应关系；方标曲线表示与S1抗原的检测结果，显示特异结合呈现剂量反应关系；正三角标曲线和倒三角标曲线分别显示与S2和RBD的检测结果，显示不结合。分析结果证明，单抗4A8识别的表位位于S1非RBD区。
2.8单抗4A8的序列分析结果如下：
重链可变区的氨基酸序列如SEQ ID NO:1所示，重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-117位氨基酸序列所示，编码重链可变区的多核苷酸序列由SEQ ID NO:2所示；轻链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第27-37、55-57、94-102位氨基酸序列所示，编码轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
实施例3 SARS-CoV-2感染时细胞模型上中和活性分析
3.1将Vero E6细胞用0.25％的胰酶消化后，用培养基(DMEM+10％FBS)稀释至3×105cells/mL浓度，接种到96孔细胞培养板中，接种体积100μL/孔，置37℃5％CO2细胞培养箱培养过夜。
3.2实验当天，将纯化单抗用培养基DMEM+2％FBS自初始浓度(4A8单抗初始浓度154ug/ml,3倍系列稀释，加入96孔培养板，体积120μL/孔；随即每孔加入120μL COVID-19病毒悬液(用DMEM+2％FBS稀释病毒，加入100TCLD50/孔)，充分混匀，置细胞培养箱孵育1h。
3.3弃去96孔板中细胞培养上清，每孔加入200μL共孵育后的病毒-抗体混合悬液；另设置存活对照(不加病毒和抗体)和死亡对照(只加病毒)，置37℃5％CO2细胞培养箱继续培养72h。
3.4 72h后弃去细胞培养上清，加入50μL结晶紫染色液室温染色30min，弃去染液，加入200μL/孔纯水，重复洗涤6次。
3.5弃尽洗液，用吸水纸拍干板孔中水份，加入100μL脱色液充分溶解，以OD620为参考，用酶标仪测OD570值；用(OD样本孔-OD死亡对照)/(OD存活对照-OD死亡对照)计算细胞活率，细胞活率和抗体浓度用GraphPad Prism 5拟合曲线，计算抗体IC50值。
3.6单抗在细胞模型上的保护效果和IC50的结果，见图9(图9横坐标表示对数浓度，纵坐标表示相对阴性对照组的保护率％)。本发明公开的单抗4A8的EC50是0.58ug/ml。
实施例4 BLI(生物膜干涉技术)测定单抗与S抗原的亲和力
使用仪器为Fortebio Octet QKe。测定的基本原理是光通过传感器的生物膜层后会发生透射与反射，反射光的频率受到生物膜层厚度的影响。一些频率的反射光与入射光会产生相长干涉现象，而另一些受到了相消干涉。这些干涉光波被光谱仪所检测到，并形成一幅干涉光谱，并以干涉光谱的相对位移强度(nm)显示。当结合到传感器表面的分子一旦有数量上的增减，传感器表面生物膜的厚度就会有变化，光谱仪便会实时地检测到干涉光谱的位移。当结合在传感器生物膜上的分子A与溶液中的分子B结合，使生物膜层厚度增加，因而产生相对位移，这个相对位移随着分子B结合量的增加而增加最后达到一个平衡状态。
实验步骤：将纯化后的单抗4A8用PBST(PBS+0.05％Tween20)稀释到5μg/mL，每孔200μL加入黑色96孔板，选用Anti-human IgG Fc Capture(AHC)Biosensors上样包被60s，随后在PBST中平衡60s，与不同浓度(500nM、250nM、125nM、62.5nM、31.25nM、16.625nM)的待测抗原S蛋白结合300s，在PBST中解离600s，最后使用软件DataAnalysis7.0分析数据(抗体与S蛋白的亲和力结果输出图见图10)。
实验结果：通过软件拟合计算得到4A8与S蛋白的结合常数(kon)为7.75×104Ms-1，解离常数(kd)为7.77×10-5s-1，平衡解离常数(KD)为1.00nM。说明4A8对于新冠病毒S蛋白具有极高的亲和力。
表3.单抗4A8与S抗原的亲和力测试结果
| KD(M) | kon(1/Ms) | kdis(1/s) | Full R^2 | |
| 4A8和nCoV S | 1.00E-09 | 7.75E+04 | 7.77E-05 | 0.999252 |
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> 高中和活性抗2019-冠状病毒病的全人源单克隆抗体及应用
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Claims (10)
1.一种抗SARS-CoV-2的全人源单克隆抗体，其特征在于，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-117位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第27-37、55-57、94-102位氨基酸序列所示。
2.根据权利要求1所述的单克隆抗体，其特征在于，所述单克隆抗体重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO: 5所示。
3.根据权利要求2所述的单克隆抗体，其特征在于，所述抗体重链恒定区的氨基酸序列如SEQ ID NO:3所示，轻链恒定区的氨基酸序列如SEQ ID NO:7或SEQ ID NO:9所示。
4.一种编码权利要求1-3任一所述单克隆抗体重链和轻链的多核苷酸，其特征在于，编码所述抗体的重链可变区的多核苷酸序列如SEQ ID NO:2所示，编码所述抗体的轻链可变区的多核苷酸序列如SEQ ID NO:6所示。
5.根据权利要求4所述的多核苷酸，其特征在于，编码所述抗体的重链恒定区的多核苷酸序列如SEQ ID NO:4所示，编码所述抗体的轻链恒定区的多核苷酸序列如SEQ ID NO:8或者SEQ ID NO:10所示。
6.一种表达权利要求5所述编码单克隆抗体重链和轻链的多核苷酸的功能元件，其特征在于，所述功能元件为线性表达框或哺乳动物表达载体。
7.一种含有权利要求6所述线性表达框的宿主细胞。
8.根据权利要求7所述的宿主细胞，其特征在于，所述细胞为Expi 293F细胞。
9.根据权利要求7所述的宿主细胞，其特征在于，所述细胞为CHO-S细胞。
10.权利要求1-3任一所述的单克隆抗体在制备COVID-19治疗药物中的应用。
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