CN112521496B - 特异性结合SARS-CoV-2 Spike RBD的单克隆抗体及其应用 - Google Patents
特异性结合SARS-CoV-2 Spike RBD的单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明公开一种特异性结合SARS‑CoV‑2 Spike RBD的单克隆抗体，为SARS‑CoV‑2 Spike RBD/2E10；本发明还公开了包含ARS‑CoV‑2 Spike RBD/2E10和SARS‑CoV‑2 Spike RBD/6E2的单克隆抗体、包含SARS‑CoV‑2 Spike RBD/2E10和HRP‑SARS‑CoV‑2 Spike RBD/6E2的单克隆抗体；同时还公开了一种表达载体，以及所述单克隆抗体的应用。所述单克隆抗体，可结合并识别SARS‑CoV‑2 Spike RBD的多肽链，用于SARS‑CoV‑2 Spike RBD的检测及药物制备。
Description
技术领域
本发明属于生物医学技术领域，具体涉及特异性结合SARS-CoV-2 Spike RBD的单克隆抗体及其应用。
背景技术
SARS-CoV-2 Spike protein (S蛋白) 是冠状病毒最重要的表面蛋白，与病毒的传染能力及发病机制等密切相关。S蛋白是一类很大的三聚体跨膜蛋白，可以被分成两个功能单位：S1和S2蛋白亚基。S1包含受体结合区域（receptor binding domain,RBD）部分，负责识别和结合细胞受体。
研究发现冠状病毒人传人的特性是由衣壳表面棘突蛋白（S蛋白）与宿主细胞表面受体的相互作用实现的。因此，病毒S蛋白与受体表面的相互作用可以定量的评价病毒的传播能力。石正丽等报道SARS-CoV-2 Spike RBD与人体细胞表面的ACE2受体结合进入细胞。ACE2是人类的一个重要的细胞表面受体，广泛分布于人的心脏、肾脏、睾丸、胃肠道、大脑和肺内。
S蛋白也是宿主中和抗体的重要作用位点，针对与SARS-CoV-2 Spike RBD抗体的研究不仅适用于新冠病毒的临床检测，还可应用于开发抗新冠病毒的药物。然而，目前尚未有SARS-CoV-2 Spike RBD浓度检测方法应用于相关疾病的临床诊断。
发明内容
针对现有技术的缺陷，本发明提供一种特异性结合SARS-CoV-2 Spike RBD的单克隆抗体及其在抗SARS-CoV-2 Spike RBD药物制备及检测中的应用。
特异性结合SARS-CoV-2 Spike RBD的单克隆抗体，所述单克隆抗体为SARS-CoV-2Spike RBD/2E10，所述SARS-CoV-2 Spike RBD/2E10识别并结合SARS-CoV-2 Spike RBD的多肽链；所述SARS-CoV-2 Spike RBD/2E10的轻链可变区的氨基酸序列如SEQ.ID.NO.1所示，所述SARS-CoV-2 Spike RBD/2E10的重链可变区的氨基酸序列如SEQ.ID.NO.2所示。
编码所述单克隆抗体的基因，编码SARS-CoV-2 Spike RBD/2E10的轻链可变区的基因序列如SEQ.ID.NO.5所示；编码SARS-CoV-2 Spike RBD/2E10的重链可变区的基因序列如SEQ.ID.NO.6所示。
特异性结合SARS-CoV-2 Spike RBD的单克隆抗体，包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2，所述SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2识别并结合SARS-CoV-2 Spike RBD的多肽链；
所述SARS-CoV-2 Spike RBD/6E2的轻链可变区的氨基酸序列如SEQ.ID.NO.3所示，所述SARS-CoV-2 Spike RBD/6E2的重链可变区的氨基酸序列如SEQ.ID.NO.4所示；
所述SARS-CoV-2 Spike RBD/2E10采用上述单克隆抗体SARS-CoV-2 Spike RBD/2E10。
编码所述特异性结合SARS-CoV-2 Spike RBD的单克隆抗体的基因，所述单克隆抗体包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2；编码SARS-CoV-2 Spike RBD/2E10的轻链可变区的基因序列如SEQ.ID.NO.5所示；编码SARS-CoV-2 Spike RBD/2E10的重链可变区的基因序列如SEQ.ID.NO.6所示；编码SARS-CoV-2Spike RBD/6E2的轻链可变区的基因序列如SEQ.ID.NO.7所示；编码SARS-CoV-2 SpikeRBD/6E2的重链可变区的基因序列如SEQ.ID.NO.8所示。
特异性结合SARS-CoV-2 Spike RBD的单克隆抗体，包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和HRP-SARS-CoV-2 Spike RBD/6E2，所述SARS-CoV-2 Spike RBD/2E10和HRP-SARS-CoV-2 Spike RBD/6E2识别并结合SARS-CoV-2 Spike RBD的多肽链；所述HRP-SARS-CoV-2 Spike RBD/6E2为HRP标记的SARS-CoV-2 Spike RBD/6E2；
所述SARS-CoV-2 Spike RBD/6E2的轻链可变区的氨基酸序列如SEQ.ID.NO.3所示，所述SARS-CoV-2 Spike RBD/6E2的重链可变区的氨基酸序列如SEQ.ID.NO.4所示；
所述 SARS-CoV-2 Spike RBD/2E10采用上述单克隆抗体SARS-CoV-2 Spike RBD/2E10。
编码所述特异性结合SARS-CoV-2 Spike RBD的单克隆抗体的基因，所述单克隆抗体包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和HRP-SARS-CoV-2 Spike RBD/6E2，所述HRP-SARS-CoV-2 Spike RBD/6E2为HRP标记的SARS-CoV-2 Spike RBD/6E2；编码SARS-CoV-2 Spike RBD/2E10的轻链可变区的基因序列如SEQ.ID.NO.5所示；编码SARS-CoV-2Spike RBD/2E10的重链可变区的基因序列如SEQ.ID.NO.6所示；编码SARS-CoV-2 SpikeRBD/6E2的轻链可变区的基因序列如SEQ.ID.NO.7所示；编码SARS-CoV-2 Spike RBD/6E2的重链可变区的基因序列如SEQ.ID.NO.8所示。
一种表达载体，所述表达载体含有上述单克隆抗体中的基因序列SEQ.ID.NO.5、SEQ.ID.NO.6 、SEQ.ID.NO.7 和SEQ.ID.NO.8。
所述单克隆抗体用于抗SARS-CoV-2的药物的制备的应用；优选地，所述单克隆抗体包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2，所述SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2识别并结合SARS-CoV-2 SpikeRBD的多肽链。
所述单克隆抗体用于SARS-CoV-2 Spike RBD检测的应用；优选地，所述单克隆抗体包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和HRP-SARS-CoV-2 Spike RBD/6E2，所述SARS-CoV-2 Spike RBD/2E10和HRP-SARS-CoV-2 Spike RBD/6E2识别并结合SARS-CoV-2 Spike RBD的多肽链；所述HRP-SARS-CoV-2 Spike RBD/6E2为HRP标记的SARS-CoV-2Spike RBD/6E2。
更优选地，所述单克隆抗体用于SARS-CoV-2 Spike RBD检测的应用，所述应用具体为SARS-CoV-2 Spike RBD诊断试剂的制备。
本发明的优点：
本发明提供的单克隆抗体，可结合并识别SARS-CoV-2 Spike RBD的多肽链，用于SARS-CoV-2 Spike RBD的检测及药物制备，能够为SARS-CoV-2 Spike RBD的检测应用于临床诊断奠定基础。
附图说明
图1 利用SDS-PAGE检测SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2单抗的纯化结果。
图2 利用western-blot法检测SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 SpikeRBD/6E2单抗与SARS-CoV-2 Spike RBD的反应结果。
图3 利用SARS-CoV-2 Spike RBD/2E10和HRP- SARS-CoV-2 Spike RBD/6E2双抗体夹心法，检测SARS-CoV-2 Spike RBD标准品得到标准曲线。
具体实施方式
本发明采用重组表达的SARS-CoV-2 Spike RBD免疫BALB/c小鼠，制备了一组分泌小鼠SARS-CoV-2 Spike RBD单克隆抗体的杂交瘤细胞株，从中筛选出能稳定分泌高亲和力单克隆抗体SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2的杂交瘤细胞株，制备腹水并纯化出单克隆抗体；提取所述杂交瘤细胞株的RNA，反转录成cDNA，PCR扩增获得所述单抗的轻链可变区序列和重链可变区序列，通过比对确认该序列的唯一性；使用HRP标记SARS-CoV-2 Spike RBD/6E2单抗；利用SARS-CoV-2 Spike RBD/2E10和HRP-SARS-CoV-2Spike RBD/6E2双抗体夹心法检测SARS-CoV-2 Spike RBD。
具体实施步骤如下：
1. SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2单抗的制备和鉴定
1.1动物免疫
利用SARS-CoV-2 Spike RBD重组蛋白免疫8周龄BALB/c小鼠（购自空军军医大学实验动物中心），初次免疫：取50μg/只SARS-CoV-2 Spike RBD-His重组蛋白与150μL/只弗氏完全佐剂等体积混合，重分乳化后，背部皮下多点注射，4周后取小鼠血清进行抗体效价检测，选择抗体效价高的个体继续背部皮下多点注射进行加强免疫，然后每2周免疫一次，共进行4次免疫，第4次免疫时不加佐剂腹腔注射抗原SARS-CoV-2 Spike RBD-His重组蛋白50μg /只，3天后取脾细胞，备用。
1.2 杂交瘤细胞制备
采用PEG1500为融合剂，将免疫小鼠脾细胞悬液与小鼠骨髓瘤细胞SP2/0按细胞数量3：1的比例进行融合。融合细胞接种至含滋养细胞（6周龄BALB/c鼠胸腺细胞）96孔细胞培养板中，采用含1%HAT、20%FBS的1640培养基进行筛选培养获得42株杂交瘤细胞。当杂交瘤克隆细胞生长至1/3-1/2底面积时，收集培养上清，SARS-CoV-2 Spike RBD-His重组蛋白以25ng/孔包被于酶标板中，Elisa法检测培养上清中的抗体，经筛选获得2株可稳定分泌抗SARS-CoV-2 Spike RBD抗体的杂交瘤细胞株2E10和6E2，所分泌的抗体分别命名为SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2。
1.3 SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2单抗纯化
BALB/c小鼠腹腔注射灭菌石蜡油500μL/只，小鼠平均分为两组，两周后腹腔分别注射2E10和6E2杂交瘤细胞1×106细胞/只，一周后抽取腹水。3000r/min离心5min，去除血细胞，上清液经15000r/min离心30min， 上清液经0.22μm过滤后，向其中缓慢加入3倍体积的醋酸盐缓冲液；向上述溶液中缓慢加入正辛酸（1mL腹水加入25 μL），室温搅拌10min, 4℃、10000rpm离心20min后弃沉淀，测上清液体积，过滤；加入上清液1/10体积的10XPBS，调节pH至7.4，按1：1比例向其中缓慢加入饱和硫酸铵，调节pH至7；4℃静置沉淀过夜；次日，将上述液体全部倒入离心管中，离心后沉淀用PBS重悬，透析过夜，SDS-PAGE检测纯化抗体，如图1所示。
由图1可知，经正辛酸-饱和硫酸铵沉淀法纯化获得较纯的 SARS-CoV-2 SpikeRBD/2E10和SARS-CoV-2 Spike RBD/6E2单克隆抗体。
1.4 SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2单抗鉴定
以SARS-CoV-2 Spike RBD为抗原，western-blot方法检测SARS-CoV-2 SpikeRBD/2E10和SARS-CoV-2 Spike RBD/6E2单抗与重组SARS-CoV-2 Spike RBD的结合，SARS-CoV-2 Spike RBD（0.5μg）蛋白煮沸后上样，26mA，转膜2.5h后，用5%脱脂牛奶封闭2h；TBST洗膜后按1：1000比例加入2E10和6E2单克隆抗体作为一抗，4℃ 孵育过夜；次日按1：2000比例加入山羊抗小鼠IgG-HRP抗体作为二抗，室温孵育1h，TBST洗膜后，用凝胶成像系统曝光成像，结果如图2所示。由图2可知，所述单抗SARS-CoV-2 Spike RBD/2E10、SARS-CoV-2Spike RBD/6E2可以与SARS-CoV-2 Spike RBD抗原结合。
1.5 抗体轻链和重链可变区的克隆
培养SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2杂交瘤细胞，收集细胞，利用Trizol法提取细胞RNA，利用抗体可变区保守引物进行PCR扩增，扩增条件：94℃ 5min；94℃ 90s, 50℃ 90s, 72℃ 2min (35 cycles)； 72℃ 10min。扩增产物连接pMD18-T载体，转化大肠杆菌感受态JM109，筛选阳性克隆，提取质粒，进行测序鉴定，测序结果进行Ig BLAST比对分析，得到单克隆抗体6E2和2E10的轻链、重链可变区编码基因序列，计算出SARS-CoV-2 Spike RBD/2E10轻链可变区氨基酸序列如SEQ.ID.NO.1所示，重链可变区氨基酸序列如SEQ.ID.NO.2所示，SARS-CoV-2 Spike RBD/6E2轻链可变区氨基酸序列如SEQ.ID.NO.3所示，重链可变区氨基酸序列如SEQ.ID.NO.4所示。
2 HRP标记SARS-CoV-2 Spike RBD/6E2单抗
取10mg纯化的SARS-CoV-2 Spike RBD/6E2抗体，置于碳酸盐缓冲液（pH=9.4）中透析。称取辣根过氧化物酶HRP 20mg（抗体：HRP=1:2）及高碘酸钠128mg，用超纯水溶解后配制预混酶溶液，室温避光放置30 min；将上述酶溶液加至抗体中，4℃透析过夜。取出透析过夜的HRP标记的抗体，分别加入320 μl硼氢化钠及等体积饱和硫酸铵溶液，4℃避光静置2 h；5000rpm离心30 min，弃上清，PBS溶解沉淀，溶液呈砖红色，置于PBS溶液中透析过夜，即获得HRP标记的6E2检测抗体。
3 SARS-CoV-2 Spike RBD/2E10和HRP- SARS-CoV-2 Spike RBD/6E2双抗体夹心法检测SARS-CoV-2 Spike RBD
用碳酸盐缓冲液稀释捕获抗体SARS-CoV-2 Spike RBD/2E10至2.5 μg/mL，进行酶标板包被，100 μL/孔，4℃过夜；2% BSA溶液37℃封闭1h， 200 μL/孔；用封闭液对SARS-CoV-2 Spike RBD从100 ng/ mL开始倍比稀释（100ng/ mL，50 ng/ mL，25ng/ mL，12.5 ng/mL，6.25 ng/ mL，3.125 ng/ mL，0），将稀释好的抗原依次加入到包被好SARS-CoV-2 SpikeRBD/2E10的酶标板中，100 μL/孔，37℃孵育1h；加入用封闭液稀释（1:1000）的HRP- SARS-CoV-2 Spike RBD/6E2， 100 μL/孔，37℃孵育1h；加入显色底物（TMB）100 μL /孔，避光孵育5 min；加终止液50 μL/孔，采用酶标仪检测每孔液体在波长为450 nm处的吸光度值，以吸光度为纵坐标y，稀释浓度（ng/ mL）为横坐标x，拟合的标准曲线如图3所示，检测条件见表1：
表1
由图3可知，单克隆抗体SARS-CoV-2 Spike RBD/2E10 和HRP- SARS-CoV-2 SpikeRBD/6E2组合进行双抗夹心实验检测SARS-CoV-2 Spike RBD具有高线性度，可用于SARS-CoV-2 Spike RBD的定量检测。
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Claims (6)
1.特异性结合SARS-CoV-2 Spike RBD的单克隆抗体，其特征在于：包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和SARS-CoV-2 Spike RBD/6E2，所述SARS-CoV-2 SpikeRBD/2E10和SARS-CoV-2 Spike RBD/6E2识别并结合SARS-CoV-2 Spike RBD的多肽链；
所述SARS-CoV-2 Spike RBD/6E2的轻链可变区的氨基酸序列如SEQ.ID.NO.3所示，所述SARS-CoV-2 Spike RBD/6E2的重链可变区的氨基酸序列如SEQ.ID.NO.4所示；
所述SARS-CoV-2 Spike RBD/2E10的轻链可变区的氨基酸序列如SEQ.ID.NO.1所示，所述SARS-CoV-2 Spike RBD/2E10的重链可变区的氨基酸序列如SEQ.ID.NO.2所示。
2.编码权利要求1所述特异性结合SARS-CoV-2 Spike RBD的单克隆抗体的基因，其特征在于：编码SARS-CoV-2 Spike RBD/2E10的轻链可变区的基因序列如SEQ.ID.NO.5所示；编码SARS-CoV-2 Spike RBD/2E10的重链可变区的基因序列如SEQ.ID.NO.6所示；编码SARS-CoV-2 Spike RBD/6E2的轻链可变区的基因序列如SEQ.ID.NO.7所示；编码SARS-CoV-2 Spike RBD/6E2的重链可变区的基因序列如SEQ.ID.NO.8所示。
3.特异性结合SARS-CoV-2 Spike RBD的单克隆抗体，其特征在于：包含两种抗体，分别为SARS-CoV-2 Spike RBD/2E10和HRP-SARS-CoV-2 Spike RBD/6E2，所述SARS-CoV-2Spike RBD/2E10和HRP-SARS-CoV-2 Spike RBD/6E2识别并结合SARS-CoV-2 Spike RBD的多肽链；所述HRP-SARS-CoV-2 Spike RBD/6E2为HRP标记的SARS-CoV-2 Spike RBD/6E2；
所述SARS-CoV-2 Spike RBD/6E2的轻链可变区的氨基酸序列如SEQ.ID.NO.3所示，所述SARS-CoV-2 Spike RBD/6E2的重链可变区的氨基酸序列如SEQ.ID.NO.4所示；
所述SARS-CoV-2 Spike RBD/2E10的轻链可变区的氨基酸序列如SEQ.ID.NO.1所示，所述SARS-CoV-2 Spike RBD/2E10的重链可变区的氨基酸序列如SEQ.ID.NO.2所示。
4.编码权利要求3所述单克隆抗体的基因，其特征在于：编码SARS-CoV-2 Spike RBD/2E10的轻链可变区的基因序列如SEQ.ID.NO.5所示；编码SARS-CoV-2 Spike RBD/2E10的重链可变区的基因序列如SEQ.ID.NO.6所示；编码SARS-CoV-2 Spike RBD/6E2的轻链可变区的基因序列如SEQ.ID.NO.7所示；编码SARS-CoV-2 Spike RBD/6E2的重链可变区的基因序列如SEQ.ID.NO.8所示。
5.一种表达载体，其特征在于：所述表达载体含有权利要求2所述单克隆抗体中的基因序列SEQ.ID.NO.5、SEQ.ID.NO.6 、SEQ.ID.NO.7 和SEQ.ID.NO.8。
6.权利要求3所述单克隆抗体用于制备SARS-CoV-2 Spike RBD诊断试剂的应用。
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| CN111978395A (zh) * | 2020-07-20 | 2020-11-24 | 四川大学 | 抗新型冠状病毒rbd结构域抗原的单克隆抗体 |
| CN112010965A (zh) * | 2020-05-15 | 2020-12-01 | 潍坊医学院 | 一种针对新冠病毒SARS-CoV-2棘突蛋白RBD区的单克隆抗体及其应用 |
| CN112250763A (zh) * | 2020-12-21 | 2021-01-22 | 三优生物医药(上海)有限公司 | 靶向SARS-CoV-2冠状病毒的抗体及其诊断和检测用途 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112010965A (zh) * | 2020-05-15 | 2020-12-01 | 潍坊医学院 | 一种针对新冠病毒SARS-CoV-2棘突蛋白RBD区的单克隆抗体及其应用 |
| CN111732655A (zh) * | 2020-07-01 | 2020-10-02 | 中国人民解放军军事科学院军事医学研究院 | 靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体及应用 |
| CN111978395A (zh) * | 2020-07-20 | 2020-11-24 | 四川大学 | 抗新型冠状病毒rbd结构域抗原的单克隆抗体 |
| CN112250763A (zh) * | 2020-12-21 | 2021-01-22 | 三优生物医药(上海)有限公司 | 靶向SARS-CoV-2冠状病毒的抗体及其诊断和检测用途 |
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