CN113603770B - 一种新冠病毒核蛋白抗体和应用 - Google Patents
一种新冠病毒核蛋白抗体和应用 Download PDFInfo
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Abstract
本发明提供了一种新冠病毒核蛋白抗体及应用，其重链可变区序列如SEQ ID NO：2所示，其轻链可变区序列如SEQ ID NO:3所示。该抗体具有广谱性。
Description
技术领域
本发明涉及一种新冠病毒核蛋白抗体和应用，属于新冠病毒检测技术领域。
背景技术
2019新型冠状病毒(简称新冠病毒，英文名SARS-CoV-2)，也称为严重急性呼吸系统综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)，是冠状病毒科乙型冠状病毒属，严重急性呼吸道综合征相关冠状病毒种的一个分型。新冠病毒是具有包膜的单股正链RNA病毒，也是已知基因组最大的RNA病毒之一，大约包含2.6-3.2万个碱基。新冠病毒在复制过程中，为了不断适应，会不断的发生突变，而在发生突变时会产生一些影响病毒传播力、致病性和免疫原性等特性的变异株。与2019年底最开始流行的新冠病毒野生型相比，各种突变的S-RBD都发生了较大的变化，此外，其N、M、E和orfab1等基因也发生了诸多变化。以目前流行的“Delta”变异病毒(B.1.617.2)为例，其N蛋白带有D63G/R203M/D377Y三个突变位点。
新型冠状病毒肺炎疫情开始以来，新冠病毒的检测主要有核酸检测、抗体检测和抗原检测等方法。但是，核酸检测和抗体检测是一种极为消耗人力和时间的检测方法，并且价格昂贵，对检测设备、环境以及检测人员都有很高的要求，只能在少数的专业机构开展，致使多数病毒携带者在感染早期并未能被及时鉴别发现。抗原检测具有的快速、准确且易于操作等优点,专业医技人员之外的其他医护人员也可以轻松掌握,并用以向患者提供快速的新冠检测服务,可以大大减轻医疗系统超负荷的状况，有利于将病毒传播的风险降低到最低限度,有助于推动全球各国实现对疫情的完全防控。
由于新冠病毒的高突变性，需要对各种突变病毒珠都有反应的广谱性抗体用于抗原检测。但是，现有的抗体，只是针对特定的病毒，难以满足其广谱性的要求。
发明内容
本发明提供了一种新冠病毒核蛋白抗体和应用，可以有效解决上述问题。
本发明是这样实现的：
一种新冠病毒核蛋白抗体，其重链可变区序列如SEQ ID NO：2所示，其轻链可变区序列如SEQ ID NO：3所示。
作为进一步改进的，其重链包括重链CDR1、重链CDR2及重链CDR3，其氨基酸序列如SEQ ID NO：4-6所示；其轻链包括轻链CDR1及轻链CDR3，其氨基酸序列如SEQ ID NO：7-8所示，其轻链CDR2序列为YTS。
作为进一步改进的，所述新冠病毒核蛋白抗体为以新冠病毒的N蛋白45-181位氨基酸多肽为免疫原制备的单克隆抗体。
一种检测新冠病毒的试剂盒，包括上述的新冠病毒核蛋白抗体。
一种新冠病毒核蛋白抗体，其重链可变区序列如SEQ ID NO：9所示，其轻链可变区序列如SEQ ID NO：10所示。
作为进一步改进的，其重链包括重链CDR1、重链CDR2及重链CDR3，其氨基酸序列如SEQ ID NO：11-13所示；其轻链包括轻链CDR1及轻链CDR3，其氨基酸序列如SEQ ID NO：14-15所示，其轻链CDR2序列为RAS。
作为进一步改进的，所述新冠病毒核蛋白抗体为以新冠病毒的N蛋白45-181位氨基酸多肽为免疫原制备的单克隆抗体。
一种检测新冠病毒的试剂盒，包括上述的新冠病毒核蛋白抗体。
一种检测新冠病毒的试剂盒，包括两种上述的新冠病毒核蛋白抗体。
本发明的有益效果是：
本发明的新冠病毒核蛋白抗体对多种新冠病毒的变异体抗原均具有反应，具有广谱性，能够实现对新冠病毒的多种突变体的有效检测。
附图说明
为了更清楚地说明本发明实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1是本发明实施例1提供的SARS-CoV-2N蛋白的45-181位的抗原纯度鉴定图。
具体实施方式
为使本发明实施方式的目的、技术方案和优点更加清楚，下面将结合本发明实施方式中的附图，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式是本发明一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。因此，以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
在本发明的描述中，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
实施例1克隆SARS-CoV-2N蛋白的45-181位的抗原
采用pET-28a载体，在多克隆位点BamHI/EcoRI中引入目的基因成功构建pET28-45-181的抗原表达质粒，而后利用大肠杆菌表达体系大量制备。
SARS-CoV-2N蛋白的45-181位氨基酸序列如下所示：
LPNNTA SWFTALTQHG KEDLKFPRGQ GVPINTNSSP DDQIGYYRRA TRRIRGGDGKMKDLSPRWYF YYLGTGPEAG LPYGANKDGI IWVATEGALN TPKDHIGTRN PANNAAIVLQ LPQGTTLPKGFYAEGSRGGS Q(SEQ ID NO：1)。
收集菌液打超声破碎，将破碎液12000rpm，10℃离心10min，留取上清。上清过镍柱纯化，利用250nm咪唑洗脱。纯化的抗原利用15％SDS-PAGE跑胶鉴定纯度，其结果如图1所示。
实施例2 Anti-SARS-CoV-2N蛋白的小鼠单克隆抗体制备
1.免疫原的准备：免疫原为实施例1制备的抗原。将抗原稀释至0.4mg/mL，取稀释后的抗原与弗氏佐剂等体积混合，使其成油包水状乳剂。初次免疫使用弗氏完全佐剂，后续加强免疫使用弗氏不完全佐剂，并且融合前72h的最后一次加强免疫不加佐剂。
2.小鼠的基础免疫：使用上述免疫原对6-8周龄BALB/c雌鼠进行皮下多点注射免疫，注射剂量为500μL/只/次，每次免疫前采集200μL眼球静脉血以备滴度测定。每隔2周加强免疫一次。用间接ELISA测定血清滴度，当小鼠血清滴度达到平台期后，小鼠停止免疫并在2周内开展融合，进行融合前3天开展加强注射。
3.融合杂交瘤的制备与筛选
3.1.饲养细胞的制备：分如下步骤进行：(i)将一只6周龄左右的BALB/c小鼠引颈处死，浸泡于75％乙醇溶液中5min；放入超净工作台，使小鼠腹部朝上；用镊子提起小鼠腹部皮肤，剪一小口，用大镊子向上下方向撕拉开皮肤，充分暴露腹部。(ii)用无菌眼科镊子提起腹膜，然后用5mL注射器向腹腔内注入适量培养液，用另一无菌眼科镊子轻微提动小鼠四肢，最后利用注射器吸出培养液于离心管中。(iii)将腹腔细胞液溶解于HAT培养液或HT培养液中，使成浓度为2×105/mL的巨噬细胞,(iv)加入96孔细胞培养板，每孔0.1mL，置培养箱培养。
3.2.小鼠骨髓瘤细胞的制备：一般融合前5天开始复苏小鼠骨髓瘤细胞，每次融合约需6瓶35cm2 Sp2/0细胞。
3.3.免疫脾细胞的制备：分如下步骤进行。(i)取待融合的BALB/C小鼠，摘除眼球放血致小鼠死亡，收集的血液制成抗血清，可作抗体检测的阳性对照。浸泡于75％乙醇溶液中5min，随即放入超净台内小鼠解剖板上，右侧卧位。(ii)无菌手术打开腹腔取出脾脏，用剪刀剪成小块放于200目细胞筛网上，再用研磨棒(注射器内芯)挤压研磨，同时用吹管滴加RPMI-1640培养液。(iii)补加适量RPMI-1640培养液，静置3-5min，取上2/3部分悬液移入50mL塑料离心管中。上述过程反复2-3次。(iv)用RPMI-1640培养液洗细胞3次(1500rpm×5min)。(v)用RPMI-1640培养液重悬细胞，计数。
3.4.使用PEG促融剂融合制备杂交瘤：分如下步骤进行。(1)融合前先将1mL的PEG-1450和50mL的RPMI-1640无血清培养基和200mL完全培养基预温至37℃。(ii)将制备的骨髓瘤细胞与脾细胞混合于50mL离心管内(1×108脾细胞+1×107骨髓瘤细胞，约10:1)，离心1500rpm×5min，离心后，轻轻弹击管底，使细胞疏松成糊状。(iii)1mL吸管吸取0.8mL(按1×108脾细胞+0.8mL PEG)加入离心管内，边加边轻轻搅拌，PEG平均在60s内加完，随即加入10mL预温至37℃的RPMI-1640完全培养液，搅拌要温和。最后补加RPMI-1640培养液至40mL，离心1000rpm×5min。(iv)弃上清液，取少量HT培养液将细胞小心吹散，将细胞移入准备好的HT培养液中，加入96孔细胞培养板，每孔0.1mL，同时每孔加入0.1mL饲养细胞，放入CO2孵育箱中培养。(v)12h后，配置适量的HAT完全培养基，每个孔中滴加0.1mL；5天后，用HT完全培养基给孔中的细胞上清进行100％的换液；大约9-14天后，吸取上清检测。
3.5.杂交瘤的筛选：间接ELISA筛选，包被抗原300ng/mL(抗原是pET28-45-181表达的蛋白)，每孔包被0.1mL，加入细胞上清50uL检测，并挑选出阳性克隆孔。
3.6.杂交瘤细胞的克隆化：采用有限稀释法，先把细胞按一定浓度进行梯度稀释，然后接种到96孔细胞培养板的每个孔中，尽可能使孔内只有一个细胞生长。杂交瘤单克隆阳性细胞株一般需要重复克隆2-3次，直到100％阳性后确认为稳定克隆株，最后获得了2株杂交瘤细胞F1F7、F1A3。
3.7.单抗腹水的生产
取BALB/c小鼠2-3只，经腹腔注入0.5mL液体石蜡油。1周后，将对数生长期的杂交瘤细胞以1500rpm离心5min，弃上清液。杂交瘤细胞用无血清培养液悬浮，并将细胞数调至(1-2)×106/mL，每只小鼠腹腔注射0.5mL。7-10d后，小鼠腹部明显膨大后，引颈处死小鼠，浸泡于75％乙醇中1min，使小鼠腹部朝上用注射针头固定四肢于小鼠解剖台板。用镊子提起小鼠腹部皮肤，剪一小口，然后从两边向小鼠背部方向剪开，用大镊子向上下方向撕开皮肤，充分暴露腹部。用无菌眼科镊子提取腹膜，将腹膜中央处剪一小口，然后用1mL吸管通过小口吸出腹腔内全部腹水。收集的腹水可以混合，置离心管离心，12000rpm，10min。离心后收集上清。
3.8.单抗腹水的纯化
经硫酸铵沉淀和Protein A亲和层析纯化(购自美国GE公司)，获得高纯度的的单克隆抗体。
实施例2:SARS-CoV-2高突变谱系以及高突变位点的自然变体的病毒的合成以及相关抗原制备
利用https://www.gisaid.org和https://ngdc.cncb.ac.cn/ncov/variation/annotation两个网站跟进查找针对全球新型冠状病毒的变异情况，制备不同的自然变体病毒株抗原。结果如下表1所示。
表1
以上56种的自然变体抗原都依据实施例1的方法进行开发制备。
实施例3:Anti-SARS-CoV-2N蛋白的小鼠单克隆抗体对高突变谱系的病毒株以及高突变的位点的病毒株的反应情况
3.1.反应板制备
将以上制备纯化的NTD1-NTD56个抗原以及野生型的抗原(pET28-1-419)用pH9.6的50mM CB缓冲液(NaHCO3/Na2CO3缓冲液，终浓度为50mM，pH值为9.6)稀释，终浓度为3μg/mL；在96孔酶标板每孔中加入100μL的包被液，37℃包被2小时后再2～8℃包被16～24小时；用PBST洗涤液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤1次；然后每孔加入200μL的封闭液(含有20％小牛血清及1％酪蛋白的pH值为7.4的20mM Na2HPO4/NaH2PO4缓冲液溶液)，放入37℃封闭2小时；弃去封闭液。干燥后装入铝箔袋2-8℃保存备用。
3.2Anti-SARS-CoV-2N蛋白的小鼠单克隆抗体的ELISA检测
取实施例1中获得的Anti-SARS-CoV-2N蛋白小鼠单克隆抗体，用含20％新生牛血清的PBS溶液稀释至1μg/mL，进行定性ELISA检测。
样品反应：取本实施例已包被抗原的反应板，每孔加入100μL已稀释的样品，置于37℃温箱反应30分钟。
酶标记物反应：完成样品反应步骤后，将反应板用PBST洗液(20mM PB7.4，150mMNaCl，0.1％Tween20)洗涤5遍，每孔加入100μL HRP标记的羊抗鼠IgG(GAM)反应液，置于37℃温箱反应30分钟。
显色反应：完成酶标记物反应步骤后，将反应板用PBST洗液(20mM PB7.4，150mMNaCl，0.1％Tween20)洗涤5遍，每孔加入TMB显色剂(购自北京万泰生物药业股份有限公司)各50μL，置于37℃温箱反应15分钟。
终止反应及读值测量：完成显色反应步骤后，在反应完的反应板中每孔加入终止液(购自北京万泰生物药业股份有限公司)50μL，并于酶标仪上检测各孔的OD450/620值。
Anti-SARS-CoV-2N蛋白的小鼠单克隆抗体与56种自然变体抗原的反应性判定：根据反应后的读值进行判定。自然变体检测值/野生型抗原检测值大于50％，则判定为对自然变体有检出，反之说明抗体对自然变体存在风险。
3.3 Anti-SARS-CoV-2N蛋白小鼠单克隆抗体的识别性质分析
结果如表2所示：
表2
结果分析：本发明筛选到了F1F7和F1A3单克隆抗体对目前收集到的新型冠状病毒的高频突变系以及高频突变位点的自然变体抗原都有很好的反应，为后续的诊断以及病毒的研究提供工具。
实施例4用胶体金检测灵敏度
4.1胶体金标记抗体制备
往商业化胶体金溶液(厂家：nanocs，货号：GP01-50-20)中添加1％0.2mol/mLK2CO3和1％待标记的蛋白，室温旋转混合反应2小时；添加1％的BSA，室温旋转混合反应30min，封闭金颗粒；16000rpm，4℃离心30min，去除上清，加入胶体金溶液的1/10体积的保存液重悬保存。将标记好胶体金颗粒，用冻干缓冲液稀释20倍后，平铺于玻璃纤维上，冷冻干燥15小时后制成胶体金结合垫备用。
4.2胶体金包被抗体制备
将NC膜粘贴于PVC底板对应位置上，制备包被板，置于50％湿度环境平衡30min。分别用5％SDS和纯水清洗划膜仪管路；将蛋白用包被缓冲液稀释至2mg/mL；将准备好的包被液，通过划膜仪喷涂至包被板上对应位置；将喷涂好的包被板至于37℃，20％湿度的恒温箱中干燥15小时后备用。
4.3胶体金评估检测方法
在干燥后的包被板上对应位置分别粘贴上滤纸、胶体金结合垫、样品垫，利用自动斩切机裁切成4mm宽度的试纸条，并将试纸条装入卡壳中，制成成品试剂备用。
利用裂解液处理新型冠状病毒抗原检测试剂国家参考品中的S编号样品(样品来源体外诊断试剂参考品，批号：370095-202001)，S编号样品分别用20mM PBS稀释成1:50、1:100、1:200、1:400、1:800分别标记成S1-S6。
分别对以上稀释的6个样品进行测定。取出40ul的裂解液加入到100ul的样品中混匀，而后取70ul裂解后的样品加入成品试剂的上样孔中，静置层析20分钟，观察检测线和质控线显色情况。
结果判定：若检测线和质控线均显色，则为阳性；若仅质控线显色，则为阴性；若质控线不显色，无论检测线显不显色，均为无效结果。阴阳性结果强弱用“-、+、++、+++”表示。结果如下表3所示。
表3
| 包被抗体 | F1F7 | F1A3 |
| 标记抗体 | F1A3 | F1F7 |
| S1 | +++ | +++ |
| S2 | +++ | +++ |
| S3 | +++ | +++ |
| S4 | ++ | ++ |
| S5 | ++ | ++ |
| S6 | + | + |
结果分析：目前F1F7/F1A3、F1A3/F1F7两个配对检测新型冠状病毒抗原检测试剂与国家参考品中的样品检测灵敏度相当，都满足需求。
实施例5利用广谱性抗体F1F7/F1A3抗体组装的抗原胶体金检测试剂进一步评估56种自然突变体和野生型的抗原反应情况
如实施例4的检测方法，开展F1F7/F1A3的抗原胶体金检测试剂的准备，以及测试。样品为实施2制备获得的自然变体的抗原和实施例1制备获得的野生型pET28-1-419的抗原。每个抗原利用20％NBS稀释至100pg/mL，而后依据实施例4的检测方法开展测试。结果如下表4所示。
表4
结果分析，对于现有自然变体的抗原都有检出，其中NTD42是当下大家关注的delta突变株，本配对检测也没有问题。
经检测，抗体F1F7包含如SEQ ID NO：2所示的重链可变区序列
EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTIHWVKQSHGESLEWIGGINPNNGDTTYNQTFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGPKDHSYLAWFAYWGQ
和SEQ ID NO：3所示的轻链可变区序列
DIQMTQTTSSLSASLGDRVTISCSASLDVSNYLNWYQQKPEGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKL。
抗体F1F7包含分别如SEQ ID NO:4-6所示的重链CDR序列CDR1 GYTFTEYT，CDR2INPNNGDT，CDR3 ARGPKDHSYLAWFAY；和SEQ ID NO:7-8所示的轻链CDR序列CDR1 LDVSNY，CDR3 QQYSKLPYT，其CDR2序列为YTS。
抗体F1A3的序列为包含如SEQ ID NO：9所示的重链可变区序列
EIQLQQTRPELVKPGASVKISCKASGFSFTDYIMFWVKQSHGKSLEWIGNITPYYGTTSYNLKFKGKATLTVDKSSSTAYMQLNSLTSADSAVYYCACGTEGDYSAMDYWGQ和SEQ ID NO：10所示的轻链可变区序列
DIVLTQSPASLAVSLGQRATISCRASESVDSYGKSFMHWYQQKPGQPPKLLISRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNADPYTFGGGTKLDIK。
抗体F1A3包含分别如SEQ ID NO:11-13所示的重链CDR序列CDR1 GFSFTDYI，CDR2ITPYYGTT，CDR3 ACGTEGDYSAMDY；和SEQ ID NO:14-15所示的轻链CDR序列CDR1ESVDSYGKSF，CDR3 QQSNADPYT，其CDR2序列为RAS。
以上所述仅为本发明的优选实施方式而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
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Claims (9)
1.一种新冠病毒核蛋白抗体，其特征在于，其重链可变区序列如SEQ ID NO：2所示，其轻链可变区序列如SEQ ID NO：3所示。
2.根据权利要求1所述的新冠病毒核蛋白抗体，其特征在于，其重链包括重链CDR1、重链CDR2及重链CDR3，其氨基酸序列如SEQ ID NO：4-6所示；其轻链包括轻链CDR1及轻链CDR3，其氨基酸序列如SEQ ID NO：7-8所示，其轻链CDR2序列为YTS。
3.根据权利要求1所述的新冠病毒核蛋白抗体，其特征在于，所述新冠病毒核蛋白抗体为以新冠病毒的N蛋白45-181位氨基酸多肽为免疫原制备的单克隆抗体。
4.一种检测新冠病毒的试剂盒，其特征在于，包括权利要求1至3任一项所述的新冠病毒核蛋白抗体。
5.一种新冠病毒核蛋白抗体，其特征在于，其重链可变区序列如SEQ ID NO：9所示，其轻链可变区序列如SEQ ID NO：10所示。
6.根据权利要求1所述的新冠病毒核蛋白抗体，其特征在于，其重链包括重链CDR1、重链CDR2及重链CDR3，其氨基酸序列如SEQ ID NO：11-13所示；其轻链包括轻链CDR1及轻链CDR3，其氨基酸序列如SEQ ID NO：14-15所示，其轻链CDR2序列为RAS。
7.根据权利要求5所述的新冠病毒核蛋白抗体，其特征在于，所述新冠病毒核蛋白抗体为以新冠病毒的N蛋白45-181位氨基酸多肽为免疫原制备的单克隆抗体。
8.一种检测新冠病毒的试剂盒，其特征在于，包括权利要求5至7任一项所述的新冠病毒核蛋白抗体。
9.一种检测新冠病毒的试剂盒，其特征在于，包括权利要求1至3任一项所述的新冠病毒核蛋白抗体和权利要求5至7任一项所述的新冠病毒核蛋白抗体。
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