CN114195889A - 一种SARS-Cov-2-N纳米抗体及其衍生蛋白和应用 - Google Patents
一种SARS-Cov-2-N纳米抗体及其衍生蛋白和应用 Download PDFInfo
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- CN114195889A CN114195889A CN202111515070.7A CN202111515070A CN114195889A CN 114195889 A CN114195889 A CN 114195889A CN 202111515070 A CN202111515070 A CN 202111515070A CN 114195889 A CN114195889 A CN 114195889A
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Abstract
本发明属于免疫学领域，涉及一种SARS‑Cov‑2‑N纳米抗体及其衍生蛋白和应用。所述的单域抗体由重链构成，重链包括重链CDR1、重链CDR2和重链CDR3；所述的重链CDR1、重链CDR2和重链CDR3的氨基酸序列为（1）‑（5）的序列组合或与其同源性高的序列。本发明使用生物基因工程技术筛选出SARS‑Cov‑2‑N纳米抗体，这些抗体初步亲和力明显，通过原核表达即具有良好的结合活性，可用于SARS‑Cov‑2的检测。
Description
技术领域
本发明属于免疫学领域，涉及一种SARS-Cov-2-N纳米抗体及其衍生蛋白和应用。
背景技术
冠状病毒(Coronaviruses)是一种包膜型无节段阳性RNA病毒，属于冠状病毒科(Coronaviridae)和无节段病毒目(Nidovirales)，是目前已知最大的正链RNA病毒，其基因组长度为26000-32000bp，成熟的冠状病毒直径为60-220nm，因在电子显微镜下呈日冕状或皇冠状，故名为冠状病毒。Beaudette和Hudson于1937年首次从禽类分离出冠状病毒，1965年Tyr-rell等将普通感冒患者鼻冲洗液接种到人胚气管细胞，检测到病毒增殖，并于1968年鉴定为人冠状病毒。根据病毒的血清学和基因组特点，现将冠状病毒亚科分为α、β、γ、δ四个属，其中β-冠状病毒又分为A、B、C、D四系。冠状病毒已在几种鸟类宿主，以及各种哺乳动物中被发现，包括骆驼、蝙蝠、蒙面棕榈果子狸、老鼠、穿山甲、狗和猫，冠状病毒经常在新的哺乳动物发现，并且可引起动物和人的呼吸道、肠道、肝脏、神经系统感染等症状，感染后病情的严重程度又因不同动物种类而异，对于冠状病毒引起的人体感冒的时间多发生在冬季和早春季节。由于冠状病毒RNA依赖的RNA多聚酶易突变，导致RNA重组率高，不仅导致冠状病毒具有各种表现型和基因型，也易产生新的能适应新宿主和生态小环境的变种，从而引发新的人冠状病毒感染疫情。目前已知的可引起人类感染的冠状病毒有以下7种：人冠状病毒229E(HCoV-229E)、人冠状病毒OC43(HCoV-OC43)、严重急性呼吸综合征冠状病毒(SARS-CoV)、人冠状病毒NL63(HCoV-NL63)和人冠状病毒HKU1(HCoV-HKU1)、中东呼吸综合征冠状病毒(MERS-CoV)、2019新型冠状病毒(2019-nCoV)。
2019年末，出现新型冠状病毒(SARSCoV-2，此前称2019-nCoV)感染疾病以来，疫情的发展引起世界关注。2020年2月11日，世界卫生组织将引起此次新冠肺炎的病毒正式命名为SARS-CoV-2。越来越多的证据表明，2019新型冠状病毒与其他已知的在蝙蝠中传播的冠状病毒存在关联，证明其与蝙蝠亚种菊头蝠(Phinolophusbat)存在关联。它是目前已知的第七种能够感染人类的冠状病毒，具有高传染性和高隐蔽性，且临床上针对病毒感染性疾病的治疗较为困难。SARSCoV-2属于冠状病毒β属，遗传物质为正链单股RNA，RNA基因组包含29891个核苷酸，与2003年的SARS冠状病毒(SARS-CoV)的同源性为82％。目前多数患者以发热、乏力、干咳为主，少数患者伴有鼻塞、流涕、咽痛和腹泻等症状，重症患者多在发病1周或2周后出现呼吸困难和低氧血症，严重的患者会快速进展为急性呼吸窘迫综合征、脓毒症休克、甚至出现难以纠正的代谢性酸中毒和出凝血功能障碍等。
目前，国内外研究机构已经广泛开展了针对新冠病毒中和抗体和针对细胞因子风暴的抗体研究工作，其研发策略主要集中于S蛋白，即采用针对S蛋白或ACE2蛋白的中和抗体结合病毒颗粒表面的S蛋白或ACE2受体来阻断S蛋白与ACE2的结合，从而阻断病毒进入细胞。但S蛋白很容易受到环境压力而发生变异，全球各地出现的新冠病毒变种即产生于新冠病毒S蛋白的变异。
有研究证实了经过筛选的N蛋白抗体可以像S蛋白抗体一样激活机体对于病毒感染的初次免疫应答，且在二次免疫应答效果上优于S蛋白抗体。通过测序研究的结果也显示，N蛋白抗体具备的更高突变频率，使其可以刺激宿主产生比S蛋白抗体更强的免疫反应。N蛋白的结构在不同冠状病毒及不同SAES-CoV-2变异毒株中具有很高的相似性，由于N蛋白自身的保守特性，其在新冠病毒的演化及变异中仍然处于保守状态，N蛋白被包裹在病毒中的特点，也使其能够免受导致S蛋白产生变化的环境压力影响。新冠病毒的核衣壳蛋白(N蛋白)是SARS-CoV-2感染后表达量最大的蛋白，在病毒的装配过程中位于病毒颗粒的核心部分，以与基因组RNA结合的形式存在，并在病毒RNA的转录和复制过程中与RNA相互作用。但N蛋白相较于其他的如S、M蛋白更保守，N蛋白不仅能够诱导体液免疫应答，也能诱导细胞免疫应答。
SARS-CoV-2病毒N蛋白全长419个氨基酸，由N端结构域(NTD)和C端结构域(CTD)以及连接两个结构域及其两侧的三段无规则柔性区(IDR)构成。其中NTD为单体，而CTD形成同源二聚体，NTD和CTD都含有较为保守的结合RNA的正电荷分布区域。N蛋白是SARS-CoV-2的核心成分，它与病毒基因组RNA结合，将RNA包装成核糖核蛋白(RNP)复合体。除了组装外，N蛋白还在病毒mRNA转录和复制中发挥重要作用，并参与免疫调节。
纳米抗体是生物医学科学家在传统抗体的基础上，运用分子生物学技术结合纳米粒子科学的概念，从而研发出的最新和最小的抗体分子。1993年，Hamers-Cazterman等在单峰驼以及双峰的亚洲驼和南美骆驼的血清中发现一种天然缺失轻链的重链抗体(HCAb)，克隆重链抗体的可变区得到只由一个重链可变区组成的单域抗体，称为VHH抗体(variabledomain of heavy chain of heavy-chain antibody)，其晶体结构呈椭圆形，直径2.5nm，长4nm，是最小的功能性抗原结合片段，又被称为纳米抗体(Nanobody)。1993年Hamers等发现，骆驼血液中的抗体有一半天然缺失轻链和重链恒定区1(CH1)，克隆这种抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，现已被重新命名为"纳米抗体"(nanobody)，它是具有完整功能的最小的抗原结合片段，分子量15KD。骆驼重链抗体可变区的纳米抗体(VHH)，其分子质量为15KDa，远远小于Fab段(60KDa)和普通抗体(150KDa)。存在于羊驼体内天然重链抗体的分子结构为：铰链区形成的链间二硫键连接两条完全相同的重链，每条重链分子有一个独特的重链可变区(VhH)、一个铰链区和CH2、CH3两个恒定区。在IgG的重链分子中，恒定区CH1是与轻链经链间二硫键相连的部位，CH1在重链抗体的基因组中也存在，但在其mRNA形成过程中被剪切，形成没有轻链和CH1区的骆驼重链抗体。
纳米抗体是抗体界的新宠，由于分子量小，可以通过简单的分子克隆技术获得双价、三价或双特异性抗体。纳米抗体由于其分子小的特点，不论在原核表达系统(大肠杆菌)，还是真核表达系统(CHO细胞、293细胞等)中进行都可以达到很高的产量。纳米抗体的迅速发展成为在抗体药物开发方面一股潜力无限的力量，代表着从今以后的抗体药物的重要发展方向。
纳米抗体与靶结合位点具有很高的亲和力和良好的穿透能力，使得它更容易与受体靶向结合，渗入到血管少的组织内。同时鉴于纳米抗体的低免疫原性，将纳米抗体对小鼠反复给药，经检测，没有引起任何体液或细胞免疫。另外，纳米抗体可以用于构建多种分子结构，从而可以进行一些分子辅助治疗。目前现有技术中缺少针对N蛋白的SARS-Cov-2-N纳米抗体。
发明内容
为了克服上述缺陷，本发明的目的是提供一种SARS-Cov-2-N纳米抗体及其衍生蛋白和应用，使用生物基因工程技术筛选出特异性针对新冠病毒SARS-CoV-2的N蛋白的SARS-Cov-2-N纳米抗体，这些抗体初步亲和力明显，通过原核表达即具有良好的结合活性，能有效检测新冠病毒。
本发明的第一方面，提供了一种SARS-Cov-2-N纳米抗体，所述的单域抗体由重链构成，重链包括重链CDR1、重链CDR2和重链CDR3；
所述的重链CDR1、重链CDR2和重链CDR3的氨基酸序列为下述(1)-(5)的一种：
(1)SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:38所示的CDR3；
(2)SEQ ID NO:27所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:36所示的CDR3；
(3)SEQ ID NO:28所示的CDR1，SEQ ID NO:33所示的CDR2，SEQ ID NO:37所示的CDR3；
(4)SEQ ID NO:30所示的CDR1，SEQ ID NO:34所示的CDR2，SEQ ID NO:39所示的CDR3；
(5)SEQ ID NO:29所示的CDR1，SEQ ID NO:34所示的CDR2，SEQ ID NO:40所示的CDR3。
即，所述的重链包括互补决定区CDR；所述互补决定区CDR包括重链CDR1、CDR2和CDR3的氨基酸序列。以上CDR序列(1)-(5)依次与SEQ ID NO.1-5对应。上述所有序列，可以替换为与该序列具有“至少80％同源性”的序列或仅一个或少数几个氨基酸替换的序列；优选为“至少85％同源性”，更优选为“至少90％同源性”，更优选为“至少95％同源性”，最优选为“至少98％同源性”。
在优选实施例中，所述的单域抗体的序列还包括框架区FR；所述框架区FR包括FR1、FR2、FR3和FR4的氨基酸序列；
所述的单域抗体的框架区FR的序列为下述(a)-(e)中的一种；
(a)SEQ ID NO:12所示的FR1，SEQ ID NO:18所示的FR2，SEQ ID NO:21所示的FR3，SEQ ID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
(b)SEQ ID NO:15所示的FR1，SEQ ID NO:19所示的FR2，SEQ ID NO:24所示的FR3，SEQ ID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
(c)SEQ ID NO:13所示的FR1，SEQ ID NO:17所示的FR2，SEQ ID NO:20所示的FR3，SEQ ID NO:25所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
(d)SEQ ID NO:11所示的FR1，SEQ ID NO:16所示的FR2，SEQ ID NO:23所示的FR3，SEQ ID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
(e)SEQ ID NO:14所示的FR1，SEQ ID NO:16所示的FR2，SEQ ID NO:22所示的FR3，SEQ ID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换。
在一个实施方案中，所述针对SARS-Cov-2-N的单域抗体与选自SEQ ID NO：1-5的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同源性，并且能够特异性结合SARS-Cov-2病毒的N蛋白。
在另一个优选实施例中，所述SARS-Cov-2-N纳米抗体与选自SEQ ID NO：1-5的氨基酸序列具有至少95％序列同源性，并且能够特异性结合SARS-Cov-2的N蛋白。
本发明的第二方面是提供针对SARS-Cov-2-N纳米抗体，所述纳米抗体分别如SEQID NO.1-5所示，或者所述单域抗体与SEQ ID NO.1-5的氨基酸序列具有至少95％序列同源性。
在一个实施方案中，编码所述SARS-Cov-2-N纳米抗体的核酸分子与选自SEQ IDNO：6-10的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同源性，并且其编码的SARS-Cov-2-N纳米抗体能够特异性结合SARS-Cov-2的N蛋白。
优选的，所述纳米抗体的编码序列分别如SEQ ID NO.6-10所示，或者与SEQ IDNO.6-10具有至少95％序列同源性。
本发明的第三个方面是提供编码前述的SARS-Cov-2-N纳米抗体的核苷酸分子，其核苷酸序列分别如SEQ ID NO：6-10所示，或者与SEQ ID NO.6-10具有至少95％序列同源性。
本发明的第四个方面是提供一种表达载体，其包含编码前述的纳米抗体的核苷酸分子或者前述的核苷酸分子。
本发明的第五个方面是提供一种宿主细胞，其可以表达出前述的SARS-Cov-2-N纳米抗体，或其包含前述的表达载体。
本发明还提供一种产生SARS-Cov-2-N纳米抗体的方法，包括步骤：(a)在适合产生纳米抗体的条件下，培养前述的宿主细胞，从而获得含所述SARS-Cov-2-N纳米抗体；(b)从所述培养物中分离或回收所述的SARS-Cov-2-N纳米抗体；以及(c)任选地，纯化和/或修饰步骤(b)中获得的SARS-Cov-2-N纳米抗体。
本发明还提供前述的SARS-Cov-2-N纳米抗体的用途，用于制备试剂、检测板或试剂盒；其中，所述试剂、检测板或试剂盒用于：检测样品中SARS-Cov-2病毒的存在和/或含量。
所述的单域抗体为VHH，其仅包含抗体重链，不包含抗体轻链。本文中，单域抗体即纳米抗体。
相对于现有技术，本发明使用生物基因工程技术筛选出SARS-Cov-2-N纳米抗体，这些抗体初步亲和力明显，通过原核表达即具有良好的结合活性，这些纳米抗体具有以下优势：
(1)这些纳米抗体的表达体系选择灵活，既可以在原核系统中表达也可以在酵母细胞或哺乳动物细胞的真核系统中表达，且其在原核表达系统中的表达成本低，可降低后期生产成本。
(2)由于纳米抗体为单结构域抗体，所以其抗体的多组合形式改造更为简单，通过基因工程的方式简单的串联即可以获得多价、多特异性的抗体。
(3)正如多篇文献报道的那样，纳米抗体的亲和力范围更为宽泛，在未进行亲和力成熟之前，其亲和力范围可以从nM级别至pM级别，为后期不同用途的抗体提供多个选择。
(4)可用于检测SARS-Cov-2，检测准确率高。
附图说明
图1人源重组SARS-Cov-2-N蛋白SDS-PAGE分析图；
图2VHH序列插入率分析；
图3靶向SARS-Cov-2-N淘选的文库富集情况；
图4SARS-Cov-2-N靶点部分原核表达抗体SDS-PAGE；
图5SARS-Cov-2-N靶点抗体抗原结合活性。
具体实施方式
下面结合实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
单域抗体(sdAb，也被研发者Ablynx称为纳米抗体或VHH)是本领域技术人员公知的。单域抗体为其互补决定区是单域多肽的一部分的抗体。因此，单域抗体包含单个互补决定区(单个CDR1、单个CDR2和单个CDR3)。单域抗体的实例为仅有重链的抗体(该抗体天然不包含轻链)、衍生自常规抗体的单域抗体和工程化抗体。
单域抗体可以衍生自任何物种，包括小鼠、人、骆驼、美洲驼、山羊、兔和牛。例如，天然存在的VHH分子可以衍生自骆驼科物种(例如骆驼、单峰骆驼、美洲驼和原驼)提供的抗体。像完整的抗体一样，单域抗体能够选择性地结合特定抗原。单域抗体可以仅含有免疫球蛋白链的可变结构域，该结构域具有CDR1、CDR2和CDR3以及框架区。
如本文所用，术语“序列同源性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同源性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同源性。
本发明中，与本发明公开的CDR1-3的序列同源性高的序列，也可以得到SARS-Cov-2-N纳米抗体。在一些实施例中，与(1)-(5)中的序列具有“至少80％同源性”的序列，或者“至少85％同源性”、“至少90％同源性”、“至少95％同源性”、“至少98％同源性”的序列都可以实现发明目的(即衍生蛋白)。
在一些实施例中，与(1)-(5)中的序列相比仅仅替换一个或少数几个氨基酸的序列，例如，包含1、2、3、4、5、6、7、8、9或10个保守氨基酸取代，也可以实现发明目的。实际上，在确定两个氨基酸序列之间的序列同源性程度或在确定单域抗体中的CDR1、CDR2和CDR3组合时，技术人员可以考虑所谓的“保守”氨基酸取代，在取代情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸，其通常可以被描述为氨基酸残基被具有相似化学结构的另一个氨基酸残基替代的氨基酸取代，并且该取代对多肽的功能、活性或其它生物学性质几乎没有或基本上没有影响。所述保守氨基酸取代在本领域是通用的，例如保守氨基酸取代是以下(a)-(d)组内的一个或少数几个氨基酸被同一组内另一个或少数几个氨基酸所取代：(a)极性带负电残基及其不带电酰胺：Asp、Asn、Glu、Gln；(b)极性带正电残基：His、Arg、Lys；(c)芳香族残基：Phe、Trp、Tyr；(d)脂肪族非极性或弱极性残基：Ala、Ser、Thr、Gly、Pro、Met、Leu、Ile、Val、Cys。特别优选的保守氨基酸取代如下：Asp被Glu取代；Asn被Gln或His取代；Glu被Asp取代；Gln被Asn取代；His被Asn或Gln取代；Arg被Lys取代；Lys被Arg、Gln取代；Phe被Met、Leu、Tyr取代；Trp被Tyr取代；Tyr被Phe、Trp取代；Ala被Gly或Ser取代；Ser被Thr取代；Thr被Ser取代；Gly被Ala或Pro取代；Met被Leu、Tyr或Ile取代；Leu被Ile或Val取代；Ile被Leu或Val取代；Val被Ile或Leu取代；Cys被Ser取代。另外，本领域技术人员知晓，单域抗体的创造性体现在CDR1-3区，而框架区序列FR1-4并非是不可改变的，FR1-4的序列可以采取本发明公开的序列的保守序列变体。
本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
本专利是通过基因工程技术制备目标蛋白以及目标蛋白的截短体形式，然后将获得的抗原蛋白免疫内蒙古阿拉善双峰驼，通过多次免疫后，获得骆驼的外周血淋巴细胞或者脾细胞，通过基因工程的方式，将驼源抗体可变区编码序列重组到噬菌体展示载体中，通过噬菌体展示技术筛选出针对抗原蛋白的特异性抗体，并进一步检测其与抗原结合的能力。
现将上述技术方案拆分详解，以具体实施例的方式描述：
实施例1：人源重组SARS-Cov-2-N蛋白的制备：
本专利中用到的人源重组SARS-Cov-2-N蛋白为公司自己表达纯化获得，SARS-Cov-2-N蛋白的表达载体设计方案具体如下：
(1)在NCBI中检索获得SARS-Cov-2-N蛋白的编码序列，其核苷酸收录号为NC_045512.2，该序列编码产生的氨基酸序列登录号为YP_009724397，Uniprot ID为P0DTC9。
(2)利用序列特异性引物，通过限制性内切酶XbaI和AgeI将编码SARS-Cov-2-N蛋白的第1～419位氨基酸的核苷酸序列克隆到载体pcDNA3.4中。
(3)将构建好的载体进行Sanger测序，比对原始序列，确认无误后，将该重组质粒进行批量抽提，去除内毒素，转染悬浮293F进行目的蛋白(SARS-Cov-2-N蛋白)的表达和纯化，人源重组SARS-Cov-2-N蛋白纯化后SDS-PAGE分析如图1所示。由图1可知，纯化后蛋白纯度高达90％，满足动物免疫需求。
实施例2：构建SARS-Cov-2-N蛋白的单域抗体文库
:将1mg步骤1中纯化获得的人源重组SARS-Cov-2-N蛋白与等体积的弗氏完全佐剂混合，免疫一只内蒙古阿拉善双峰驼，每周免疫一次，共连续免疫7次，除首次免疫外，其余六次均是用1mgSARS-Cov-2-N蛋白与弗氏不完全佐剂等体积混合进行动物免疫，该免疫过程是为了集中刺激骆驼使其产生针对SARS-Cov-2-N蛋白的抗体。
动物免疫结束后，抽取骆驼外周血淋巴细胞150mL，并提取细胞的RNA。利用提取的总RNA合成cDNA，并通过套式PCR反应以cDNA为模板扩增VHH(抗体重链可变区)。
然后利用限制性内切酶分别酶切pMECS载体和VHH片段，然后将酶切后的片段和载体链接。将连接后的片段电转化至感受态细胞TG1中，构建SARS-Cov-2-N蛋白的噬菌体展示文库并测定库容，文库的库容大小约为1×109，同时，通过菌落PCR鉴定检测文库在目的片段的正确插入率，结果如图2所示。
结果显示，从文库中随机挑选的30个菌落进行PCR扩增后，有28个克隆可以扩增出大小为1110bp(预测大小)的条带，有2个克隆扩增出条带不正确，故正确插入率为28÷30×100％≈93.3％。
实施例3：筛选抗SARS-Cov-2-N蛋白的单域抗体
取200μL步骤2中的重组TG1细胞至2×TY培养基中培养，期间加入40μL辅助噬菌体VCSM13侵染TG1细胞，并培养过夜以扩增噬菌体，次日利用PEG/NaCl沉淀噬菌体，离心收集扩增噬菌体。
将稀释在100mMpH8.3的NaHCO3中的SARS-Cov-2-N蛋白500μg偶联在酶标板上，4℃放置过夜，同时设立阴性对照孔；第二天加入200μL的3％的脱脂乳，室温封闭2h；封闭结束后，加入100μl扩增后噬菌体文库(大约2×1011个噬菌体颗粒)，室温作用1h；作用1小时后，用PBS+0.05％Tween-20洗15遍，以洗掉未结合的噬菌体。
用终浓度为25mg/mL的胰蛋白酶将与SARS-Cov-2-N蛋白特异性结合的噬菌体解离下，并感染处于对数生长期的大肠杆菌TG1细胞，37℃培养1h，产生并收集噬菌体用于下一轮的筛选，相同筛选过程重复1轮，逐步得到富集，当富集倍数达到10倍以上时，富集效果如图3所示。
图3中，P/N＝生物淘选中阳性孔洗脱下的噬菌体感染TG1细菌后生长的单克隆细菌数/阳性孔洗脱下的噬菌体感染TG1细菌后生长的单克隆细菌数，该参数在富集发生后会逐渐增大；I/E＝生物淘选中每轮加入阳性孔的噬菌体总量/生物淘选中每轮从阳性孔洗脱出的噬菌体总量，该参数在富集发生后会逐渐趋近于1。
实施例4：酶联法筛选抗SARS-Cov-2-N的特异性阳性克隆
根据上述实施例3中的筛选方法对抗SARS-Cov-2-N蛋白的单域抗体进行3轮筛选，抗SARS-Cov-2-N蛋白的噬菌体富集因子达到10以上，筛选结束后，从筛选获得的阳性克隆中挑选384个单菌落分别接种于含100μg/mL氨苄青霉素的2×TY培养基的96深孔板中，并设置空白对照，37℃培养至对数期后，加入终浓度为1mM的IPTG，28℃培养过夜。
利用渗透涨破法获得粗提抗体；将SARS-Cov-2-N重组蛋白分别释至100mMpH8.3的NaHCO3中并将100μg蛋白在酶标板(ELISA板)中4℃包被过夜。将获得的抗体粗提液取100uL转移至加入抗原的ELISA板上，室温孵育1h；用PBST洗去未结合的抗体，加入100μl经1:2000稀释后的MouseAnti-HAtagAntibody(HRP)(鼠抗HA辣根过氧化物酶标记抗体，ThermoFisher)，在室温孵育1h；用PBST洗去未结合的抗体，加入辣根过氧化物酶显色液，37℃下反应15min后，加入终止液，于酶标仪上450nm波长处，读取吸收值。
当样品孔OD值大于对照孔5倍以上时，判定为阳性克隆孔；将阳性克隆孔的菌转摇在含有100μg/mL氨苄青霉素的LB培养基中以便提取质粒并进行测序。
根据序列比对软件VectorNTI分析各个克隆株的基因序列，把CDR1，CDR2和CDR3序列相同的株视为同一克隆株，而序列不同的株视为不同克隆株，最终获得特异性针对SARS-Cov-2-N蛋白的单域抗体(分别为纳米抗体1B2、1C3、1C11、1E4、2B9，依次对应SEQ ID NO.1-5，以及未示出序列的单域抗体1A10、1A6、1B5、1B7、1C1、1D7、1F4、1F7、1H4、2A2、2C12、2C3、2D2、2E2、2F11、1C4、1C7、1D2、1D5)。
其抗体的氨基酸序列为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4结构，构成整个VHH。获得的单域抗体重组质粒可以在原核系统中进行表达，最终获得单域抗体蛋白。
5种单域抗体的CDR序列、FR序列、氨基酸序列分别如表1、表2、表3所示。
表1 5种单域抗体的CDR序列
表2 5种单域抗体的FR序列
表3 5种单域抗体的氨基酸序列
实施例5：抗SARS-Cov-2-N蛋白的单域抗体在宿主大肠杆菌中的纯化及表达。
将实施例4中测序分析所获得不同克隆株的质粒(pMECS-VHH)电转化到大肠杆菌HB2151中，并将其涂布在LB+amp+glucose即含有氨苄青霉素和葡萄糖的培养平板上，37℃培养过夜；挑选单个菌落接种在5mL含有岸边青霉素的LB培养液中，37℃摇床培养过夜。
接种1mL的过夜培养菌种至330mLTB培养液中，37℃摇床培养，培养到OD600nm值达到0.6-0.9时，加入1MIPTG，28℃摇床培养过夜；离心，收集大肠杆菌，利用渗透涨破法，获得抗体粗提液；
通过镍柱亲和层析法纯化出抗体，纯化后的单域抗体，如图4所示，包括VHH1～20。图4中VHH4、9、15、17、20分别对应单域抗体1B2、1C3、1C11、1E4、2B9，其余单域抗体的序列未示出(为技术效果不够好或者无需在本发明申请中予以保护的单域抗体)。
实施例6：SARS-COV-2-N蛋白的特异性单域抗体的结合量效曲线测定
(1)包被50μL 1μg/mL SARS-COV-2-N，4℃过夜。
(2)洗板；加入200μL5％牛奶，37℃封闭1h。
(3)将VHH稀释至2μg/mL，然后5倍梯度稀释抗体共8个浓度梯度。这里的VHH指实施例5制得的1B2、1C3、1C11、1E4、2B9纳米抗体，以及其余未示出序列的纳米抗体。
(4)洗板；加入50μL经步骤(3)稀释的纳米抗体，两复孔，37℃孵育1h。
(5)洗板；加入50μL鼠抗HA标签HRP二抗，37℃孵育30min。
(6)洗板(多洗几次)；加入50μL预先恢复常温的TMB，避光常温反应15min。
(7)加入50μL终止液(1N HCl)，酶标仪读数保存。
(8)绘制曲线，计算EC50，如图5和表4所示，可见本发明的5种SARS-COV-2-N纳米抗体对新冠病毒N蛋白的结合效力和特异性优异。
表4各纳米抗体EC50值
| 1A10 | 1A6 | 1B2 | 1B5 | 1B7 | 1C1 | |
| EC50 | 55.34 | 6.694 | 16.06 | 5.041 | 33.11 |
| 1D7 | 1E4 | 1F4 | 1F7 | 1H4 | 2A2 | |
| EC50 | ～0.3650 | 2.818 | ～85.06 | ～1.507e-005 | ～0.002526 | 0.6320 |
| 2B9 | 2C12 | 2C3 | 2D2 | 2E2 | 2F11 | |
| EC50 | 1.885 | 10.18 | 18.62 | ～0.03121 | 64.46 |
| 1C11 | 1C3 | 1C4 | 1C7 | 1D2 | 1D5 | |
| EC50 | 2.957 | 2.456 | ～30.65 | 17.68 | ～28.46 | ～6.625e+015 |
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节。
序列表
<110> 南京融捷康生物科技有限公司
<120> 一种SARS-Cov-2-N纳米抗体及其衍生蛋白和应用
<130> GY-03-2021-28
<141> 2021-12-13
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Thr Ala Thr Tyr Tyr Cys
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<213> 人工序列(Artificial Sequence)
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Trp Asp Gln Gly Thr Gln Val Thr Val Ser Ser
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<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 27
<211> 8
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<213> 人工序列(Artificial Sequence)
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Gly Phe Ser Phe Asp Thr Ser Tyr
1 5
<210> 28
<211> 8
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<213> 人工序列(Artificial Sequence)
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Gly Gly Thr Phe Ser Arg Asn Cys
1 5
<210> 29
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gly Tyr Ile Phe Arg His Tyr Tyr
1 5
<210> 30
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Gly Tyr Ile Thr Arg His Tyr Tyr
1 5
<210> 31
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gly Tyr Thr Tyr Thr Ser Gly Cys
1 5
<210> 32
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ile Cys Asn Thr Asp Gly Thr Thr
1 5
<210> 33
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Ile Cys Ser Ser Asp Gly Thr Thr
1 5
<210> 34
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Ile Gly Gly Asp Gly Ser Thr
1 5
<210> 35
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Ile Arg Thr Asp Gly Ser Thr
1 5
<210> 36
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ala Asp Gly Ile Ser Arg Cys Thr Val Val Arg Gly Val Leu Arg
1 5 10 15
Arg His Gly Tyr
20
<210> 37
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ala Asp Leu Asn Arg Arg Trp Gly Gly Pro Tyr
1 5 10
<210> 38
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ala Gly Arg Thr Pro Tyr Glu Leu Ala Ser Gly Gly Lys Asn
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<210> 39
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ala Ser Thr Ile Pro Gly Ala Tyr Asp Thr Pro Trp Leu Ser Arg
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Arg Gln Tyr Asn Phe
20
<210> 40
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ala Ser Thr Val Pro Gly Ala Tyr Gly Thr Trp Trp Leu Ser Arg
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Arg Gln Tyr Asn Tyr
20
Claims (9)
1.一种SARS-Cov-2-N纳米抗体，其特征在于：所述的单域抗体由重链构成，重链包括重链CDR1、重链CDR2和重链CDR3；
所述的重链CDR1、重链CDR2和重链CDR3的氨基酸序列为下述（1）-（5）的一种：
（1）SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:38所示的CDR3；
（2）SEQ ID NO:27所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:36所示的CDR3；
（3）SEQ ID NO:28所示的CDR1，SEQ ID NO:33所示的CDR2，SEQ ID NO:37所示的CDR3；
（4）SEQ ID NO:30所示的CDR1，SEQ ID NO:34所示的CDR2，SEQ ID NO:39所示的CDR3；
（5）SEQ ID NO:29所示的CDR1，SEQ ID NO:34所示的CDR2，SEQ ID NO:40所示的CDR3。
2. 根据权利要求1所述的SARS-Cov-2-N纳米抗体，其特征在于：所述针对SARS-Cov-2-N纳米抗体与选自SEQ ID NO：1-5的氨基酸序列具有至少95％序列同源性，并且能够特异性结合SARS-Cov-2病毒的N蛋白。
3.根据权利要求1所述的SARS-Cov-2-N纳米抗体，其特征在于：所述的单域抗体的框架区FR的序列为下述（a）-（e）中的一种；
（a）SEQ ID NO:12所示的FR1，SEQ ID NO:18所示的FR2，SEQ ID NO:21所示的FR3，SEQID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
（b）SEQ ID NO:15所示的FR1，SEQ ID NO:19所示的FR2，SEQ ID NO:24所示的FR3，SEQID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
（c）SEQ ID NO:13所示的FR1，SEQ ID NO:17所示的FR2，SEQ ID NO:20所示的FR3，SEQID NO:25所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
（d）SEQ ID NO:11所示的FR1，SEQ ID NO:16所示的FR2，SEQ ID NO:23所示的FR3，SEQID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换；
（e）SEQ ID NO:14所示的FR1，SEQ ID NO:16所示的FR2，SEQ ID NO:22所示的FR3，SEQID NO:26所示的FR4，或其变体，所述变体在所述FR中包含至多3个氨基酸的替换。
4. 一种SARS-Cov-2-N纳米抗体，其特征在于：所述单域抗体分别如SEQ ID NO.1-5所示，或者所述单域抗体与SEQ ID NO.1-5的氨基酸序列具有至少95％序列同源性。
5. 根据权利要求4所述的一种SARS-Cov-2-N纳米抗体，其特征在于：所述单域抗体的编码序列分别如SEQ ID NO.6-10所示，或者与SEQ ID NO.6-10具有至少95％序列同源性。
6. 一种编码权利要求1-5任意一项所述的SARS-Cov-2-N纳米抗体的核苷酸分子，其特征在于：其核苷酸序列分别如SEQ ID NO：6-10所示，或者与SEQ ID NO.6-10具有至少95％序列同源性。
7.一种表达载体，其特征在于，其包含编码权利要求1-5任意一项所述的纳米抗体的核苷酸分子或者权利要求6所述的核苷酸分子。
8.一种宿主细胞，其特征在于，其可以表达出权利要求1-5任意一项所述的SARS-Cov-2-N纳米抗体，或其包含权利要求7所述的表达载体。
9. 权利要求1-5任意一项所述的SARS-Cov-2-N纳米抗体的用途，其特征在于，用于制备试剂、检测板或试剂盒； 其中，所述试剂、检测板或试剂盒用于：检测样品中新冠病毒SARS-Cov-2的存在和/或含量。
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| CN111875700A (zh) * | 2020-07-28 | 2020-11-03 | 武汉华美生物工程有限公司 | 抗sars-cov-2病毒n蛋白的单链抗体及其用途 |
| CN112079920A (zh) * | 2020-09-18 | 2020-12-15 | 北京华大蛋白质研发中心有限公司 | 一种用于检测SARS-CoV-2病毒N蛋白的单克隆抗体及其应用 |
| CN112225797A (zh) * | 2020-09-24 | 2021-01-15 | 杭州医学院 | 一种抗SARS-CoV-2核衣壳蛋白的单克隆抗体及应用 |
| CN112940110A (zh) * | 2021-04-14 | 2021-06-11 | 中山大学 | 抗新型冠状病毒n蛋白单克隆抗体及其应用 |
| CN113249337A (zh) * | 2021-07-15 | 2021-08-13 | 天津一瑞生物科技股份有限公司 | 鼠抗新型冠状病毒n蛋白杂交瘤细胞株，单克隆抗体及应用 |
| CN113603786A (zh) * | 2021-08-26 | 2021-11-05 | 深圳市亚辉龙生物科技股份有限公司 | 特异性结合SARS-CoV-2 S蛋白和N蛋白的双特异性抗体 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111875700A (zh) * | 2020-07-28 | 2020-11-03 | 武汉华美生物工程有限公司 | 抗sars-cov-2病毒n蛋白的单链抗体及其用途 |
| CN112079920A (zh) * | 2020-09-18 | 2020-12-15 | 北京华大蛋白质研发中心有限公司 | 一种用于检测SARS-CoV-2病毒N蛋白的单克隆抗体及其应用 |
| CN112225797A (zh) * | 2020-09-24 | 2021-01-15 | 杭州医学院 | 一种抗SARS-CoV-2核衣壳蛋白的单克隆抗体及应用 |
| CN112940110A (zh) * | 2021-04-14 | 2021-06-11 | 中山大学 | 抗新型冠状病毒n蛋白单克隆抗体及其应用 |
| CN113249337A (zh) * | 2021-07-15 | 2021-08-13 | 天津一瑞生物科技股份有限公司 | 鼠抗新型冠状病毒n蛋白杂交瘤细胞株，单克隆抗体及应用 |
| CN113603786A (zh) * | 2021-08-26 | 2021-11-05 | 深圳市亚辉龙生物科技股份有限公司 | 特异性结合SARS-CoV-2 S蛋白和N蛋白的双特异性抗体 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114773459A (zh) * | 2022-06-16 | 2022-07-22 | 深圳大学 | 抗新型冠状病毒及其变异株的纳米抗体及其制备方法与应用 |
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