CN114716542B - 一种针对新冠病毒刺突蛋白的单克隆抗体及应用 - Google Patents
一种针对新冠病毒刺突蛋白的单克隆抗体及应用 Download PDFInfo
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Abstract
本发明公开了一种针对新冠病毒S蛋白的单链抗体，此抗体是通过噬菌体展示库技术筛选获得，获得的单链抗体，经ELISA鉴定，可以与新冠病毒SARS‑CoV‑2的S蛋白特异性结合，具有较高的病毒抑制率。本发的方法是利用免疫后小鼠的脾脏细胞构建了噬菌体单链抗体展示库，该方法扩充了研究与诊断的资源，另外，本发明筛选到的具有与新冠病毒S蛋白高结合力的鼠源单链抗体，还可以用于病毒的检测与诊断等方面的应用。
Description
技术领域
本申请涉及生物医学领域，特别是涉及一种针对新型冠状病毒的单克隆抗体。
背景技术
新型冠状病毒囊膜上的刺突蛋白(spike protein,又称S protein或S蛋白)是侵染细胞过程中最重要的蛋白。刺突蛋白包括S1和S2两个亚基。S1中的受体结合区(receptorbinding domain,RBD)与血管紧张素转化酶II(ACE2)分子互作，决定病毒的宿主范围和特异性；S2含有膜融合过程所需要的基本元件，实现病毒与细胞的融合。开发针对刺突蛋白的中和抗体是目前对抗新型冠状病毒的一种有效手段。单链抗体(single-chain variablefragment,scFv)是由抗体重链的可变区与轻链的可变区在一段肽链的连接下构成。由于其分子量小，穿透力强，半衰期短等特点，在疾病的临床诊断、治疗、预防等方面具有重要作用和广阔的应用前景。
中国专利CN 112940111A利用纳米抗体文库，以2019新型冠状病毒的Spike S1+S2ECD为靶点，经四轮淘筛获得一种针对新型冠状病毒的纳米抗体。经ELISA检测，该抗体对2019新型冠状病毒的Spike S1+S2 ECD靶点和Spike RBD靶点均具有较高的亲和力。中国专利CN 113402603A以新型冠状病毒S1为靶点，利用噬菌体展示筛选抗体库的技术，通过四轮固相淘筛，公开了一种针对新冠病毒S1蛋白的禽源单克隆单链抗体，该单克隆抗体与新型冠状病毒刺突蛋白S1亚基特异性结合，具有一定识别病毒的能力。中国专利CN 113264998A公告了一种针对新型冠状病毒S1蛋白的人源单克隆抗体。该专利用COVID-19康复期患者外周血中分离的B细胞构建了噬菌体单链抗体展示库。该库以新冠病毒S1蛋白进行包板筛选，并对三轮淘筛后得到的单克隆抗体进行鉴定。经流式细胞术检测与S1蛋白有较好的亲和性，并且对SARS-CoV-2假病毒有较好的中和效果。
目前虽然筛选出了许多针对新型冠状病毒的抗体，但面对病毒的不断突变及实际产品开发中遇到的诸多问题，我们需要更广阔的抗体文库资源和亲和能力更高的抗体来应对挑战。
发明内容
针对现有技术的问题，本申请提供了一种抗新型冠状病毒S蛋白的单克隆抗体及其制备方法和应用。
本申请的第一个方面是提供一种抗新型冠状病毒S蛋白的单克隆抗体，所述抗体能与新型冠状病毒刺突蛋白特异性结合，本公开将抗体命名为S-pro-ab-750，S-pro-ab-753的单克隆抗体。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析上面抗体序列的CDR区的氨基酸序列。本领域普通技术人员可以理解，抗体的CDR区负责抗体对抗原的结合特异性。在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定哪些残基包含特定CDR，而不依赖于该序列自身之外的任何实验数据。以下列举IMGT这种CDR编号系统定义的CDR的合适的氨基酸残基作为比较。
所述抗体S-pro-ab-750的重链可变区(VH)为：
重链互补决定区1(H-CDR1)：GYSFTTYW(SEQ ID NO:2)
重链互补决定区2(H-CDR2)：IDPSDSVI(SEQ ID NO:4)
重链互补决定区3(H-CDR3)：ARLDSTGPYTWFLY(SEQ ID NO：6)
所述抗体S-pro-ab-750的轻链可变区(VL)为：
轻链互补决定区1(L-CDR1)：QSVDYNGISY(SEQ ID NO:9)
轻链互补决定区2(L-CDR2)：TAS(SEQ ID NO:11)
轻链互补决定区3(L-CDR3)：QQNIEDPLT(SEQ ID NO:13)
所述抗体S-pro-ab-753的重链可变区(VH)为：
重链互补决定区1(H-CDR1)：GFNIKDTY(SEQ ID NO:16)
重链互补决定区2(H-CDR2)：IDPANGNT(SEQ ID NO:18)
重链互补决定区3(H-CDR3)：ASPRALLLRYYAMDY(SEQ ID NO:20)
所述抗体S-pro-ab-753的轻链可变区(VL)为：
轻链互补决定区1(L-CDR1)：QSVDYDGDSY(SEQ ID NO:23)
轻链互补决定区2(L-CDR2)：AAS(SEQ ID NO:25)
轻链互补决定区3(L-CDR3)：QQSNEDPFT(SEQ ID NO:27)
S-pro-ab-750的重链可变区的框架区H-FR与轻链可变区的框架区L-FR如下：H-FR1的序列如SEQ NO:1所示，H-FR2的序列如SEQ NO:3所示，H-FR3的序列如SEQ NO:5所示，H-FR4的序列如SEQ NO:7所示，L-FR1的序列如SEQ NO:8所示，H-FR2的序列如SEQ NO:10所示，L-FR3的序列如SEQ NO:12所示，L-FR4的序列如SEQ NO:14所示。
H-FR1：QVQLQQSGPQLVRPGASVKISCKTS(SEQ ID NO:1)
H-FR2：MHWVKQRPGQGLEWIGM(SEQ ID NO:3)
H-FR3：RLNQKFKDKATLTVNKSSSTAYMQLSSPTSEDSAVYYC(SEQ ID NO:5)
H-FR4：WGQGTLVTVSS(SEQ ID NO:7)
L-FR1：DIVLTQSPASLAVSLGQRATIFCRAS(SEQ ID NO:8)
L-FR2：IHWFQQKPGQPPKLLIF(SEQ ID NO:10)
L-FR3：NLESGVPARFSGSGSESDFTLTIDPVEADDAATYYC(SEQ ID NO:12)
L-FR4：FGAGTKLELK(SEQ ID NO:14)
S-pro-ab-753的重链可变区的框架区H-FR与轻链可变区的框架区L-FR如下：H-FR1的序列如SEQ NO:15所示，H-FR2的序列如SEQ NO:17所示，H-FR3的序列如SEQ NO:19所示，H-FR4的序列如SEQ NO:21所示，L-FR1的序列如SEQ NO:22所示，H-FR2的序列如SEQ NO:24所示，L-FR3的序列如SEQ NO:26所示，L-FR4的序列如SEQ NO:28所示。
H-FR1：QVQLQQSGAELVKPGASVKLSCTAS(SEQ ID NO:15)
H-FR2：IHWVKQRPEQGLEWIGR(SEQ ID NO:17)
H-FR3：KYDPNFQGKATITADTSSNTAYLHLSSLTSEDTAVYYC(SEQ ID NO:19)
H-FR4：WGQGTSVTVSS(SEQ ID NO:21)
L-FR1：DIVLTQSPASLAVSLGQRATISCKAS(SEQ ID NO:22)
L-FR2：MNWYQQKPGQPPKLLIY(SEQ ID NO:24)
L-FR3：NLESGIPARFSGSGSGTDFTLTVNPVEADDVATYYC(SEQ ID NO:26)
L-FR4：FGSGTKLEIK(SEQ ID NO:28)
在一些实施例中，所述抗体包含：(1)重链可变区的高可变区CDR。HCDR包含HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO.2或16所示的氨基酸序列；HCDR2包含SEQ IDNO.4或18所示的氨基酸序列；HCDR3包含SEQ ID NO.6或20所示的氨基酸序列。(2)轻链可变区的高可变区CDR。LCDR包含LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO.9或23所示的氨基酸序列；LCDR2包含SEQ ID NO.11或25所示的氨基酸序列；LCDR3包含SEQ ID NO.13或27所示的氨基酸序列。
在一些实施例中，所述抗体还包含重链可变区的框架区FR与轻链可变区的框架区FR。重链可变区的框架区FR包含HFR1、HFR2、HFR3和HFR4，其中HFR1包含SEQ ID NO.1或15所示的氨基酸序列；HFR2包含SEQ ID NO.3或17所示的氨基酸序列；HFR3包含SEQ ID NO.5或19所示的氨基酸序列；HFR4包含SEQ ID NO.7或21所示的氨基酸序列。轻链可变区的框架区FR包含LFR1、LFR2、LFR3和LFR4，其中LFR1包含SEQ ID NO.8或22所示的氨基酸序列；LFR2包含SEQ ID NO.10或24所示的氨基酸序列；LFR3包含SEQ ID NO.12或26所示的氨基酸序列；LFR4包含SEQ ID NO.14或28所示的氨基酸序列。
在一些实施例中，所述抗体包括:(1)重链可变区，其包含下述序列或由下述序列组成：SEQ ID NO:29或31所示的氨基酸序列，和(2)轻链可变区，其包含下述序列或由下述序列组成：SEQ ID NO:30或32所示的氨基酸序列。
本申请的第二个方面是提供一种分离的核酸分子，所述核酸分子编码上述任一单克隆抗体。
本申请的第三个方面是提供一种包括上述的核酸分子的表达载体。
本申请中的表达载体是指可将编码鼠源scFv的多聚核苷酸插入其中并使scFv得到表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质在宿主细胞内得以表达。载体的种类包括本领域熟知的细菌、噬菌体、酵母、植物细胞病毒、哺乳动物细胞病毒(如腺病毒、逆转录病毒)或其他载体。在表达载体中，除了含有复制起点外，还应含有标记基因和其他翻译调控元件。
本申请的第四个方面涉及一种宿主细胞，此宿主细胞包含上述的核酸分子或上述的表达载体。
表达单克隆抗体的宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO，COS，293细胞或Bowes黑素瘤细胞的动物细胞等。
本申请的第五个方面是提供一种筛选抗新型冠状病毒单克隆抗体的方法，该方法包括如下步骤：
a)抗原免疫小鼠。
b)设计引物，获取VH与VL，连接得到scFv基因。
c)获得重组噬菌粒，将单链抗体基因片段与噬菌粒表达载体连接。
d)构建噬菌体抗体细菌文库。
e)扩增噬菌体抗体文库。
f)筛选获得抗新型冠状病毒单克隆抗体。
步骤a)中所述小鼠免疫抗原为S三聚体蛋白，相比使用S单体蛋白免疫小鼠能引起较强的免疫反应，诱导产生较高滴度的中和抗体。
步骤a)中所述小鼠免疫可采用佐剂配合，采用的佐剂可以是本领域已知或常规使用的佐剂，例如：铝盐，弗氏佐剂等。
步骤a)中所述小鼠免疫中S蛋白与佐剂组分比为1～5：1。
步骤a)中所述小鼠免疫首次免疫为10-100ug，加强免疫一般为首次免疫剂量的20％-50％。
步骤b)设计的引物包含重链可变区5’端引物和重链可变区3’端引物，κ链可变区5’端引物和3’端引物。
步骤b)设计的引物引入了酶切位点，包括但不限于SfiI和NotⅠ。
本申请的第六个方面是提供一种制备抗新型冠状病毒单克隆抗体的方法，其特征在于该方法包括如下步骤：
a)获得表达载体。将上述抗新型冠状病毒scFv基因克隆至表达载体上，构建重组表达载体。
本申请中的表达载体是指可将编码鼠源scFv的多聚核苷酸插入其中并使scFv得到表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。载体的种类包括本领域熟知的细菌质料、噬菌体、酵母质料、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。原则上，只要能在宿主体内复制和稳定，任何载体都可以用。在表达载体中，除了含有复制起点外，还可含有标记基因和其他翻译调控元件。
b)获得转化细胞。将步骤a)所述重组真核表达载体转化宿主细胞。
表达scFv抗体的宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞：真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO，COS，293细胞或Bowes黑素瘤细胞的动物细胞等。
c)培养诱导重组菌株表达。
d)获得抗新型冠状病毒单克隆抗体。提取c)中质粒，使用质粒转染细胞。培养后收集上清，获得抗体。
本申请的第七个方面是提供了一种纯化抗体蛋白的方法：
a)使用镍柱纯化抗体上清。
b)使用脱盐柱置换洗脱液。
本申请的第八个方面是提供了如上述的基于新型冠状病毒S蛋白的抗体或表达载体在制备检测和/或治疗新型冠状病毒感染的试剂中的应用。
与现有技术相比，本申请采用上述技术方案，具有如下技术效果：
本申请利用新型冠状病毒刺突蛋白免疫小鼠建立鼠源噬菌体抗体库，使用刺突蛋白进行包板淘筛，ELISA验证获得的潜在抗体后，将得到的阳性抗体与新型冠状病毒假病毒共孵育，鉴定其中和活性。筛选出的全新的抗体能够特异性识别和靶向新型冠状病毒S蛋白，原倍浓度抗体对假病毒的抑制率最高可达84％，中和活性较好，适合用于新型冠状病毒的检测，治疗与预防产品的开发。
附图说明
图1示出了间接ELISA法检测小鼠血清的特异性抗体滴度。
图2示出了相关引物序列。
图3示出了PCR扩增VH和VL后琼脂糖凝胶电泳检测结果。
图4示出了VH和VL重叠延伸PCR产物琼脂糖凝胶电泳检测结果。
图5示出了pCANTAB-5E载体质粒图谱。
图6示出了PCR产物scFv和载体质粒pCANTAB-5E双酶切产物琼脂糖凝胶电泳。
图7示出了阳性克隆测序比对结果。
图8示出了ELISA法检测不同多克隆针对S蛋白亲和性的结果。
图9示出了ELISA法检测不同单克隆针对S蛋白亲和性的结果。
图10示出了WB检测上清中抗体表达结果。
图11示出了银染鉴定抗体上清纯化后效果。
图12示出了银染鉴定抗体上清脱盐后效果。
图13示出了抗体S-pro-ab-750抑制病毒曲线。
图14示出了抗体S-pro-ab-753抑制病毒曲线。
具体实施方式
具体实施方式：下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1针对SARS-COV-2刺突蛋白的噬菌体抗体文库构建
1.1小鼠免疫
新冠S蛋白抗原使用前加铝盐佐剂乳化，抗原与佐剂比例为3:1。第一次免疫取50μg乳化后的抗原在小鼠背部皮下进行3点注射。对照组用PBS加佐剂免疫小鼠。3周后进行第一次加强免疫，取25μg乳化后的抗原对小鼠进行背部皮下3点注射。3周后进行第二次加强免疫，取25μg乳化后的抗原对小鼠进行背部皮下3点注射。最后一次加强免疫10d后眼眶采血分离血清做ELISA检测抗体效价，2d后处死小鼠取脾脏分脾细胞。
1.2 ELISA检测血清抗体效价
将SARS-CoV-2-RBD蛋白稀释至0.2μg/mL包被酶标板，4℃过夜，加PBST(含0.05％Tween-20)洗板3次。加入封闭液室温封闭2h，PBST洗板3次。将小鼠血清梯度稀释，向酶标板中加入100μL血清室温孵育1.5h，设置对照组，PBST洗板3次。加入100μL羊抗鼠抗体(1:2000稀释)室温孵育1.5h，PBST洗板3次。加入显色液室温孵育10min，加入终止液终止显色反应，于酶标仪450nm处检测吸光度。通过间接ELISA法检测小鼠血清的特异性抗体滴度，将小鼠血清分别稀释500、1000、20000、40000、80000、160000、320000倍，设置用PBS加佐剂免疫的小鼠为阴性对照组，未免疫小鼠为空白对照组，检测吸光值，若吸光值大于PBS孔的2.1倍，即视为阳性。结果如附图1所示，第1组免疫后小鼠血清抗体效价最高，终抗体效价为3.2×105。
1.3鼠脾细胞的分离
脱颈处死小鼠，取出脾脏组织，置于RPMI-1640培养基中润洗一遍。将脾脏转移至70μm细胞滤网上，充分研磨脾脏。加入PBS多次冲洗滤网，直至溶液由红色变为白色。400×g离心5min，弃上清，加入PBS重悬细胞。400×g离心5min，弃上清，加入5mL红细胞裂解液重悬细胞，室温孵育5min。加入PBS终止反应，400×g离心5min，弃上清，加RPMI-1640培养基重悬细胞，用台盼蓝染色计数，取1×107个细胞。
1.4鼠脾细胞总RNA提取
400×g离心5min弃上清，取细胞沉淀。加入1mL RZ裂解液重悬细胞。加入氯仿，振荡15s，室温放置3min。12000rpm离心10min，样品分成三层，将无色水相转移至新管中。加入0.5倍体积无水乙醇，转移至吸附柱中，离心1min。加入去蛋白液离心1min。加入漂洗液，室温静置2min，离心1min，重复此步。将吸附柱放入收集管中，离心2min去除残留液体。将吸附柱开盖通风晾干2min，转入新的1.5mL EP管中，加入ddH2O洗脱，室温放置2min，离心1min，收集RNA样本立即做反转录实验。
1.5反转录合成cDNA
在PCR管中配制如下混合液：ddH2O 6μL；4×gDNA Wiper Mix 4μL；模板RNA4μL，置于PCR仪中42℃反应2min。加入4μL5×HiScript II于PCR仪中50℃反应15min然后85℃反应5s进行逆转录反应。将cDNA模板置于-20℃保存。
1.6 PCR扩增获取VH与VL
以上一步反转录获得的鼠源cDNA为模板，上下游引物配对见表1(详细信息见图2)，按表2和表3中反应体系和程序进行PCR扩增反应。PCR反应结束后，将产物进行1.5％琼脂糖凝胶电泳检测，VH和VL扩增结果如附图3所示，条带大小在300-350bp之间，符合理论值。使用诺唯赞DNA纯化试剂盒进行产物纯化。重叠延伸PCR结束后，胶回收拼接产物。用1.5％琼脂糖凝胶电泳进行检测，拼接结果如附图4所示，条带大小在750bp左右，符合理论值。用Qubit测量拼接产物浓度，产物浓度为67.6ng/μL，置于-20℃保存。
表1引物配对
表2 PCR扩增反应体系
表3 PCR扩增反应程序
1.7 scFv与pCANTAB-5E载体双酶切
pCANTAB-5E载体质粒从淼灵生物公司购买，具体图谱见附图5。拿到质粒后，转化大肠杆菌TG1，挑取单克隆菌落，接种在2×YT培养基中，37℃摇床摇菌14h。提取质粒，用Qubit测量其浓度，浓度为768ng/μL。产物于-20℃保存。
按表4配制双酶切反应体系，先加入SfiI内切酶于金属浴中50℃反应2h，再加入NotI-HF内切酶金属浴中37℃反应2h。酶切结束后，进行1％琼脂糖凝胶电泳鉴定酶切结果，切取目的条带，进行胶回收反应。1％琼脂糖凝胶电泳检测胶回收产物，结果如附图6，与理论值相符合。用Qubit测量浓度，载体浓度为10ng/μL，拼接产物浓度为30ng/μL。胶回收产物置于-20℃保存。
表4双酶切反应体系
1.8连接重组
按表5进行连接反应，混匀后将体系置于金属浴中25℃连接5h。重组产物于-20℃保存。
表5连接反应体系
1.9电转化建库
冰上解冻电转感受态菌液TG1。预冷0.1cm电击杯。加入2μL DNA重组产物，冰上孵育1min。将重组产物和菌液混合物转移至电击杯中，1.8kV脉冲4～5ms。立即加入1mL 37℃预热的SOC培养基。37℃摇床振荡培养1h。取10μL菌液10倍梯度稀释(10-1～10-6)，取100μL稀释后的菌液涂SOB平板，37℃过夜倒置培养。第2d数平板上菌落个数，计算库容，细菌文库容量约为1.2×107。将阳性克隆送去测序，测序结果进行序列比对，检测库多样性，结果如附图7所示，序列均不相同，且长度为750bp左右，含有scFv基因的完整序列，证明scFv基因已成功连接到pCANTAB-5E质粒载体中，说明噬菌体抗体库构建成功。剩余菌液涂SOB平板，第2d将菌落重悬在2×YT培养基中，加入终浓度为20％的甘油，分装于EP管中，置于-80℃冰箱保存，即为噬菌体抗体细菌文库。
实施例2噬菌体抗体文库富集、筛选与鉴定
2.1噬菌体抗体文库扩增与纯化
取1mL冻存菌液接种于2×YT培养基中，37℃摇床振荡培养至OD600值为0.5。加入辅助噬菌体M13K07(辅助噬菌体:菌＝20:1)，37℃摇床振荡培养1h。加入Kana(终浓度为50μg/mL)和IPTG(终浓度为0.2μM)，30℃摇床振荡培养过夜。8000×g 15min离心菌液，去除沉淀。加入1/4菌液体积的预冷的PEG/NaCl，冰上静置30min，12000×g离心10min，弃去上清。加入1mL PBS重悬沉淀，于-80℃保存。
取10μL纯化后噬菌体测滴度：XL1-Blue菌液加入2×YT培养基中，37℃摇床振荡培养至OD600为0.5。加入PBS 10倍梯度稀释噬菌体，10-1～10-8。取菌液加稀释后的噬菌体，37℃孵育30min，涂2×YT-Amp平板，37℃倒置培养过夜。第2d数菌落个数计算滴度，纯化后噬菌体抗体文库滴度为1×1010pfu/mL。
2.2淘筛
包板：第1轮淘筛每孔包被2μg重组SARS-CoV-2-S蛋白；第2轮包被500ng蛋白；第3轮包被200ng蛋白。用PBS溶解蛋白加入酶标板中4℃过夜，PBST洗板3次。加入封闭液室温封闭2h，PBST洗板3次。加入噬菌体抗体上清，室温孵育1h，加PBST洗板10次。加入胰蛋白酶(10μg/mL)洗脱噬菌体，37℃孵育30min，将洗脱液转移至5mL EP管中。
取TG1菌液加入2×YT培养基中，37℃摇床振荡培养至OD600为0.5。向洗脱的噬菌体中加入TG1菌液，37℃孵育30min，摇床37℃振荡培养30min。加入2×YT培养基和10×GA，摇床37℃振荡培养至OD600为0.5。加入辅助噬菌体，37℃孵育30min，摇床37℃振荡培养30min。3000×g离心5min，吸弃上清。加入2×YT-AK培养基，摇床30℃振荡培养过夜。3000×g离心5min，将上清转移至新EP管中进行下一轮淘筛，置于-80℃冰箱保存。
2.3 ELISA鉴定重组噬菌体抗体
将重组SARS-CoV-2-S蛋白加入PBS稀释至浓度为1.33μg/mL包被酶标板，4℃过夜。加入封闭液室温封闭2h。加入200μL噬菌体抗体上清溶液室温孵育1.5h，设置空白对照组，PBS、培养基为阴性对照组。加入Anti-M13抗体(1:200稀释)室温孵育1.5h。加入显色液37℃孵育20min，加入50μL终止液终止显色反应，于酶标仪450nm处检测吸光度。将阳性克隆株OD值用GraphPad Prism作图，结果如附图8所示，显示有22个不同的抗体多克隆株对SARS-CoV-2-S蛋白有一定的亲和性。
2.4挑取阳性单克隆株
挑取5个吸光值最高的阳性克隆株感染OD600为0.5的TG1菌液，37℃孵育30min，涂2×YT-Amp平板，37℃倒置培养过夜。从每个平板上各挑取8个单克隆，加入2×YT培养基，37℃摇床振荡培养至OD600为0.5。加入辅助噬菌体，37℃孵育30min，摇床37℃振荡培养30min。3000×g离心5min，吸弃上清。加入2×YT-AK培养基，摇床30℃振荡培养过夜。从每管中各取1mL过夜培养菌液于-80℃保存，剩余菌液3000×g离心5min，将上清转移至EP管中-80℃保存。
2.5 ELISA鉴定噬菌体抗体单克隆
将重组SARS-CoV-2-S蛋白加入PBS稀释至1.33μg/mL包被酶标板，4℃过夜。加入封闭液室温封闭2h。加入200μL噬菌体抗体上清溶液室温孵育1.5h，设置对照组。加入Anti-M13抗体(1:200稀释)室温孵育1.5h。加入显色液37℃孵育20min，加入50μL终止液终止显色反应，于酶标仪450nm处检测吸光度。将阳性单克隆株OD值用GraphPad Prism作图，结果如附图9所示，显示有13个不同的抗体单克隆株对SARS-CoV-2-S蛋白有一定的亲和性，挑取10个吸光值最高的单克隆菌液送去测序。
2.6抗体表达
将测序获得的两株抗体序列(一株抗体长度750bp，命名为S-pro-ab-750；另一株抗体长度753bp，命名为S-pro-ab-753)分别克隆至pcDNA3.1(+)-C-6His真核表达载体上。拿到质粒后，转化大肠杆菌DH5α，挑取单克隆菌落，接种在LB培养基中，37℃摇菌12h。提取质粒，用Qubit测量质粒浓度，pcDNA3.1-S-pro-ab-750质粒浓度为59ng/μL，pcDNA3.1-S-pro-ab-753质粒浓度为184ng/μL。置于-20℃保存。
将293T细胞以5×106个/mL细胞密度接种于T-25cm2培养瓶中。待细胞汇合度至85％，更换培养基。取两个EP管，A管中加入20μL P3000，10μg质粒和DME/F-12培养基(补齐至终体积为250μL)；B管中加入242μL DME/F-12培养基和7.5μLLipofactamine3000。将A、B管溶液混合，室温孵育10min，加入细胞培养瓶中，置于37℃恒温、5％CO2浓度的细胞培养箱中培养。转染48h、72h后分别收取培养上清，400×g离心5min去除细胞沉淀，将上清转移至EP管中，-80℃保存。
2.7 WB鉴定上清中抗体表达
将抗体上清液与5×SDS-PAGE蛋白上样缓冲液混匀，金属浴99℃煮沸10min变性蛋白。配制12％下层胶，点样蛋白Marker和样品。上层胶电压80V电泳15min，下层胶电压120V电泳50min。切除多余胶，裁剪滤纸和NC膜。将滤纸、NC膜和胶浸没于转膜缓冲液中，转至转膜仪中，电压15V转膜20min。加入封闭液(5％脱脂奶粉溶于PBST)，摇床上室温封闭2h。按1:10000稀释His-tag抗体，4℃过夜。将ECL显色液滴加于NC膜上，检测目的蛋白。结果如附图10所示，两株抗体在48h、72h均有表达，蛋白大小35KD左右，第1、2泳道抗体有两条带可能是二聚体结构。
2.8镍柱纯化抗体上清
加600μL平衡缓冲液平衡镍柱，2900rpm离心2min。分次加入600μL抗体上清，1600rpm离心5min，收集液体分装于EP管中。加入600μL洗涤液洗2次，2900rpm离心2min。将吸附柱转移至新1.5mL EP管中，加300μL洗脱液洗脱2次，2900rpm离心2min，收集洗脱液，于-80℃保存。
2.9银染鉴定抗体上清纯化效果
将抗体上清液与5×蛋白上样缓冲液混匀，金属浴99℃煮沸10min变性蛋白。配制12％下层胶。点样蛋白Marker和样品。上层胶80V电泳15min，下层胶120V电泳50min。将凝胶放入固定液中(50％乙醇、10％乙酸和40％纯水)，4℃过夜。加入30％乙醇，室温摇床上10min。加入纯水，室温摇床上10min。加入银染增敏液，室温摇床上2min。加入纯水洗2次，每次室温摇床上1min。加入银溶液，室温摇床上10min。加入纯水洗1次，室温摇床上1min。加入银染显色液，室温摇床上摇动3min至出现较理想的预期蛋白条带。加入银染终止液，室温摇床上5min。结果如附图11所示，两株抗体纯化后杂蛋白均减少，70KD左右蛋白为BSA，镍柱纯化无法去除。
2.10脱盐置换洗脱液
掰断脱盐柱尾部并松开盖子，将柱子置于50mL离心管中。1000×g离心2min去除储存液。向柱子中加入5mL PBS缓冲液，1000×g离心2min去除缓冲液，重复此步2次。将柱子置于一个新的50mL离心管中，去掉盖子并缓慢地将样品加到柱中心，1000×g离心2min收集样品，于-80℃保存。
2.11银染鉴定抗体上清脱盐效果
将抗体上清液与5×蛋白上样缓冲液混匀，金属浴99℃煮沸10min变性蛋白。配制12％下层胶。点样蛋白Marker和样品。上层胶80V电泳15min，下层胶120V电泳50min。将凝胶放入固定液中，4℃过夜。加入30％乙醇，室温摇床上10min。加入纯水，室温摇床上10min。加入银染增敏液，室温摇床上2min。加入纯水洗2次，每次室温摇床上1min。加入银溶液，室温摇床上10min。加入纯水洗1次，室温摇床上1min。加入银染显色液，室温摇床上摇动3min至出现较理想的预期蛋白条带。加入银染终止液，室温摇床上5min。结果如附图12所示，两株抗体置换洗脱液后杂蛋白也有一定程度的减少，但抗体浓度降低。
2.12抗体中和活性检测
将SARS-CoV-2-Fluc假病毒从-80℃转至冰上解冻，使用含10％FBS的DME培养基稀释至1×104TCID50/mL。将抗体进行3倍比梯度稀释，取96孔细胞培养板向每孔中加入90μL稀释后的抗体溶液，向每孔中再加入90μL稀释后的假病毒溶液，设置细胞对照组和病毒对照组。将96孔细胞培养板置于37℃培养箱中孵育1h。将细胞培养板中混合液转入96孔白板中，设置3个复孔，50μL/孔。将293T-ACE2细胞稀释至4×106个/mL，每孔50μL细胞悬液加入96孔白板中，置于37℃培养箱中培养。培养24h后，向每孔中加入25μL 37℃预热的含有10％FBS的DME培养基。继续培养48h，取出96孔白板，每孔加入5μL荧光素酶检测底物，混匀后用酶标仪检测化学发光值。
抗体抑制病毒曲线如图13和图14所示，原倍浓度S-pro-ab-750抗体病毒抑制率达84％，25μL抗体可以中和205TCID50新冠假病毒；原倍浓度S-pro-ab-753抗体病毒抑制率达55％，25μL抗体可中和135TCID50新冠假病毒。
2.13抗体氨基酸的检测
本实施例中对具体实施方式中获得的抗体S-pro-ab-750和S-pro-ab-753的氨基酸结构序列进行检测。
其中S-pro-ab-750的氨基酸序列为:SEQ ID NO:33。
S-pro-ab-753的氨基酸序列为：SEQ ID NO:34。
上述实施例中载体、基因和耗材均可从商业途径中得到。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
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<110> 武汉滨会生物科技股份有限公司
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<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 14
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
1 5 10
<210> 15
<211> 25
<212> PRT
<213> 人工序列(artificial sequence)
<400> 15
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser
20 25
<210> 16
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 16
Gly Phe Asn Ile Lys Asp Thr Tyr
1 5
<210> 17
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 17
Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly
1 5 10 15
Arg
<210> 18
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 18
Ile Asp Pro Ala Asn Gly Asn Thr
1 5
<210> 19
<211> 38
<212> PRT
<213> 人工序列(artificial sequence)
<400> 19
Lys Tyr Asp Pro Asn Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr
1 5 10 15
Ser Ser Asn Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 20
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<400> 20
Ala Ser Pro Arg Ala Leu Leu Leu Arg Tyr Tyr Ala Met Asp Tyr
1 5 10 15
<210> 21
<211> 11
<212> PRT
<213> 人工序列(artificial sequence)
<400> 21
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
1 5 10
<210> 22
<211> 26
<212> PRT
<213> 人工序列(artificial sequence)
<400> 22
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser
20 25
<210> 23
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 23
Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr
1 5 10
<210> 24
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 24
Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
1 5 10 15
Tyr
<210> 25
<211> 3
<212> PRT
<213> 人工序列(artificial sequence)
<400> 25
Ala Ala Ser
1
<210> 26
<211> 36
<212> PRT
<213> 人工序列(artificial sequence)
<400> 26
Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
1 5 10 15
Thr Asp Phe Thr Leu Thr Val Asn Pro Val Glu Ala Asp Asp Val Ala
20 25 30
Thr Tyr Tyr Cys
35
<210> 27
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 27
Gln Gln Ser Asn Glu Asp Pro Phe Thr
1 5
<210> 28
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 28
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
1 5 10
<210> 29
<211> 121
<212> PRT
<213> 人工序列(artificial sequence)
<400> 29
Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Thr Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Asp Pro Ser Asp Ser Val Ile Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asn Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Asp Ser Thr Gly Tyr Pro Thr Trp Phe Leu Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 30
<211> 111
<212> PRT
<213> 人工序列(artificial sequence)
<400> 30
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Phe Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn
20 25 30
Gly Ile Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Phe Thr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Glu Ser Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ile
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 31
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<400> 31
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Asn Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Pro Arg Ala Leu Leu Leu Arg Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 32
<211> 111
<212> PRT
<213> 人工序列(artificial sequence)
<400> 32
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 33
<211> 251
<212> PRT
<213> 人工序列(artificial sequence)
<400> 33
Met Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro
1 5 10 15
Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr
20 25 30
Thr Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
35 40 45
Trp Ile Gly Met Ile Asp Pro Ser Asp Ser Val Ile Arg Leu Asn Gln
50 55 60
Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asn Lys Ser Ser Ser Thr
65 70 75 80
Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Leu Asp Ser Thr Gly Tyr Pro Thr Trp Phe Leu Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr
130 135 140
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile
145 150 155 160
Phe Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn Gly Ile Ser Tyr Ile
165 170 175
His Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Phe
180 185 190
Thr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
195 200 205
Gly Ser Glu Ser Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp
210 215 220
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ile Glu Asp Pro Leu Thr
225 230 235 240
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
245 250
<210> 34
<211> 251
<212> PRT
<213> 人工序列(artificial sequence)
<400> 34
Met Ala Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro
1 5 10 15
Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys
20 25 30
Asp Thr Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu
35 40 45
Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro
50 55 60
Asn Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr
65 70 75 80
Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Ser Pro Arg Ala Leu Leu Leu Arg Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr
130 135 140
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile
145 150 155 160
Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met
165 170 175
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr
180 185 190
Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
195 200 205
Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Asn Pro Val Glu Ala Asp
210 215 220
Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Phe Thr
225 230 235 240
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
245 250
Claims (7)
1.抗体或抗原结合片段，特异性结合新型冠状病毒刺突蛋白，其中，所述抗体或者抗原结合片段包含：
(1)重链可变区的高可变区CDR；其包含HCDR1、HCDR2和HCDR3，其中HCDR1为SEQ IDNO.16所示的氨基酸序列；HCDR2为SEQ ID NO.18所示的氨基酸序列；HCDR3为SEQ ID NO.20所示的氨基酸序列；
(2)轻链可变区的高可变区CDR；其包含LCDR1、LCDR2和LCDR3，其中LCDR1为SEQ IDNO.23所示的氨基酸序列；LCDR2为SEQ ID NO.25所示的氨基酸序列；LCDR3为SEQ ID NO.27所示的氨基酸序列。
2.权利要求1所述的抗体或抗原结合片段，其中所述抗体或抗原结合片段包含：
(1)重链可变区的框架区FR；其包含HFR1、HFR2、HFR3和HFR4，其中HFR1为SEQ ID NO.15所示的氨基酸序列；HFR2为SEQ ID NO.17所示的氨基酸序列；HFR3为SEQ ID NO.19所示的氨基酸序列；HFR4为SEQ ID NO.21所示的氨基酸序列；
(2)轻链可变区的框架区FR；其包含LFR1、LFR2、LFR3和LFR4，其中LFR1为SEQ ID NO.22所示的氨基酸序列；LFR2为SEQ ID NO.24所示的氨基酸序列；LFR3为SEQ ID NO.26所示的氨基酸序列；LFR4为SEQ ID NO.28所示的氨基酸序列。
3.权利要求1或2任一所述的抗体或抗原结合片段，其包括:
(1)重链可变区；所述重链可变区的氨基酸序列如SEQ ID NO.31所示；及
(2)轻链可变区；所述轻链可变区的氨基酸序列如SEQ ID NO.32所示。
4.一种编码权利要求1或2中任一所述抗体或抗原结合片段的核酸。
5.一种载体，其包含权利要求4的核酸。
6.一种宿主细胞，其包含权利要求5的载体。
7.权利要求3所述的抗体或抗原结合片段或权利要求4所述的核酸或权利要求5所述的载体在制备检测和/或治疗新型冠状病毒感染的试剂中的应用。
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