CN114702575B - 抗SARS-CoV-2 S蛋白的纳米抗体、重组纳米抗体、重组载体、重组菌及应用 - Google Patents
抗SARS-CoV-2 S蛋白的纳米抗体、重组纳米抗体、重组载体、重组菌及应用 Download PDFInfo
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- CN114702575B CN114702575B CN202210077156.4A CN202210077156A CN114702575B CN 114702575 B CN114702575 B CN 114702575B CN 202210077156 A CN202210077156 A CN 202210077156A CN 114702575 B CN114702575 B CN 114702575B
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Abstract
本发明提供了抗SARS‑CoV‑2 S蛋白的纳米抗体、重组纳米抗体、重组载体、重组菌及应用，涉及生物医药技术领域。本发明所述纳米抗体包括C4和C9，C4和C9均为驼源，可特异性识别SARS‑CoV‑2 S蛋白。本发明将所述C4和C9分别与人IgG Fc段进行融合，可获得重组C4和重组C9两种重组纳米抗体，并且经验证，所述重组C4和重组C9均能够特异性的识别SARS‑CoV‑2 S蛋白，都能够应用于新冠病毒的诊断试剂或抗病毒药物的制备。
Description
技术领域
本发明属于生物医药技术领域，具体涉及抗SARS-CoV-2 S蛋白的纳米 抗体、重组纳米抗体、重组载体、重组菌及应用。
背景技术
2019新型冠状病毒(SARS-CoV-2)是2019年在人体中发现的冠状病毒 新毒株。SARS-CoV-2病毒通过其刺突蛋白即S蛋白与人血管紧张素转换酶 -2(ACE2)结合，从而入侵宿主细胞。SARS-CoV-2病毒感染后，肺泡巨噬 细胞和上皮细胞会释放大量促炎细胞因子和趋化因子；单核细胞和中性粒细 胞会被募集到感染部位并清除含有病毒颗粒和感染细胞的渗出液，导致炎症 反应失控。在这个过程中，由于显著的淋巴细胞数量减少和功能失调，适应 性免疫难以有效启动。失控的病毒感染会导致严重的巨噬细胞浸润，进一步 加重肺损伤。同时，播散的SARS-CoV-2病毒也可直接攻击其它器官，免疫 反应可导致***性的炎症风暴，同时还有微循环障碍，这些因素一起作用最 终引发病毒性脓毒症。
纳米抗体是由比利时科学家于1993年在Nature中首次报道，在羊驼外 周血液中存在的一种天然缺失轻链的抗体(VHH)，1995年又在护士鲨、斑 纹须鲨、银鲛等软骨鱼中发现，是目前已知的可结合目标抗原的最小单位。 VHH分子量只有15KD，因此也被称作纳米抗体(Nanobody，Nb)。纳米抗 体具有独特优势，包括分子量小、可溶性好、稳定性强、亲和力高、免疫原 性低、体内组织渗透性好，极易穿过血管或组织到达靶部位等，具有很大发 展空间，纳米抗体的应用也非常广泛，临床上既可用于肿瘤治疗，也可作为 诊断工具。
因此，寻求更为有效的抗体用于SARS-CoV-2病毒感染后的治疗显得尤 为迫切。
发明内容
有鉴于此，本发明的目的在于提供抗SARS-CoV-2 S蛋白的纳米抗体、 重组纳米抗体、重组载体、重组菌及应用，所述纳米抗体和重组纳米抗体均 能够特异性识别SARS-CoV-2 S蛋白。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一组抗SARS-CoV-2 S蛋白的纳米抗体，所述纳米抗体包 括C4和C9，所述C4的氨基酸序列如SEQ ID NO.1所示，所述C9的氨基 酸序列如SEQ ID NO.2所示。
本发明还提供了一组抗SARS-CoV-2 S蛋白的重组纳米抗体，所述重组 纳米抗体包括上述纳米抗体的任一种和人IgG Fc段。
优选的，所述重组纳米抗体包括重组C4和重组C9，所述重组C4的氨 基酸序列如SEQ ID NO.5所示，所述重组C9的氨基酸序列如SEQ ID NO.6 所示。
本发明还提供了表达上述重组纳米抗体的重组载体，所述重组载体包括 编码所述重组纳米抗体的核苷酸序列和基础载体。
优选的，所述编码重组C4的核苷酸序列如SEQ ID NO.8所示，编码重 组C9的核苷酸序列如SEQ ID NO.9所示。
优选的，所述基础载体包括pET-30a。
本发明还提供了上述重组载体的构建方法，包括以下步骤：将编码所述 重组纳米抗体的的核苷酸序列***所述基础载体的NdeI和XhoI位点之间， 得所述重组载体。
本发明还提供了表达上述重组纳米抗体的重组菌，所述重组菌的基础菌 包括大肠杆菌。
优选的，所述大肠杆菌的菌株包括Arctic Express。
本发明还提供了上述纳米抗体、上述重组纳米抗体、上述重组载体或上 述重组菌在制备抗病毒药物或病毒感染诊断试剂中的应用。
有益效果：本发明提供了一组抗SARS-CoV-2 S蛋白的纳米抗体，所述 纳米抗体包括C4和C9，C4和C9均为驼源，可特异性识别SARS-CoV-2 S 蛋白。本发明将所述C4和C9分别与人IgG Fc段进行融合，可获得重组C4 和重组C9两种重组纳米抗体，并且经验证，所述重组C4和重组C9均能够 特异性的识别SARS-CoV-2 S蛋白，都能够应用于新冠病毒的诊断试剂或抗 病毒药物的制备。
附图说明
图1为纳米抗体的第一轮DNA电泳图，从左到右凝胶孔的DNA条带 分别是：第六道为1000bp的Marker(条带大小依次为：1000、700、500、 400、300、200、100bp)，第一、二、三及五道为PCR产物，条带约为700bp， 第四道为空；
图2为纳米抗体的第二轮DNA电泳图，从左到右凝胶孔的DNA条带 分别是：第一道为1000bp的Marker(条带大小同上)，第二、四及六道为 PCR产物，条带约为400bp，第三及五道为空；
图3为用噬菌体的酶联免疫方法(phage-ELISA)筛选特异性单个阳性 克隆的模式图：其中1是将SARS-CoV-2 S蛋白包被在酶标板上，2是噬菌 体上清，3是鼠抗km13107抗体，4是山羊抗鼠IgG(AP)的抗体，5是TMB 显色液；
图4为纯化的重组纳米抗体，其中，左图为重组C4纳米抗体破碎后处 理样品、流出样品和洗脱样品的SDS-PAGE电泳染色图；右图为重组C9纳 米抗体破碎后处理样品、流出样品和洗脱样品的SDS-PAGE电泳染色图；
图5为纯化的重组纳米抗体的westernblot图，其中，左图为重组C4纳 米抗体，右图为重组C9纳米抗体(M：蛋白质分子质量标准1：纯化后抗 SARS-CoV-2 S蛋白纳米抗体)；
图6为SARS-CoV-2 S蛋白的western blot图，其中，左图一抗为重组 C4纳米抗体，右图一抗为重组C9纳米抗体。
具体实施方式
本发明提供了一组抗SARS-CoV-2 S蛋白的纳米抗体，所述纳米抗体包 括C4和C9，所述C4的氨基酸序列如SEQ ID NO.1所示，所述C9的氨基 酸序列如SEQ ID NO.2所示。
本发明所述C4的氨基酸序列如SEQ ID NO.1所示，且所述C4包括4 个框架区FR(FR1、FR2、FR3和FR4)和3个抗原决定簇互补区CDR(CDR1、 CDR2和CDR3)。本发明所述C4的框架区FR的氨基酸序列优选如下：
FR1:QVQLQESGGGSVQAGGSIVLSCAAF(SEQ ID NO.10)；
FR2:MAWWRQGSDKVREQVAN(SEQ ID NO.11)；
FR3:SYSRSVEGRFTISRDNAKNTLTLQMNELKPEDTDMYYC(SEQ ID NO.12)；
FR4:WGPGTQVTVSS(SEQ ID NO.13)；
对应的核苷酸序列优选如下：
FR1:CAGGTCCAACTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGC TGGAGGGTCTATAGTACTCTCCTGCGCGGCCTTT(SEQ ID NO.14)；
FR2:ATGGCCTGGTGGCGCCAGGGTTCAGACAAGGTGCGCGAACA GGTCGCAAAT(SEQ IDNO.15)；
FR3:AGCTACTCACGCTCCGTAGAGGGCCGATTCACCATCTCCCGA GACAACGCCAAGAACACTCTGACTCTCCAAATGAACGAATTGAAACCT GAGGACACTGACATGTACTACTGT(SEQ ID NO.16)；
FR4:TGGGGCCCGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO.17)。
本发明所述C4的抗原决定簇互补区CDR的氨基酸序列，优选如下：
CDR1:GYSGTPLC(SEQ ID NO.18)；
CDR2:IDSDGTT(SEQ ID NO.19)；
CDR3:AAVEGSAGEIYCSGGY(SEQ ID NO.20)；
对应的核苷酸序列，优选如下：
CDR1:GGATACAGCGGCACGCCCTTGTGC(SEQ ID NO.21)；
CDR2:ATTGATAGTGATGGGACGACA(SEQ ID NO.22)；
CDR3:GCAGCTGTGGAGGGCTCCGCCGGCGAAATTTATTGCAGTGG TGGTTAC(SEQ ID NO.23)。
本发明中所述C9的氨基酸序列如SEQ ID No.2所示，其内优选包括4 个框架区FR(FR1、FR2、FR3和FR4)和3个抗原决定簇互补区CDR(CDR1、CDR2和CDR3)。
本发明所述C9的框架区FR的氨基酸序列，优选如下：
FR1:QVQLQESGGGSVGLGGSMRLTCTIS(SEQ ID NO.24)；
FR2:MGWYRQAPGKWREGVAD(SEQ ID NO.25)；
FR3:GYIDSVRGRFIISRDNDKNILYLQMNSLKPEDTAMYYC(SEQ ID NO.26)；
FR4:WGQGTQVTVSS(SEQ ID NO.27)；
对应的核苷酸序列，优选如下：
FR1:CAGGTCCAACTGCAGGAGTCTGGGGGAGGCTCGGTGGGGCT TGGAGGGTCTATGAGACTCACCTGCACAATCTCC(SEQ ID NO.28)；
FR2:ATGGGTTGGTACCGCCAGGCTCCAGGGAAATGGCGCGAGGG GGTCGCAGAC(SEQ IDNO.29)；
FR3:GGCTACATAGACTCCGTGAGGGGCCGATTCATCATCTCCCGGG ACAACGACAAGAACATTCTGTATCTCCAAATGAACAGCCTAAAACCTG AGGACACCGCCATGTACTACTGT(SEQ ID NO.30)；
FR4:TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO.31)。
本发明所述C9的抗原决定簇互补区CDR的氨基酸序列，优选如下：
CDR1:AVIDDYC(SEQ ID NO.32)；
CDR2:IAESGTP(SEQ ID NO.33)；
CDR3:AARLRGSCSTDPHNFGY(SEQ ID NO.34)；
对应的核苷酸序列，优选如下：
CDR1:GCCGTAATCGACGACTACTGT(SEQ ID NO.35)；
CDR2:ATTGCCGAATCTGGCACCCCA(SEQ ID NO.36)；
CDR3:GCGGCTAGATTACGTGGCTCGTGTAGCACGGACCCCCACAA CTTTGGTTAC(SEQ IDNO.37)。
本发明对所述C4和C9的获取方法并没有特殊限定，实施例中优选通 过用噬菌体表面展示技术构建天然驼源纳米抗体噬菌体展示文库，然后以生 物素化的SARS-CoV-2 S蛋白为基础进行筛选，获得SARS-CoV-2 S蛋白特 异性的纳米抗体基因序列。在本发明中，所述天然驼源纳米抗体噬菌体展示 文库的构建方法，优选包括以下步骤：1)提取骆驼脾脏组织总RNA、反转 录所述总RNA获得cDNA；2)以所述cDNA为模板进行巢式PCR扩增获 得重链抗体的可变区片段；3)分别酶切所述重链抗体的可变区片段和 pcantab5e噬菌体载体后，进行连接获得连接产物；4)将所述连接产物转入 感受态细胞中获得天然驼源纳米抗体噬菌体展示文库。本发明对步骤1)所 述总RNA的提取方法和反转录的方法并没有特殊限定，利用本领域的常规 方法进行提取和反转录即可。本发明在得到所述cDNA后，以所述cDNA为模板进行巢式PCR扩增获得重链抗体的可变区片段，所述巢式PCR优选包 括两轮PCR；第一轮PCR用于扩增重链抗体引导肽和抗体CH2之间的片段， 所述第一轮PCR的引物序列优选的如SEQ ID NO.38和SEQ ID NO.39所示； 第二轮PCR用于扩增重链抗体FR1区和长、短铰链区之间的片段，所述第 二轮PCR的引物序列优选的如SEQ ID NO.40和SEQ ID NO.41所示。本发 明在获得所述重链抗体的可变区片段后，分别酶切所述重链抗体的可变区片 段和pcantab5e噬菌体载体后，进行连接获得连接产物。本发明在获得所述 连接产物后，将所述连接产物转入感受态细胞中获得天然驼源纳米抗体噬菌 体展示文库。本发明所述感受态细胞优选为大肠杆菌感受态细胞TG1；所述 转入的方法优选为电转化。本发明在所述转化后，优选还包括辅助噬菌体救 援，本发明对所述电转化和辅助噬菌体援助的具体方法没有特殊限定，采用 本领域的常规方法即可。
本发明还提供了编码上述C4和C9的基因序列，其中编码C4的基因的 核苷酸序列优选如SEQ ID NO.3所示；编码C9的基因的核苷酸序列优选如 SEQ ID NO.4所示。
本发明还提供了一组抗SARS-CoV-2 S蛋白的重组纳米抗体，所述重组 纳米抗体包括上述纳米抗体的任一种和人IgG Fc段。
本发明将C4与人IgG Fc段连接，得到重组C4纳米抗体(简称重组C4)； 将C9与人IgG Fc段连接，得到重组C9纳米抗体(简称重组C9)，且本发 明所述连接优选为利用连接片段进行，所述连接片段的编码基因的核苷酸序 列优选如SEQ ID NO.7所示：GGTGGTGGTGGTAGT。本发明所述人IgG Fc 段的编码基因核苷酸序列优选如SEQ ID NO.42所示。
本发明所述重组C4的氨基酸序列优选如SEQ ID NO.5所示；编码所述 重组C4的基因的核苷酸序列优选如SEQ ID NO.8所示。
本发明所述重组C9的氨基酸序列优选如SEQ ID NO.6所示；编码所述 重组C9的基因的核苷酸序列优选如SEQ ID NO.9所示。
本发明还提供了表达上述重组纳米抗体的重组载体，所述重组载体包括 编码所述重组纳米抗体的核苷酸序列和基础载体。
本发明所述基础载体优选包括pET-30a，并且将上述SEQ ID NO.8所述 的基因或SEQ ID NO.9所示的基因均***所述pET-30a的NdeI和XhoI位点 之间。
本发明还提供了上述重组载体的构建方法，包括以下步骤：将编码所述 重组纳米抗体的的核苷酸序列***所述基础载体的NdeI和XhoI位点之间， 得所述重组载体。
本发明对所述***的方法并没有特殊限定，优选利用双酶切的方法。
本发明还提供了表达上述重组纳米抗体的重组菌，所述重组菌的基础菌 包括大肠杆菌。
本发明所述大肠杆菌的菌株优选包括Arctic Express。本发明优选将所述 重组载体转入所述大肠杆菌Arctic Express中获得所述重组菌。本发明对所 述转入的方法并没有特殊限定，采用本领域常规的转入方法即可。
本发明还提供了上述纳米抗体、上述重组纳米抗体、上述重组载体或上 述重组菌在制备抗病毒药物或病毒感染诊断试剂中的应用。
本发明利用所述重组菌可制备所述重组纳米抗体，且所述纳米抗体和重 组纳米抗体都可特异性识别SARS-CoV-2 S蛋白，可用于制备抗病毒药物或 病毒感染诊断试剂。
下面结合实施例对本发明提供的抗SARS-CoV-2 S蛋白的纳米抗体、重 组纳米抗体、重组载体、重组菌及应用进行详细的说明，但是不能把它们理 解为对本发明保护范围的限定。
实施例1
针对于天然驼源纳米抗体基因库的构建
(1)Trizol法提取骆驼脾脏组织总RNA，利用TIANGEN公司提供的 RNA纯化试剂盒纯化，按照Thermo Scientific ReverAid First Strand cDNA Synthesis Kits试剂盒反转录得到cDNA。
(2)以cDNA为模板，用巢式PCR扩增得到重链抗体的可变区片段；
第一轮PCR：
上游引物：5’-GTCCTGGCTGCTCTTCTACAAAG-3’(SEQ ID No.38)
下游引物：5’-GGTACGTGCTGTTGAACTGTTCC-3’(SEQ ID No.39)
20μL反应体系：cDNA2μL、Mix 10μL、上游引物1μL、下游引物1μL 和余量ddH2O；
PCR扩增反应：95℃5min；95℃30s，55℃30s，72℃45s，32个循环； 72℃10min。
扩增结果如图1所示，纳米抗体基因电泳条带约为700bp。
以第一轮PCR产物为模板，进行第二轮PCR：
上游引物：5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGT CTGGGG-3'(SEQ IDNO.40)；
下游引物：5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCT GGGTCCCC-3'(SEQ IDNO.41)；
50μL反应体系：700bp产物2μL、Mix 25μL、上游引物1μL、下游引物 1μL和余量ddH2O；
PCR扩增反应：94℃5min；94℃40s，55℃40s，72℃40s，25个循环； 72℃10min。
扩增结果如图2所示，纳米抗体基因电泳条带约为400bp。
(3)使用限制性内切酶(购自Takara公司)SifI和Not I酶切pcantab5e 噬菌体载体及VHH片段(C4的VHH片段SEQ ID NO.1；C9的VHH片段 SEQ ID NO.2)，并用T4 DNA连接酶(购自Takara公司)连接两个片段。
将连接产物电转化至电转感受态细胞TG1中，构建天然驼源纳米抗体 噬菌体展示文库，经辅助噬菌体救援后，其库容量达9.0×1013。
其中辅助噬菌体救援步骤如下：
①取100μL的文库，接种于50mL的2×YT/Amp/Glu培养基中，37℃， 200rpm，震荡培养至对数期OD600约为0.4～0.5。
②向培养液中加入感染复数为20:1的辅助噬菌体M13KO7，混匀后37℃ 静置30min。
③将培养液在室温，9000rpm，离心10min，弃上清沉淀菌体，用200mL 的2×YT/Amp/Kana培养液重悬，37℃200rpm，培养过夜。
④将培养液于4℃，9000rpm，离心10min取上清，加入1/5体积的 PEG/NaCl，4℃静置6h。
⑤9000rpm离心20min弃上清，用PBS(1mL)重悬沉淀，得到重组噬 菌体抗体库，分装到1.5ml Ep管中，4℃保存。
与此同时，通过菌落PCR检测文库的***率，引物使用第二轮PCR引 物，退火温度55℃，结果显示***率达到95％以上(目的片段***率＝含目 的片段的菌落数/所有菌落数)。
(4)针对抗SARS-CoV-2 S蛋白纳米抗体的筛选过程：
将噬菌体文库(1×1012个噬菌体)与50μL链霉亲和素磁珠在转动台上 室温孵育1h后，收集噬菌体抗体；在2个已用2％PBSM封闭过的1ml离心 管中，分别加入500μL预先削减的噬菌体抗体，再向一个离心管中加入500μL 用PBS稀释的5μg生物素化的SARS-CoV-2 S蛋白，另一个离心管中加入 500μL的PBS缓冲液作为阴性对照，在转动台上室温孵育1h，后加入预先 封闭的50μL的链霉亲和素磁珠，在转动台上室温孵育30min，收集磁珠。 用PBST洗涤磁珠7次，PBSM洗涤2次，PBS洗涤1次。加入pH2.7的Glycine 进行洗脱，pH9.1的1mol/LTris-HCl进行中和。将上述中和液加入到5mL 处于对数生长期的TG1(OD600为0.5)中，产生并纯化噬菌体用于下一轮的 筛选，经过4轮筛选，阳性克隆将不断得到富集，从而达到了利用噬菌体展 示技术筛选抗体库中SARS-CoV-2 S蛋白特异性抗体的目的。
噬菌体的酶联免疫方法(phage-ELISA)筛选特异性单个阳性克隆：
筛选原理模式图如图3所示，具体方法如下：
首先制备VHH噬菌体单克隆上清：从3～4轮筛选后的固体平板上，随 机挑取90个单菌落并接种于含有100μg/mL的氨苄青霉素及2％的葡萄糖的 2×YT培养基中，220rpm 37℃培养过夜，次日取50μL菌液至新的96深孔板 中，每孔加入800μL含有100μg/mL的氨苄青霉素及2％的葡萄糖的2×YT 培养基，生长至对数期后，加入感染复数为20:1的辅助噬菌体M13K07，37℃ 侵染30min，10000rpm离心5min，弃上清，用800μL含有100μg/mL的氨 苄青霉素及50μg/mL的卡那青霉素的新鲜2×YT培养基重悬菌体，37℃， 220rpm培养12h，次日，将菌液10000rpm，离心5min，其上清即为VHH 噬菌体单克隆上清。
用包被液将SARS-CoV-2 S蛋白稀释至10μg/mL，每孔加入100μL，4℃ 包被过夜，并设立阴性和阳性对照。次日，用PBST洗涤三次，用2％PBSM 于37℃封闭2h，用PBST洗涤三次，加入200μL预处理的VHH噬菌体单克 隆上清，37℃孵育1h。加入1:5000的用0.1％的PBST稀释的鼠源性抗 M13KO7/HRP的二抗，37℃孵育1h，洗去未结合的抗体，加入TMB显色液， 于酶标仪上在450nm波长处读取吸光度值。当样品孔OD值大于对照孔OD 两倍以上时判定为阳性对照孔，取阳性菌液进行基因测序。
使用DNAMAN软件进行序列分析及blast比对，把CDR1、CDR2和 CDR3序列相同的菌株视为同一克隆株。最终采用氨基酸序列SEQ ID NO.1、 SEQ ID NO.2所示的纳米抗体序列做后续实验。
实施例2
重组纳米抗体在宿主菌大肠杆菌中的表达及纯化
(1)将实施例1中测序分析所获得的纳米抗体序列连接人源性IgG Fc 段并亚克隆pET-30a质粒载体中，并转化至大肠杆菌Arctic Express，挑取转 化平板上的单克隆接种于含50μg/mLKan的3mL LB培养液的试管中，37℃ 220rpm振摇过夜；(2)次日按1:100接种于50μg/mLKan的30mL LB培养 液中，37℃220rpm振摇至菌体OD600为0.6～0.8，加入IPTG至终浓度为 0.5mM，20℃220rpm振摇过夜，诱导融合蛋白表达；(3)收集菌体并进行 超声破碎，得到包涵体蛋白粗体液，后经Ni柱亲和纯化得到融合蛋白。
图4是纯化的重组纳米抗体，其中左图为重组C4纯化过程中的SDS-PAGE电泳染色图：其中泳道M为蛋白分子标准，泳道1-2分别是破碎 后处理样品、流出样品，泳道3-4是洗脱样品；右图为重组C9纯化过程中 的SDS-PAGE电泳染色图：其中泳道M为蛋白分子标准，泳道1-2分别是 破碎后处理样品、流出样品，泳道3-4是洗脱样品。图5是纯化的重组纳米 抗体的westernblot图，其中左图为重组C4，右图为重组C9。
实施例3
重组纳米抗体的特异性验证：
用SARS-CoV-2 S蛋白做westernblot(其中一抗用纯化的重组纳米抗体， 二抗为抗人IgG Fc/HRP)。
westernblot结果如图6所示，左图一抗为重组C4，右图一抗为重组C9)： 其中泳道M是蛋白分子标准，泳道1、2均为SARS-CoV-2 S蛋白。由此说 明，说明本发明提供的重组纳米抗体可以特异性识别SARS-CoV-2 S蛋白。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普 通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润 饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东医科大学
<120> 抗SARS-CoV-2 S蛋白的纳米抗体、重组纳米抗体、重组载体、重组菌及应用
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
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Ser Ile Val Leu Ser Cys Ala Ala Phe Gly Tyr Ser Gly Thr Pro Leu
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Cys Met Ala Trp Trp Arg Gln Gly Ser Asp Lys Val Arg Glu Gln Val
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Ala Asn Ile Asp Ser Asp Gly Thr Thr Ser Tyr Ser Arg Ser Val Glu
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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Thr Leu
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Gln Met Asn Glu Leu Lys Pro Glu Asp Thr Asp Met Tyr Tyr Cys Ala
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Ala Val Glu Gly Ser Ala Gly Glu Ile Tyr Cys Ser Gly Gly Tyr Trp
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Gly Pro Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 2
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gly Leu Gly Gly
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Ser Met Arg Leu Thr Cys Thr Ile Ser Ala Val Ile Asp Asp Tyr Cys
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Trp Arg Glu Gly Val Ala
35 40 45
Asp Ile Ala Glu Ser Gly Thr Pro Gly Tyr Ile Asp Ser Val Arg Gly
50 55 60
Arg Phe Ile Ile Ser Arg Asp Asn Asp Lys Asn Ile Leu Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala
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Arg Leu Arg Gly Ser Cys Ser Thr Asp Pro His Asn Phe Gly Tyr Trp
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Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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<210> 3
<211> 366
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tatagtactc 60
tcctgcgcgg cctttggata cagcggcacg cccttgtgca tggcctggtg gcgccagggt 120
tcagacaagg tgcgcgaaca ggtcgcaaat attgatagtg atgggacgac aagctactca 180
cgctccgtag agggccgatt caccatctcc cgagacaacg ccaagaacac tctgactctc 240
caaatgaacg aattgaaacc tgaggacact gacatgtact actgtgcagc tgtggagggc 300
tccgccggcg aaatttattg cagtggtggt tactggggcc cggggaccca ggtcaccgtc 360
tcctca 366
<210> 4
<211> 366
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
caggtccaac tgcaggagtc tgggggaggc tcggtggggc ttggagggtc tatgagactc 60
acctgcacaa tctccgccgt aatcgacgac tactgtatgg gttggtaccg ccaggctcca 120
gggaaatggc gcgagggggt cgcagacatt gccgaatctg gcaccccagg ctacatagac 180
tccgtgaggg gccgattcat catctcccgg gacaacgaca agaacattct gtatctccaa 240
atgaacagcc taaaacctga ggacaccgcc atgtactact gtgcggctag attacgtggc 300
tcgtgtagca cggaccccca caactttggt tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 5
<211> 359
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Ile Val Leu Ser Cys Ala Ala Phe Gly Tyr Ser Gly Thr Pro Leu
20 25 30
Cys Met Ala Trp Trp Arg Gln Gly Ser Asp Lys Val Arg Glu Gln Val
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Ala Asn Ile Asp Ser Asp Gly Thr Thr Ser Tyr Ser Arg Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Thr Leu
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Gln Met Asn Glu Leu Lys Pro Glu Asp Thr Asp Met Tyr Tyr Cys Ala
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Ala Val Glu Gly Ser Ala Gly Glu Ile Tyr Cys Ser Gly Gly Tyr Trp
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Gly Pro Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu
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Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
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Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
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Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 6
<211> 359
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gly Leu Gly Gly
1 5 10 15
Ser Met Arg Leu Thr Cys Thr Ile Ser Ala Val Ile Asp Asp Tyr Cys
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Trp Arg Glu Gly Val Ala
35 40 45
Asp Ile Ala Glu Ser Gly Thr Pro Gly Tyr Ile Asp Ser Val Arg Gly
50 55 60
Arg Phe Ile Ile Ser Arg Asp Asn Asp Lys Asn Ile Leu Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala
85 90 95
Arg Leu Arg Gly Ser Cys Ser Thr Asp Pro His Asn Phe Gly Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu
115 120 125
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
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Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
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Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 7
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggtggtggtg gtagt 15
<210> 8
<211> 1077
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tatagtactc 60
tcctgcgcgg cctttggata cagcggcacg cccttgtgca tggcctggtg gcgccagggt 120
tcagacaagg tgcgcgaaca ggtcgcaaat attgatagtg atgggacgac aagctactca 180
cgctccgtag agggccgatt caccatctcc cgagacaacg ccaagaacac tctgactctc 240
caaatgaacg aattgaaacc tgaggacact gacatgtact actgtgcagc tgtggagggc 300
tccgccggcg aaatttattg cagtggtggt tactggggcc cggggaccca ggtcaccgtc 360
tcctcaggtg gtggtggtag tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 420
tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 480
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 540
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 600
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 660
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 720
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 780
tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 840
gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 900
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctatagc 960
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1020
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtcccc gggtaaa 1077
<210> 9
<211> 1077
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
caggtccaac tgcaggagtc tgggggaggc tcggtggggc ttggagggtc tatgagactc 60
acctgcacaa tctccgccgt aatcgacgac tactgtatgg gttggtaccg ccaggctcca 120
gggaaatggc gcgagggggt cgcagacatt gccgaatctg gcaccccagg ctacatagac 180
tccgtgaggg gccgattcat catctcccgg gacaacgaca agaacattct gtatctccaa 240
atgaacagcc taaaacctga ggacaccgcc atgtactact gtgcggctag attacgtggc 300
tcgtgtagca cggaccccca caactttggt tactggggcc aggggaccca ggtcaccgtc 360
tcctcaggtg gtggtggtag tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 420
tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 480
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 540
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 600
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 660
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 720
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 780
tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 840
gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 900
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctatagc 960
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1020
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtcccc gggtaaa 1077
<210> 10
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Ile Val Leu Ser Cys Ala Ala Phe
20 25
<210> 11
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ala Trp Trp Arg Gln Gly Ser Asp Lys Val Arg Glu Gln Val Ala
1 5 10 15
Asn
<210> 12
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ser Tyr Ser Arg Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Thr Leu Gln Met Asn Glu Leu Lys Pro Glu Asp
20 25 30
Thr Asp Met Tyr Tyr Cys
35
<210> 13
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 14
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tatagtactc 60
tcctgcgcgg ccttt 75
<210> 15
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggcctggt ggcgccaggg ttcagacaag gtgcgcgaac aggtcgcaaa t 51
<210> 16
<211> 114
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agctactcac gctccgtaga gggccgattc accatctccc gagacaacgc caagaacact 60
ctgactctcc aaatgaacga attgaaacct gaggacactg acatgtacta ctgt 114
<210> 17
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tggggcccgg ggacccaggt caccgtctcc tca 33
<210> 18
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Gly Tyr Ser Gly Thr Pro Leu Cys
1 5
<210> 19
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Ile Asp Ser Asp Gly Thr Thr
1 5
<210> 20
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Ala Ala Val Glu Gly Ser Ala Gly Glu Ile Tyr Cys Ser Gly Gly Tyr
1 5 10 15
<210> 21
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggatacagcg gcacgccctt gtgc 24
<210> 22
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
attgatagtg atgggacgac a 21
<210> 23
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gcagctgtgg agggctccgc cggcgaaatt tattgcagtg gtggttac 48
<210> 24
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gly Leu Gly Gly
1 5 10 15
Ser Met Arg Leu Thr Cys Thr Ile Ser
20 25
<210> 25
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Trp Arg Glu Gly Val Ala
1 5 10 15
Asp
<210> 26
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gly Tyr Ile Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn
1 5 10 15
Asp Lys Asn Ile Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 27
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 28
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
caggtccaac tgcaggagtc tgggggaggc tcggtggggc ttggagggtc tatgagactc 60
acctgcacaa tctcc 75
<210> 29
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atgggttggt accgccaggc tccagggaaa tggcgcgagg gggtcgcaga c 51
<210> 30
<211> 114
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ggctacatag actccgtgag gggccgattc atcatctccc gggacaacga caagaacatt 60
ctgtatctcc aaatgaacag cctaaaacct gaggacaccg ccatgtacta ctgt 114
<210> 31
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
tggggccagg ggacccaggt caccgtctcc tca 33
<210> 32
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Ala Val Ile Asp Asp Tyr Cys
1 5
<210> 33
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Ile Ala Glu Ser Gly Thr Pro
1 5
<210> 34
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Ala Ala Arg Leu Arg Gly Ser Cys Ser Thr Asp Pro His Asn Phe Gly
1 5 10 15
Tyr
<210> 35
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gccgtaatcg acgactactg t 21
<210> 36
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
attgccgaat ctggcacccc a 21
<210> 37
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gcggctagat tacgtggctc gtgtagcacg gacccccaca actttggtta c 51
<210> 38
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gtcctggctg ctcttctaca aag 23
<210> 39
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ggtacgtgct gttgaactgt tcc 23
<210> 40
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
tcgcggccca gccggcccag gtccaactgc aggagtctgg gg 42
<210> 41
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ataagaatgc ggccgctgag gagacggtga cctgggtccc c 41
<210> 42
<211> 696
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60
gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgggg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaaa 696
Claims (10)
1.一组抗SARS-CoV-2S蛋白的纳米抗体，所述纳米抗体包括C4和C9，所述C4的氨基酸序列如SEQ ID NO.1所示，所述C9的氨基酸序列如SEQ ID NO.2所示。
2.一组抗SARS-CoV-2S蛋白的重组纳米抗体，所述重组纳米抗体包括权利要求1所述纳米抗体的任一种和人IgG Fc段。
3.根据权利要求2所述重组纳米抗体，其特征在于，所述重组纳米抗体包括重组C4和重组C9，所述重组C4的氨基酸序列如SEQ ID NO.5所示，所述重组C9的氨基酸序列如SEQ IDNO.6所示。
4.表达权利要求2或3所述重组纳米抗体的重组载体，其特征在于，所述重组载体包括编码所述重组纳米抗体的核苷酸序列和基础载体。
5.根据权利要求4所述重组载体，其特征在于，所述编码重组C4的核苷酸序列如SEQ IDNO.8所示，编码重组C9的核苷酸序列如SEQ ID NO.9所示。
6.根据权利要求4所述重组载体，其特征在于，所述基础载体包括pET-30a。
7.权利要求4～6任一项所述重组载体的构建方法，其特征在于，包括以下步骤：将编码所述重组纳米抗体的的核苷酸序列***所述基础载体的NdeI和XhoI位点之间，得所述重组载体。
8.表达权利要求2或3所述重组纳米抗体的重组菌，所述重组菌的基础菌包括大肠杆菌。
9.根据权利要求8所述重组菌，其特征在于，所述大肠杆菌的菌株包括ArcticExpress。
10.权利要求1所述纳米抗体、权利要求2或3所述重组纳米抗体、权利要求4～6任一项所述重组载体或权利要求8或9所述重组菌在制备抗病毒药物或病毒感染诊断试剂中的应用。
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