CN115124615A - 抗新型冠状病毒rbd结构域抗原的纳米抗体 - Google Patents
抗新型冠状病毒rbd结构域抗原的纳米抗体 Download PDFInfo
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Abstract
本发明公开了一种氨基酸序列如SEQ ID NO.1～8所示的抗新型冠状病毒RBD结构域抗原的纳米抗体，属于生物制品领域。本发明的纳米抗体，与新型冠状病RBD的亲和力很强，在新型冠状病毒的检测试剂中有潜在应用；特别是其中的N5纳米抗体，具有良好的阻断新型冠状病毒RBD与其受体结合的病毒中和能力，可以有效阻止病毒感染细胞，有望成为治疗新型冠状病毒的有效药物。
Description
技术领域
本发明属于生物制品领域，具体涉及抗新型冠状病毒RBD结构域抗原的纳米抗体。
背景技术
截至目前，SARS-CoV2病毒引发的肺炎尚无特效药物，目前的研究已经证实，新型冠状病毒表面的刺突糖蛋白(Spike蛋白)在三维空间构象抗原性上与SARS抗原性非常接近；基于病毒的结构研究发现SARS-CoV2病毒上的受体结合域蛋白(receptor bindingdomain,RBD)能够特异性的识别人血管紧张素转化酶(ACE2)，这也是新冠病毒Spike蛋白介导细胞侵染的结构基础及分子机制。
基于该研究背景，研发能够高度特异性的识别该区域的治疗性抗体将有望阻止新型冠状病毒通过RBD与人ACE2蛋白进行结合，继而阻止病毒的侵入，因此，发展针对新型冠状病毒RBD结构域抗原的治疗性抗体，将有望为新型冠状病毒的诊断及治疗提供新的策略。
目前的抗体研发大多采用单克隆抗体技术，这类由哺乳动物(包括人)的抗体由两条重链与两条轻链组成，其中，重链的Fc区域可以确保抗体在机体内有较长的半衰期，保证较为充足的免疫保护。然而，传统的抗体药物具有一些缺点:(1)分子量较大，很难穿透组织、细胞以及血脑屏障等；(2)抗体在制备、保存和运输过程中容易发生聚集和沉淀；(3)容易发生某些抗抗体反应；(4)筛选到具有生物活性的高亲和力抗体较为困难。
而新型纳米抗体(Nanobody)能够很好的避开传统抗体的不足。纳米抗体相比传统抗体，只含有一个重链可变区，也可称为“VHH”抗体。相关研究指出，纳米抗体只有传统抗体的十分之一，但完全具备抗体的活性，它即能够有效的识别及靶向抗原，又具备很强的穿透能力，能够穿透组织和细胞到达病变部位；同时，纳米抗体具有较好的稳定性，能够耐受较强的温度和酸碱性等强刺激，具备较低的免疫源性和较高的抗原亲和力；加上它能够在体外进行化学及生物学的相关修饰，因此可以作为良好的抗体药物。就纳米抗体的结构而言，与传统抗体相比，它同样含有3个互补决定区(CDR，包括CDR1、CDR2和CDR3)，共同组成抗原结合部位，其中CDR3对抗原的亲和力起到了关键作用。但不同的是，其CD3的长度大于人抗体VH，这就使得CD3能够形成凸环状结构，因此能够深入抗原内部并更好的结合抗原，这也是其亲和力更高的原因。同时，VHH的FR2的疏水残基能够被亲水残基所取代，使得其水溶性更好，不易形成聚集体。同时，相关研究证实，在抗感染方面，纳米抗体能够通过阻断病毒-细胞结合、病毒入侵及包被过程，防止病毒的扩散。
目前尚无抗新型冠状病毒抗原(尤其是RBD结构域抗原)的纳米抗体面世。
发明内容
本发明要解决的问题是：提供一种抗新型冠状病毒RBD结构域抗原的纳米抗体。
本发明的技术方案如下：一种抗新型冠状病毒的纳米抗体，所述纳米抗体的氨基酸全长序列如SEQ ID NO.1～8任意一项所示。
本发明还提供了编码权利要求1所述新型冠状病毒抗体的核酸。
进一步地，上述核酸的序列如SEQ ID NO.9～16所示；优选地，编码氨基酸序列为SEQ ID NO.1的纳米抗体的核酸序列为SEQ ID NO.8；
编码氨基酸序列为SEQ ID NO.2的纳米抗体的核酸序列为SEQ ID NO.9；
编码氨基酸序列为SEQ ID NO.3的纳米抗体的核酸序列为SEQ ID NO.10；
编码氨基酸序列为SEQ ID NO.4的纳米抗体的核酸序列为SEQ ID NO.11；
编码氨基酸序列为SEQ ID NO.5的纳米抗体的核酸序列为SEQ ID NO.12；
编码氨基酸序列为SEQ ID NO.6的纳米抗体的核酸序列为SEQ ID NO.13；
编码氨基酸序列为SEQ ID NO.7的纳米抗体的核酸序列为SEQ ID NO.14。
编码氨基酸序列为SEQ ID NO.8的纳米抗体的核酸序列为SEQ ID NO.16。
本发明还提供了一种重组核酸载体，所述载体包括上述的核酸。
进一步地，上述核酸载体是真核表达载体。
本发明还提供了一种重组细胞，所述重组细胞内含有上述的载体。
本发明还提供了上述纳米抗体在制备抗新型冠状病毒的药物中的用途。
本发明还提供了一种抗新型冠状病毒的药物，所述药物以上述纳米抗体为活性成分。
本发明还提供了上述的纳米抗体在新型冠状病毒检测试剂中的用途。
本发明还提供了一种新型冠状病毒检测试剂，它含有上述的纳米抗体。
本发明的纳米抗体，与新型冠状病RBD的亲和力很强，在新型冠状病毒的检测试剂中有潜在应用；特别是其中的N5纳米抗体，具有良好的阻断新型冠状病毒RBD与其受体结合的病毒中和能力，可以有效阻止病毒感染细胞，有望成为治疗新型冠状病毒的有效药物。
本发明术语：“新型冠状病毒”指SARS-Cov-2。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1：SARS-CoV2病毒表面SPIKE蛋白的RBD结构域SDS-PAGE图。
图2：PCR扩增经RBD抗原免疫羊驼后产生的VHH目的基因琼脂糖凝胶图。
图3：抗体序列比对图。
图4：抗体R1的纯化图。
图5：抗体R2的纯化图。
图6：抗体R3的纯化图。
图7：抗体R5的纯化图。
图8：抗体R8的纯化图。
图9：抗体R10的纯化图。
图10：抗体R22的纯化图。
图11：通过Biocore测量本发明纳米抗体N1、N3、N5、N8、N10、N22 对不同突变体的亲和力和阻断能力的结果。
图12：纳米抗体浓度为1μg/ml，流式细胞仪表征纳米抗体的竞争力结果。
图13：纳米抗体浓度为2μg/ml，流式细胞仪表征纳米抗体的竞争力结果。
图14：纳米抗体浓度为4μg/ml，流式细胞仪表征纳米抗体的竞争力结果。
图15：纳米抗体N1、N2、N3、N5、N8、N10的假病毒中和实验结果。
具体实施方式
实施例1抗新型冠状病毒RBD结构域抗原的纳米抗体的制备
一、RBD抗原的制备
本实验针对小鼠使用的免疫抗原为SARS-CoV2病毒表面SPIKE蛋白的 RBD结构域，采用昆虫表达系统，由于其为分泌蛋白，离心后收集培养基上清，使用镍柱进行分离纯化。纯化后的蛋白质使用12.5％的SDS-PAGE胶进行定量分析(见图1)。然后将纯化得到的蛋白质分装冷冻干燥-80C保存。(避免反复冻融导致蛋白质降解)。
二、RBD抗原免疫羊驼后采集血清及抗血清效价检测
利用RBD作为抗原一共对羊驼进行4次免疫，将羊驼的颈部淋巴结处作为抗原的注射位置，在羊驼颈部左右两个淋巴结处分别注射一半样品，操作过程中应特别注意整个过程低温保存蛋白质和佐剂，避免其反复冻融继而导致的蛋白质的降解。用250ug RBD与250ul完全弗氏佐剂的乳化混合物对羊驼进行初次免疫，在第14天、28天、42天用RBD抗原与250ul不完全弗氏佐剂加强免疫3次。
第三次和第四次免疫一周后，采集血液检测抗血清滴度；第四次免疫一周后，采集100ml血液用于噬菌体抗体库构建。采集的血液分离出血清用于检测针对RBD蛋白的抗体效价，未免疫前的血清作对照。
具体步骤如下：
(1)包被抗原，每孔500ng重组RBD蛋白，用PBS缓冲液稀释，4℃过夜；
(2)每孔加入200μl浓度为2.5％脱脂奶粉，26℃封闭1h；
(3)用PBS洗涤3次，免疫前、后的羊驼血清先稀释到1000，再进行 2倍逐级稀释，吸取100μL加入到包被有RBD蛋白的孔中，26℃孵育1h；
(4)PBS洗涤3次后，加入1:3000稀释的兔抗羊驼的多抗100μl，26℃孵育1h；
(5)每孔加入HRP(辣根过氧化物酶)标记的羊抗兔多抗(1:5000) 100μL，26℃孵育1h；洗板3次，加100μl的TMB(3,3',5,5'-四甲基联苯胺)反应30min；
(6)每孔滴加50μl的3M硫酸终止，读取OD450nm。RBD重组蛋白作为免疫原免疫羊驼，第四次免疫后，收集羊驼外周血并分离血清，通过间接ELISA的方法检测血清中RBD重组蛋白特异性抗体的效价。
第四次免疫后，骆驼血清RBD重组蛋白特异性抗体的效价≥1:128000。可用于下一步噬菌体展示文库的构建。
三、VHH噬菌体库构建与淘选
收集经RBD抗原免疫后羊驼的外周血100ml，注意新鲜的血液样品应保 存在18℃-20℃之间，取血后2-6小时内应尽快完成淋巴细胞的分离，采用淋 巴细胞分离液(GEFicoll-Paque Plus)分离获得羊驼的PBMC，采用Trizol提 取total RNA，同时利用oligodT反转为cDNA,通过引物扩增，以及分子克 隆等技术，将羊驼的VHH基因克隆至真核表达载体上，转化TG1细菌，得到 VHH噬菌体库。为了进一步鉴定RBD-VHH噬菌体库是否构建成功，设计相 对应的引物，通过PCR扩增经过RBD抗原免疫羊驼后产生的VHH目的基 因，可以看出目的条带为5 0 0b p，大小与预期相符(图2展示了部分结 果)，可以进一步说明该RBD-VHH噬菌体抗体文库中含有VHH目的基因。 挑选33个菌落扩增后进行测序，并分析序列多样性，将DNA序列翻译成 氨基酸序列，随后进行序列比对以及预测功能分区情况，结果见图3。检测 的结果发现该构建得到一个RBD-VHH噬菌体抗体文库的库容为 (1.3X(9)cfu，阳性率90％，有效插入率(In frame rate)为100％。
随后进行文库救援，采用M13KO7噬菌体进行辅助救援，首先用 VHH-phen2ed转化的细菌，进行噬菌体抗体库的复苏，并用PEG/NaCl进行沉淀。将包被有4μg/ml的RBD-His蛋白进行4次富集噬菌体抗体库。将富集的噬菌体，洗脱、转化、涂板、挑取单克隆进行噬菌体与RBD蛋白ELISA 的结合鉴定，将结合读值与Blank对照比值大于5的单克隆进行基因测序，并克隆至表达载体pcDNA3.1，转染293T细胞表达生产RBD纳米单抗。
将淘选后的抗体文库与RBD蛋白进行结合检测。通过RBD蛋白淘选成功地富集了具有较高结合力的RBD-VHH噬菌体文库。通过筛选，我们发现了7个具有较高结合力的VHH抗体，并进行了基因测序，序列如下。
N1(SEQ ID NO.9)
CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAG ACTCTCCTGTACAGCCTCTGGAAGCGGTTTCAGTAACAATAACATGGCCTGGTACCG CCAGGCTCCAGGGAAGGCGCGCGAGTGGGTCGCAGCTGTTAGTAGTAGTGGTAACA CAAACTATGCGGCCTCCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG AACACGGTGTATCTGCAAATGAGCAGCCTGAAACCTGAAGACACAGCCGTCTATTA CTGTTATGCAGTGGCACCGGAGGGTGGTGACTCCTGGGGCCAGGGGACCCAGGTCA CCGTCTCCTCA
N2(SEQ ID NO.10)
CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGA GACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGAGACGTTGCCATGACCTGGTACC GCCAGGCTCCAGGGAAGCAGCGCGAGTTCGTCGCAGCTATTTCTACGAGTGGTAGT CCAAGCTACGCAAACTCCGTGAACGGCCGATTCACCATCGCCAGAGACGACGCCAA GAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATT ACTGTCACCTAGCTAGCTCTAGGGGGAATGACTACTGGGGCCAGGGGACCCAGGTC ACTGTCTCCTCA
N3(SEQ ID NO.11)
CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGA GACTCTCCTGTCGAGCCTCTGGAAGTGTCTTTACCATGGGCTGGCACCGCCAGGCTC CAGGGAACGGGCGCGAGTTCGTCGCGTCAATTACTCGTGATGGTAACGCAAATTATA AAGGCTCCGTGAAGGACCGATTCACCATCTCCAGAGACAACGGCAAAAACACCTTG TATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTATCGAC TACAACACCAGCACCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
N5(SEQ ID NO.12)
CAGGTGCAGCTCGTGGAGTCTGGGGGAGGACCGGTGCAGGCTGGGGGCTCTCTGA GACTCTCCTGTGCAGCCGCTGGAAGCGCCCTCAGTATCTATCGCATGGGCTGGTACC GCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCATTTATTACTACTGGTGGCAGC ACGGACCACATAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAA GAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATT ACTGTAATTCAGATCCGCCCTGGGTCCTAGGGGCCGACTGGGGCCAGGGGACCCAG GTCACTGTCTCCTCA
N8(SEQ ID NO.13)
CAGGTGCAGCTCGTGGAGTCTGGGGGCGGCTTGGTGCAGGCTGGGGGGTCTCTGAG ACTCTCCTGTCGAGCCTCTGGAAGTGTCTTTAACATGGGCTGGCACCGCCAGGCTC CAGGGAAGCAGCGCGGTTCGTCGCGACAATTAGTCAAGATGGACGCACAAATTAT AAAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAACACCT TGTATCTGCAGATGAACAGCCTGAAACCTGAGACACAGCCGTCTATTACTGTATCG ACTACCACACCAGCACCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
N10(SEQ ID NO.14)
CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGGAGGCTGGGGGGTCTCTGA GACTCTCCTGTGCAGCCTCTGGAATCATCTTCAGTATCAATAACATGGCCTGGTACCG CCAGGCTCCAGGGAAGCCGCGCGAGTGGGTCGCAGCTGTTAGTAGTAGTGGTAACA CAAACTATGCGGCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG AACACGTTGTATCTACAAATGAGCAGCCTGAAACCTGAAGACACAGCCGTCTATTAC TGCTATGCAGTGGCACCGGAGGGAGGTGACTCCTGGGGCCAGGGGACCCAGGTCA CCGTCTCCTCA
N22(SEQ ID NO.15)
CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGA GACTCTCCTGTCGAGCCTCTGGAACTGTCTTTAACATGGGCTGGCACCGCCAGGCT CCAGGGCAGCAGCGCGGTTCGTCGCGTCAATTAGTCATGATGGGCGCACAAATTAT GCAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACACCTT GTATCTGCAAATGAACAGCCTGAAACCTGAGACACAGCCGTCTATTACTGTATCGA CTACCACACCAGTCTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
N28(SEQ ID NO.16)
ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCCACAGCTACCGGCGTGCACAGC CAGGTGCAGCTGGTGGAAAGCGGCGGCGGTCTCGTGCAAGCTGGCGGCTCCCTGA GACTGAGCTGCGCCGCTAGCGGCGCCACCTTTCCATTCAACAGCATGGCCTGGTGG CGGCAGGCCCCTGGCAAGCAGAGAGAGTGGGTCGCCAGCATCACATCTGGCGGAA GCACCAATTACGCCGAGAGCGTGAAAGGCCGGTTCGCCATCAGCGGCGACAACGCC AAGGGCACAGTGTACGTGCAGATGAACTACCTGAAGCCCGAGGACACCGCCGTGTA TTACTGCTACTACGTGTCTGGAAGAGGCGGAGATTACTGGGGACAGGGCACCCAGG TGACCGTGTCCAGCCTCGAGCTGGTTCCTAGAGGCTCTCACCACCACCACCACCATC ACCACTGA
各纳米抗体的氨基酸序列如下：
N1(SEQ ID NO.1)：
QVQLVESGGGLVQPGGSLRLSCTASGSGFSNNNMAWYRQAPGKAREWV AAVSSSGNTNYAASAKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCYAV APEGGDSWGQGTQVTVSS
N2(SEQ ID NO.2)：
QVQLVESGGGLVQAGGSLRLSCAASGSIFRDVAMTWYRQAPGKQREFVA AISTSGSPSYANSVNGRFTIARDDAKNTVYLQMNSLKPEDTAVYYCHLAS SRGNDYWGQGTQVTVSS
N3(SEQ ID NO.3)：
QVQLVESGGGLVQAGGSLRLSCRASGSVFTMGWHRQAPGNGREFVASIT RDGNANYKGSVKDRFTISRDNGKNTLYLQMNSLKPEDTAVYYCIDYNTST WGQGTQVTVSS
N5(SEQ ID NO.4)：
QVQLVESGGGPVQAGGSLRLSCAAAGSALSIYRMGWYRQAPGKQRELVA FITTGGSTDHIDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNSDPP WVLGADWGQGTQVTVSS
N8(SEQ ID NO.5)：
QVQLVESGGGLVQAGGSLRLSCRASGSVFNMGWHRQAPGKQREFVATIS QDGRTNYKGSVKGRFTISRDNGKNTLYLQMNSLKPEDTAVYYCIDYHTST WGQGTQVTVSS
N10(SEQ ID NO.6)：
QVQLVESGGGLVEAGGSLRLSCAASGIIFSINNMAWYRQAPGKPREWVAA VSSSGNTNYAASVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYYCYAVAP EGGDSWGQGTQVTVSS
N22(SEQ ID NO.7)：
QVQLVESGGGLVQAGGSLRLSCRASGTVFNMGWHRQAPGQQREFVASIS HDGRTNYAGSVKGRFTISRDNTKNTLYLQMNSLKPEDTAVYYCIDYHTSL WGQGTQVTVSS
N28(SEQ ID NO.8)：
QVQLVESGGGLVQAGGSLRLSCAASGATFPFNSMAWWRQAPGKQREWV ASITSGGSTNYAESVKGRFAISGDNAKGTVYVQMNYLKPEDTAVYYCYY VSGRGGDYWGQGTQVTVSS
本发明的纳米抗体可以按照上述方法获得，也可以通过本领域常规技术手段，按照上述序列合成获得。
四、纳米抗体真核表达与纯化
将获得的上述8个VHH基因片段克隆到真核表达载体，构建无肉毒素的重组质粒，转化到昆虫表达系统(Hi-5)进行表达纯化，纯化分为镍柱亲和层析及分子筛(ColonnesSuperdex 75 10/300GL)两步纯化，纯化结果见图4～11。
以下通过实验例说明本发明的有益效果。
实验例1、本发明纳米抗体中和实验测试结果
1.实验方法
为了检验本发明纳米抗体是否具备如期良好潜在的中和新型冠状病毒的效应，通过综合运用流式细胞术、表面等离子共振技术(SPR)、假病毒中和等技术进行全面多角度地检测。
2.实验结果
表面等离子共振实验结果(图12)表明，RBD蛋白与ACE2受体的亲和力即KD值为173nM,而经过测试我们所制备的纳米抗体N1、N3、N5、N10、 N22与RBD的亲和力分别高达0.56nM,1.4nM,5.3nM,0.59nM和0.19nM，远远高于RBD与ACE2受体的结合能力。据此可以推测，该系列纳米抗体有望阻断新冠病毒通过ACE2受体入侵宿主细胞，具备潜在的中和新冠病毒的能力。
为了进一步检测纳米抗体是否具备有效的中和能力，我们分别以1ug/ml、 2ug/ml、4ug/ml的各类纳米抗体与新冠病毒刺突蛋白(S蛋白)共同孵育，然后通过流式细胞术实验进行测定。结果如图13～15所示，相比于其他纳米抗体，纳米抗体N5在各浓度下均可以更有效竞争性地阻断S蛋白与ACE2受体的结合，无独有偶，假病毒中和实验结果表明，在诸多纳米抗体当中，N5具有最强的假病毒中和效果，IC50达到0.023μg/mL。
综上所述，本但的纳米抗体与RBD蛋白具有很强的结合能力，在新型冠状病毒的检测领域有潜在应用价值；综合流式细胞术以及假病毒中和实验结果表明，其中的纳米抗体N5具有很强的病毒中和能力，有望成为治疗新型冠状病毒的有效药物。
SEQUENCE LISTING
<110> 四川一埃科技有限公司
<120> 抗新型冠状病毒RBD结构域抗原的纳米抗体
<130> GYKH1680-2022P0114857CC
<160> 16
<170> PatentIn version 3.5
<210> 1
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Ser Gln Asp Gly Arg Thr Asn Tyr Lys Gly Ser Val Lys Gly Arg Phe
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<213> N1
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caaatgagca gcctgaaacc tgaagacaca gccgtctatt actgctatgc agtggcaccg 300
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cagcgcggtt cgtcgcgtca attagtcatg atgggcgcac aaattatgca ggctccgtga 180
agggccgatt caccatctcc agagacaaca ccaagaacac cttgtatctg caaatgaaca 240
gcctgaaacc tgagacacag ccgtctatta ctgtatcgac taccacacca gtctctgggg 300
ccaggggacc caggtcaccg tctcctca 328
<210> 16
<211> 456
<212> DNA
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atgggctggt cctgtatcat cctgttcctg gtggccacag ctaccggcgt gcacagccag 60
gtgcagctgg tggaaagcgg cggcggtctc gtgcaagctg gcggctccct gagactgagc 120
tgcgccgcta gcggcgccac ctttccattc aacagcatgg cctggtggcg gcaggcccct 180
ggcaagcaga gagagtgggt cgccagcatc acatctggcg gaagcaccaa ttacgccgag 240
agcgtgaaag gccggttcgc catcagcggc gacaacgcca agggcacagt gtacgtgcag 300
atgaactacc tgaagcccga ggacaccgcc gtgtattact gctactacgt gtctggaaga 360
ggcggagatt actggggaca gggcacccag gtgaccgtgt ccagcctcga gctggttcct 420
agaggctctc accaccacca ccaccatcac cactga 456
Claims (10)
1.一种抗新型冠状病毒的纳米抗体，其特征在于：所述纳米抗体的氨基酸全长序列如SEQ ID NO.1～8任意一项所示。
2.编码权利要求1所述抗新型冠状病毒的纳米抗体的核酸。
3.如权利要求2所述的核酸，其特征在于，所述核酸的序列如SEQ ID NO.9～16所示。
4.一种重组核酸载体，其特征在于：所述载体包括权利要求2或3所述的核酸。
5.如权利要求4所述的核酸载体，其特征在于：所述核酸载体是真核表达载体。
6.一种重组细胞，其特征在于：所述重组细胞内含有权利要求5所述的载体。
7.权利要求1所述的纳米抗体在制备抗新型冠状病毒的药物中的用途。
8.一种抗新型冠状病毒的药物，其特征在于：所述药物以权利要求1所述的纳米抗体为活性成分。
9.权利要求1所述的纳米抗体在新型冠状病毒检测试剂中的用途。
10.一种新型冠状病毒检测试剂，其特征在于，它含有权利要求1所述的纳米抗体。
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| CN115124615B (zh) | 2023-06-16 |
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