技术领域Technical field
本发明属于生物技术、免疫检测和生物医药领域，具体涉及特异性纳米抗体或抗原结合片段及其在SARS-CoV-2的检测、诊断、预防、治疗中的用途，尤其涉及在SARS-CoV-2野生型毒株和/或B.1.1.7突变株(即α突变株)的检测、诊断、预防、治疗中的用途。The invention belongs to the fields of biotechnology, immune detection and biomedicine, and specifically relates to specific nanobodies or antigen-binding fragments and their use in the detection, diagnosis, prevention and treatment of SARS-CoV-2, especially in the detection, diagnosis, prevention and treatment of SARS-CoV-2. 2. Use in the detection, diagnosis, prevention and treatment of wild-type strains and/or B.1.1.7 mutant strains (i.e. alpha mutant strains).
背景技术Background technique
新型冠状病毒SARS-CoV-2是一种β冠状病毒属RNA病毒。该病毒具有传播性强、致死率高和突变速度快等特点，。SARS-CoV-2会引起呼吸道感染，从而导致一些患者出现病毒性肺炎和急性呼吸窘迫综合征(ARDS)。同时还会引发细胞因子风暴，引起多器官损伤。新冠病毒原始毒株被分离至今，在全球传播过程中不断出现的新型突变株病毒如D614G突变株、B.1.1.7突变株、B.1.351突变株、B.1.429突变株、P.1突变株、B.1.617.2突变株等，不仅大大增强了病毒的传播性和致死率，造成了全球大多数国家的疫情反复爆发，同时也造成了疫苗保护力的不断降低。The new coronavirus SARS-CoV-2 is an RNA virus of the betacoronavirus genus. The virus has the characteristics of strong transmissibility, high fatality rate and rapid mutation rate. SARS-CoV-2 causes respiratory infections, leading to viral pneumonia and acute respiratory distress syndrome (ARDS) in some patients. At the same time, it can also trigger a cytokine storm and cause multiple organ damage. Since the original strain of the new coronavirus was isolated, new mutant strains of the virus have continued to appear during the global spread, such as the D614G mutant strain, the B.1.1.7 mutant strain, the B.1.351 mutant strain, the B.1.429 mutant strain, and the P.1 mutation. strains, B.1.617.2 mutant strains, etc., not only greatly enhanced the transmissibility and fatality rate of the virus, causing repeated outbreaks in most countries around the world, but also caused the continuous reduction of vaccine protection.
在COVID-19患者治疗中使用一些小分子药物和干扰素进行抗病毒治疗，然而临床结果均显示无效或仅能在病毒感染的早期能起到有限的治疗效果,同时还会伴随一系列严重的药物副作用。已有研究证明抗体治疗策略是治疗冠状病毒患者，尤其是中晚期患者的最佳解决方案。利用含有大量中和抗体的COVID-19愈后患者血清治疗新冠病毒患者是行之有效的治疗策略。但患者血清疗法的局限性在于康复者血浆较难获得，数量较少，不能满足庞大患者群体需求，所以需要替代性的工程抗体进行治疗。Some small molecule drugs and interferons are used for antiviral treatment in the treatment of COVID-19 patients. However, clinical results have shown that they are ineffective or can only have limited therapeutic effect in the early stage of viral infection. They are also accompanied by a series of serious diseases. Medication Side Effects. Studies have proven that antibody treatment strategies are the best solution for treating coronavirus patients, especially those in the intermediate and advanced stages. Using serum from post-COVID-19 patients containing large amounts of neutralizing antibodies to treat patients with COVID-19 is an effective treatment strategy. However, the limitation of patient serum therapy is that plasma from recovered patients is difficult to obtain and the quantity is small, which cannot meet the needs of a large patient population. Therefore, alternative engineered antibodies are needed for treatment.
纳米抗体(Nanobody)是只含有重链抗体抗原结合域VHH的单域抗体，与传统多克隆抗体、单克隆抗体和单链抗体相比具有诸多明显优势，比如体积小，可以穿过常规抗体无法进入的组织和器官(如鞘膜、脊髓、大脑等)中；稳定性强，无需冷链运输和冷藏保存；免疫原性低，易于进行人源化改造。本发明将SARS-CoV-2病毒表面突刺蛋白(Spike蛋白，即S蛋白)做为靶标，通过构建噬菌体展示纳米抗体免疫文库、生物淘选研制出可同时识别新冠病毒Wild Type原始株和B.1.1.7突变株的高特异性、高亲和力纳米抗体，为新冠肺炎的机理研究、临床诊断和治疗打下基础，以及将来有可能出现的新型冠状病毒再次爆发的进行战略储备都具有重要意义。Nanobodies are single-domain antibodies that only contain the heavy chain antibody antigen-binding domain VHH. Compared with traditional polyclonal antibodies, monoclonal antibodies, and single-chain antibodies, they have many obvious advantages, such as small size and the ability to pass through structures that conventional antibodies cannot. It enters the tissues and organs (such as the sheath, spinal cord, brain, etc.); it has strong stability and does not require cold chain transportation and refrigerated storage; it has low immunogenicity and is easy to carry out humanized transformation. The present invention uses the SARS-CoV-2 virus surface spike protein (Spike protein, i.e. S protein) as a target, and develops a phage display nanobody immune library that can simultaneously identify the original strain of the new coronavirus Wild Type and B. The highly specific and high-affinity nanobodies of the 1.1.7 mutant strains are of great significance to lay the foundation for mechanism research, clinical diagnosis and treatment of new coronavirus pneumonia, as well as strategic reserves for possible new coronavirus outbreaks in the future.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种针对冠状病毒的广谱型、高亲和力抗体，该抗体可有效检测、阻断、和治疗冠状病毒尤其是SARS-CoV-2病毒原始株及其突变株。The technical problem to be solved by the present invention is to provide a broad-spectrum, high-affinity antibody against coronavirus, which can effectively detect, block, and treat coronavirus, especially the original strain of SARS-CoV-2 virus and its mutant strain. .
在本发明的具体实施方案中，提供了抗冠状病毒例如SARS-CoV-2的纳米抗体，其可以和原始株(Wild Type)，英国突变株(B1.1.7)S蛋白的S1亚基(也称为S1蛋白)结合，亲和力均达到皮摩尔级别。In specific embodiments of the present invention, Nanobodies against coronaviruses such as SARS-CoV-2 are provided, which can interact with the S1 subunit (also known as S1) of the S protein of the original strain (Wild Type) and the British mutant strain (B1.1.7). Called S1 protein), the affinity reaches the picomolar level.
在本发明的具体实施方案中，提供了抗冠状病毒例如SARS-CoV-2的纳米抗体，其能够有效阻断SARS-CoV-2假病毒对hACE2过表达的293T细胞的感染，半有效中和浓度达到纳摩尔级别。In specific embodiments of the present invention, Nanobodies against coronaviruses such as SARS-CoV-2 are provided, which can effectively block the infection of hACE2-overexpressing 293T cells by SARS-CoV-2 pseudovirus and semi-effectively neutralize Concentrations reach nanomolar levels.
在本发明的具体实施方案中，可以进行多种基于抗原/抗体反应的酶联免疫分析检测方法的建立和检测产品的开发。In specific embodiments of the present invention, a variety of enzyme-linked immunoassay detection methods based on antigen/antibody reactions can be established and detection products can be developed.
在本发明的具体实施方案中，可以进行同种或多种基于纳米抗体的多价化等基因工程改造。In specific embodiments of the present invention, genetic engineering such as multivalency based on the same or multiple Nanobodies can be carried out.
在本发明的具体实施方案中，提供了针对冠状病毒例如SARS-CoV-2的纳米抗体，所述纳米抗体包含如下的氨基酸序列和功能特性：In a specific embodiment of the invention, Nanobodies against coronaviruses such as SARS-CoV-2 are provided, the Nanobodies comprising the following amino acid sequences and functional properties:
i)SEQ ID NO:1-8所示的氨基酸序列；或者所述抗体可具有SEQ ID NO:9-13任一所示的高度可变区CDR1氨基酸序列；SEQ ID NO:14-18任一所示的高度可变区CDR2氨基酸序列；和SEQ ID NO:19-26任一所示的高度可变区CDR3氨基酸序列；i) The amino acid sequence shown in SEQ ID NO: 1-8; or the antibody may have the highly variable region CDR1 amino acid sequence shown in any one of SEQ ID NO: 9-13; any one of SEQ ID NO: 14-18 The highly variable region CDR2 amino acid sequence shown; and the highly variable region CDR3 amino acid sequence shown in any one of SEQ ID NO: 19-26;
ii)所述纳米抗体与冠状病毒例如SARS-CoV-2病毒Wild Type原始株和B.1.1.7株具有纳摩尔级别的亲和力；ii) The Nanobody has nanomolar-level affinity to coronaviruses such as the original SARS-CoV-2 Wild Type strain and the B.1.1.7 strain;
iii)所述纳米抗体有效阻断SARS-CoV-2假病毒对hACE2过表达的293T细胞的感染。iii) The Nanobody effectively blocks the infection of hACE2-overexpressing 293T cells by SARS-CoV-2 pseudovirus.
本发明还提供一种含有所述编码所述抗体的核酸分子的生物材料，所述生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体或工程菌。The present invention also provides a biological material containing the nucleic acid molecule encoding the antibody, and the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector or engineered bacteria.
本发明还提供所述抗体的以下任一应用：The invention also provides any of the following applications of the antibody:
1)用于冠状病毒例如SARS-CoV-2病毒原始株及其突变株相关的科学研究；1) For scientific research related to coronaviruses such as the original strain of the SARS-CoV-2 virus and its mutant strains;
2)用于冠状病毒例如SARS-CoV-2病毒原始株及其突变株表面S蛋白的检测；2) Used for the detection of surface S protein of coronaviruses such as the original strain of SARS-CoV-2 virus and its mutant strains;
3)用于研制冠状病毒例如SARS-CoV-2病毒原始株及其突变株检测试剂或ELISA检测试剂。3) Used to develop detection reagents or ELISA detection reagents for coronaviruses such as the original strain of the SARS-CoV-2 virus and its mutant strains.
本发明中，分析检测时，向包被有冠状病毒例如所述SARS-CoV-2病毒Wild Type原始株和B.1.1.7突变株抗原的酶标板的各孔中加入不同浓度所述纳米抗体，由于每个孔中的固相抗原含量均一致，因此当结合在固相抗原上的抗体少，加入的酶标二抗与被结合的纳米抗体结合量少，最后加入底物液和显色液，显色反应浅，用酶标仪检测的OD值低；反之，当所述纳米抗体和固相抗原结合多时，则所测的OD值高，根据加入的纳米抗体量和对应孔的OD值绘制纳米抗体和SARS-CoV-2的结合曲线。In the present invention, during analysis and detection, different concentrations of the nanoparticles are added to each well of an enzyme plate coated with the antigen of a coronavirus, such as the SARS-CoV-2 Wild Type original strain and the B.1.1.7 mutant strain. Antibody. Since the solid-phase antigen content in each well is the same, when there are less antibodies bound to the solid-phase antigen, the added enzyme-labeled secondary antibody will bind less to the bound nanobody. Finally, add substrate solution and display Color liquid, the color reaction is light, and the OD value detected with a microplate reader is low; conversely, when the Nanobody and the solid-phase antigen are combined more, the measured OD value is high. According to the amount of Nanobody added and the size of the corresponding well, The OD value is used to draw the binding curve of Nanobodies and SARS-CoV-2.
具体地，本发明提供了以下技术方案：Specifically, the present invention provides the following technical solutions:
1、一种抗体或其抗原结合片段，其氨基酸序列包含由SEQ ID NO:9-13任一所示的CDR1、由SEQ ID NO:14-18任一所示的CDR2、由SEQ ID NO:19-26任一所示的CDR3；1. An antibody or an antigen-binding fragment thereof, the amino acid sequence of which comprises CDR1 represented by any one of SEQ ID NO:9-13, CDR2 represented by any one of SEQ ID NO:14-18, or CDR2 represented by any one of SEQ ID NO:14-18, or CDR2 represented by any one of SEQ ID NO:14-18. CDR3 shown in any one of 19-26;
优选地，所述抗原结合片段例如为Fv、Fab、Fab'、scFv、F(ab')2、多价化或多特异片段。Preferably, the antigen-binding fragment is, for example, a Fv, Fab, Fab', scFv, F(ab')2 , multivalent or multispecific fragment.
2、根据项目1所述的抗体或抗原结合片段，其氨基酸序列如SEQ ID NO:1-8任一所示；2. The antibody or antigen-binding fragment according to item 1, whose amino acid sequence is as shown in any one of SEQ ID NO: 1-8;
或者所述抗体或抗原结合片段是包含将SEQ ID NO:1-8任一所示序列自N末端起第1位至124位氨基酸进行截短所获得的序列的抗体，或者是将SEQ ID NO:1-8任一所示序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的抗体或抗原结合片段。Alternatively, the antibody or antigen-binding fragment is an antibody comprising a sequence obtained by truncating any of the sequences shown in SEQ ID NO: 1-8 from the 1st to the 124th amino acid from the N terminus, or is an antibody obtained by truncating SEQ ID NO: 1-8. Antibodies or antigen-binding fragments with the same function obtained by substitution and/or deletion and/or addition of one or several amino acid residues in any of the sequences shown in 1-8.
3、基因工程抗体，其包含项目1或2所述的抗体或抗原结合片段；优选地，所述基因工程抗体为人源化抗体、嵌合抗体、多价化或多特异性抗体。3. Genetically engineered antibodies, which comprise the antibodies or antigen-binding fragments described in item 1 or 2; preferably, the genetically engineered antibodies are humanized antibodies, chimeric antibodies, multivalent or multispecific antibodies.
4、融合蛋白，其包含项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体；优选地，融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。4. Fusion protein, which includes the antibody or antigen-binding fragment described in Item 1 or 2 or the genetically engineered antibody described in Item 3; preferably, the fusion protein also includes a tag polypeptide, a detection protein or an auxiliary protein.
5、偶联物，其包含项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白；优选地，所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP，或其组合。5. Conjugate, which contains the antibody or antigen-binding fragment described in Item 1 or 2 or the genetically engineered antibody described in Item 3 or the fusion protein described in Item 4; Preferably, the conjugate also contains a detectable Labels, contrast agents, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, liposomes, viral coat proteins or VLPs, or combinations thereof.
6、一种核酸分子，其编码如项目1-2所述的抗体或抗原结合片段、如项目3所述的基因工程抗体、如项目4所述的融合蛋白或如项目5所述的偶联物，其中所述核酸分子为RNA、DNA或cDNA。6. A nucleic acid molecule encoding an antibody or antigen-binding fragment as described in Item 1-2, a genetically engineered antibody as described in Item 3, a fusion protein as described in Item 4, or a coupling as described in Item 5 Object, wherein said nucleic acid molecule is RNA, DNA or cDNA.
7、一种表达载体，其包含项目6所述的核酸分子；7. An expression vector comprising the nucleic acid molecule described in item 6;
任选地，所述表达载体可以是DNA、RNA、病毒载体、质粒、表达盒、转座子、其他基因转移系统、或其组合；Optionally, the expression vector can be DNA, RNA, viral vectors, plasmids, expression cassettes, transposons, other gene transfer systems, or combinations thereof;
优选地，所述表达载体包括病毒载体，如噬菌体载体、慢病毒、腺病毒、AAV病毒、逆转录病毒、其他蛋白表达系统、或其组合。Preferably, the expression vector includes viral vectors, such as phage vectors, lentiviruses, adenoviruses, AAV viruses, retroviruses, other protein expression systems, or combinations thereof.
8、宿主细胞，其包含项目7所述的表达载体；其中，所述宿主细胞是用于表达外源蛋白的宿主细胞，例如原核表达细胞、真核表达细胞、转基因细胞系；优选地，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞、植物细胞、动物细胞。8. Host cell, which contains the expression vector described in item 7; wherein the host cell is a host cell used to express foreign proteins, such as prokaryotic expression cells, eukaryotic expression cells, and transgenic cell lines; preferably, the host cell The host cells include prokaryotic cells, yeast cells, insect cells, plant cells, and animal cells.
9、组织样本或培养物，其通过培养项目8所述的宿主细胞获得。9. Tissue samples or cultures obtained by culturing the host cells described in item 8.
10、蛋白或抗原结合片段，其从项目9所述的组织样本或培养物中分离获得。10. Protein or antigen-binding fragment isolated from the tissue sample or culture described in item 9.
11、制备项目1-2所述的抗体或抗原结合片段、如项目3所述的基因工程抗体、如项目4所述的融合蛋白或如项目5所述的偶联物的方法，包括从项目9所述的组织样本或培养物中分离/回收目的蛋白或抗原结合片段。11. Methods for preparing the antibodies or antigen-binding fragments described in Item 1-2, the genetically engineered antibodies described in Item 3, the fusion proteins described in Item 4, or the conjugates described in Item 5, including from items Isolate/recover the target protein or antigen-binding fragment from the tissue sample or culture described in 9.
12、药物组合物，其包含项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物作为活性成分；例如，所述药物组合物为吸入式雾化药物、粘膜或表皮外用型药物、皮下注射型药物、血管输入型药物、或其组合；优选地，所述药物还包括药用赋形剂或载体。12. Pharmaceutical composition, which contains the antibody or antigen-binding fragment described in Item 1 or 2 or the genetically engineered antibody described in Item 3 or the fusion protein described in Item 4 or the conjugate described in Item 5 as an active ingredient; For example, the pharmaceutical composition is an inhaled aerosol drug, a mucosal or epidermal external drug, a subcutaneous injection drug, a vascular infusion drug, or a combination thereof; preferably, the drug also includes pharmaceutical excipients or carriers .
13、含有项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物的产品；例如，所述产品为口罩或空气净化器滤芯，环境、物体或人体表面消毒剂，或其组合；优选地，所述产品涂布在净化器滤芯中或溶解于消毒剂中用于雾化喷洒或表面擦拭。13. Products containing the antibody or antigen-binding fragment described in item 1 or 2, the genetically engineered antibody described in item 3, the fusion protein described in item 4, or the conjugate described in item 5; for example, the product is Mask or air purifier filter element, environmental, object or human surface disinfectant, or a combination thereof; preferably, the product is coated in the purifier filter element or dissolved in the disinfectant for atomized spraying or surface wiping.
14、项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物在制备用于预防、治疗和/或诊断冠状病毒感染的产品或药物中的用途。14. The antibody or antigen-binding fragment described in Item 1 or 2 or the genetically engineered antibody described in Item 3 or the fusion protein described in Item 4 or the conjugate described in Item 5 is used in preparation for prevention, treatment and/or Use in products or medicines for diagnosing coronavirus infections.
15、项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物在制备用于以下功能的产品中的应用：15. The use of the antibody or antigen-binding fragment described in Item 1 or 2 or the genetically engineered antibody described in Item 3 or the fusion protein described in Item 4 or the conjugate described in Item 5 in the preparation of products for the following functions application:
1)检测冠状病毒抗原，尤其是SARS-CoV-2病毒原始株及其突变株；1) Detect coronavirus antigens, especially the original strain of SARS-CoV-2 virus and its mutant strains;
2)阻断冠状病毒感染，尤其是SARS-CoV-2病毒原始株及其突变株；2) Block coronavirus infection, especially the original strain of SARS-CoV-2 virus and its mutant strains;
3)消杀冠状病毒颗粒，尤其是SARS-CoV-2病毒原始株及其突变株；3) Eliminate coronavirus particles, especially the original strain of SARS-CoV-2 virus and its mutant strains;
4)诊断冠状病毒引起的相关疾病，尤其是SARS-CoV-2病毒原始株及其突变株；4) Diagnose related diseases caused by coronavirus, especially the original strain of SARS-CoV-2 virus and its mutant strains;
5)治疗冠状病毒引起的相关疾病，尤其是SARS-CoV-2病毒原始株及其突变株；5) Treat related diseases caused by coronavirus, especially the original strain of SARS-CoV-2 virus and its mutant strains;
6)进行冠状病毒相关的基础科学研究，尤其是SARS-CoV-2病毒原始株及其突变株。6) Conduct basic scientific research related to coronaviruses, especially the original strain of the SARS-CoV-2 virus and its mutant strains.
在本发明的具体实施方案中，所述冠状病毒包括HCoV-NL63、SARS-CoV-1、SARS-CoV-2、HCoV-229E、MERS-CoV、HCoV-OC43、HCoV-HKU1或其他具有相似表面S蛋白结构的冠状病毒。In specific embodiments of the invention, the coronavirus includes HCoV-NL63, SARS-CoV-1, SARS-CoV-2, HCoV-229E, MERS-CoV, HCoV-OC43, HCoV-HKU1 or others with similar surface S protein structure of coronavirus.
在本发明的具体实施方案中，SARS-CoV-2病毒的突变株包括D614G突变株、B.1.1.7突变株、B.1.351突变株、B.1.429突变株、P.1突变株、B.1.617.2突变株等。In specific embodiments of the invention, mutant strains of SARS-CoV-2 virus include D614G mutant strain, B.1.1.7 mutant strain, B.1.351 mutant strain, B.1.429 mutant strain, P.1 mutant strain, B .1.617.2 mutant strain, etc.
在本发明的具体实施方案中，所述标签多肽中所包含纯化标签、检测标签、鉴定标签、偶联标签、功能验证标签等功能多肽，例如His标签、HA标签、Flag标签、c-Myc标签、Avi标签等。In specific embodiments of the present invention, the tag polypeptide includes purification tags, detection tags, identification tags, coupling tags, functional verification tags and other functional polypeptides, such as His tag, HA tag, Flag tag, c-Myc tag , Avi tags, etc.
在本发明的具体实施方案中，所述融合蛋白中所包含的检测蛋白包括荧光蛋白、荧光素标记蛋白、过氧化物酶等功能蛋白，例如FPs蛋白、HRP蛋白、Alexa Fluor标记蛋白、FITC标记蛋白等。In specific embodiments of the invention, the detection proteins included in the fusion protein include functional proteins such as fluorescent proteins, fluorescein-labeled proteins, peroxidases, etc., such as FPs proteins, HRP proteins, Alexa Fluor-labeled proteins, FITC-labeled proteins, etc. Protein etc.
在本发明的具体实施方案中，所述融合蛋白中所包含的辅助蛋白为用于辅助折叠、辅助表达、辅助溶解、屏蔽毒性蛋白等功能的蛋白，例如GST蛋白、MBP蛋白、SUMO蛋白、NusA蛋白。In a specific embodiment of the present invention, the auxiliary protein included in the fusion protein is a protein used to assist folding, assist expression, assist dissolution, shield toxic proteins and other functions, such as GST protein, MBP protein, SUMO protein, NusA protein.
技术效果Technical effect
本发明提供的用于抗冠状病毒例如SARS-CoV-2的抗体，有效克服目前冠状病毒例如SARS-CoV-2康复患者血清来源少，成本高，结构不稳定等缺点，具有高亲和力、高灵敏度、高中和能力、高产量、高稳定性，低成本并能进行大批量快速生产。本发明提供的抗体除了可以用于初期感染阻断、早期感染诊断、中晚期感染治疗外，还可以用于科研工具和体外快速检测，例如生产ELISA检测/诊断试剂盒、胶体金检测/诊断试剂盒。The antibodies provided by the present invention for use against coronaviruses such as SARS-CoV-2 can effectively overcome the shortcomings of current coronaviruses such as SARS-CoV-2, such as low serum sources, high cost, and unstable structure, and have high affinity and sensitivity. , high mixing ability, high output, high stability, low cost and capable of rapid mass production. In addition to being used for early infection blocking, early infection diagnosis, and mid-late infection treatment, the antibodies provided by the present invention can also be used for scientific research tools and in vitro rapid detection, such as the production of ELISA detection/diagnostic kits and colloidal gold detection/diagnostic reagents. box.
利用本发明的所述抗体建立的检测方法例如ELISA检测方法能准确灵敏地检测样品中是否含有冠状病毒例如SARS-CoV-2病毒。样品的前处理过程简单，耗时少，能同时检测大量的样品，样品检测成本远低于传统的核酸检测方法。本发明所述的抗体应用于胶体金检测/诊断试剂盒可以迅速且准确检测样品中是否含有冠状病毒例如SARS-CoV-2病毒，对解决大规模人群感染和环境、货物样品污染筛查鉴定具有重要的现实意义。Detection methods established using the antibodies of the present invention, such as ELISA detection methods, can accurately and sensitively detect whether a sample contains coronavirus, such as SARS-CoV-2 virus. The sample pre-processing process is simple, less time-consuming, and can detect a large number of samples at the same time. The sample detection cost is much lower than traditional nucleic acid detection methods. The antibody of the present invention can be used in a colloidal gold detection/diagnostic kit to quickly and accurately detect whether a sample contains coronavirus, such as SARS-CoV-2 virus, and is useful for solving large-scale population infections and environmental and cargo sample pollution screening and identification. important practical significance.
附图说明Description of drawings
为了更清楚地说明本发明或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，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的附图。In order to more clearly illustrate the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are the drawings of the present invention. For some embodiments, those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting creative efforts.
图1为本发明所述纳米抗体与SARS-CoV-2病毒Wild Type原始株和B.1.1.7突变株S1蛋白的结合曲线；Figure 1 is the binding curve of the Nanobody of the present invention to the S1 protein of the SARS-CoV-2 Wild Type original strain and the B.1.1.7 mutant strain;
图2为本发明所述纳米抗体与SARS-CoV-2病毒Wild Type原始株和B.1.1.7突变株S1蛋白的亲和力曲线图(以抗体A1为例)；Figure 2 is an affinity curve diagram of the Nanobody of the present invention and the S1 protein of the original strain of SARS-CoV-2 Wild Type virus and the B.1.1.7 mutant strain (taking antibody A1 as an example);
图3为本发明所述纳米抗体对SARS-CoV-2病毒Wild Type原始株和B.1.1.7突变株假病毒的中和能力检测；Figure 3 is the detection of the neutralizing ability of the Nanobody of the present invention against the original strain of SARS-CoV-2 virus Wild Type and the B.1.1.7 mutant strain pseudovirus;
图4显示了本发明所述抗体的序列及其CDR区；Figure 4 shows the sequence of the antibody of the present invention and its CDR region;
图5为实施例1中所使用的pComb3Xss的质粒图谱。Figure 5 is a plasmid map of pComb3Xss used in Example 1.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明中的附图，对本发明中的技术方案进行清楚、完整地描述，显然，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。基于本发明中的实施例，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例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用仪器等未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。所述方法如无特别说明均为常规方法，所述原材料如无特别说明均能从公开商业途径而得。If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. The instruments used, etc., if the manufacturer is not indicated, are all conventional products that can be purchased through regular channels. Unless otherwise stated, the methods are conventional methods, and the raw materials can be obtained from public commercial sources unless otherwise stated.
若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。Unless otherwise specified, the examples all follow conventional experimental conditions, such as Sambrook et al.'s Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or conditions recommended by the manufacturer's instructions.
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention.
下述实施例中的实验方法，如无特殊说明，均为常规方法。The experimental methods in the following examples are all conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。The quantitative experiments in the following examples were repeated three times, and the results were averaged.
根据本发明的一些优选实施例，所述纳米抗体可以按如下方法进行制备：将原始毒株SARS-CoV-2蛋白作为免疫原免疫实验动物骆驼，提取外周血淋巴细胞的总RNA，经反转录及巢式PCR，克隆出纳米抗体重链(VHH)基因片段，通过酶切连接，将基因片段克隆至噬菌粒载体，高效电转化至大肠杆菌，经辅助噬菌体拯救，构建得到噬菌体纳米抗体库，筛选出SARS-CoV-2纳米抗体，将其进行表达纯化，得到灵敏度高的SARS-CoV-2纳米抗体，并且与流行的突变毒株有较高的交叉反应。制备的纳米抗体分子小，可溶性强，耐高温，易纯化，易表达。According to some preferred embodiments of the present invention, the Nanobody can be prepared as follows: using the original strain SARS-CoV-2 protein as an immunogen to immunize experimental animal camels, extracting total RNA from peripheral blood lymphocytes, and inverting After recording and nested PCR, the Nanobody heavy chain (VHH) gene fragment was cloned. Through enzyme digestion and ligation, the gene fragment was cloned into the phagemid vector, and efficiently electrotransformed into E. coli. After rescue by auxiliary phage, the phage Nanobody was constructed. library, screen out SARS-CoV-2 Nanobodies, express and purify them, and obtain SARS-CoV-2 Nanobodies with high sensitivity and high cross-reactivity with popular mutant strains. The prepared nanobody has small molecules, strong solubility, high temperature resistance, easy purification, and easy expression.
根据本发明的一些优选实施例，所述SARS-CoV-2病毒wild type原始株S蛋白和RBD蛋白作为免疫原，SARS-CoV-2病毒wild type原始株S1蛋白和B.1.1.7突变株S1蛋白作为包被的抗原，均购买于北京义翘神州生物有限公司。According to some preferred embodiments of the present invention, the S protein and RBD protein of the wild type original strain of SARS-CoV-2 virus are used as immunogens, and the S1 protein and B.1.1.7 mutant strain of the wild type original strain of SARS-CoV-2 virus are used as immunogens. S1 protein, as the coated antigen, was purchased from Beijing Yiqiao Shenzhou Biological Co., Ltd.
所述酶标板为96孔酶标板，包被抗原的包被浓度为1ug/mL。The enzyme-labeled plate is a 96-well enzyme-labeled plate, and the coating concentration of the coated antigen is 1ug/mL.
所述酶标记的二抗为辣根过氧化物酶标记的抗HA标签抗体，浓度为0.1μg/mL。购自Abcam公司，商品编号:ab1265。The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled anti-HA tag antibody with a concentration of 0.1 μg/mL. Purchased from Abcam Company, product number: ab1265.
所述显色液A液由过氧化脲1g、柠檬酸10.3g、Na2HPO4·12H2O35.8g、吐温-20 100μL和蒸馏水1000mL配制而成，pH值5。The chromogenic solution A is prepared from 1g of carbamide peroxide, 10.3g of citric acid, 5.8g of Na2 HPO4 ·12H2 O3, 100 μL of Tween-20 and 1000 mL of distilled water, with a pH value of 5.
所述显色液B液由四甲基联苯胺700mg、DMSO 40mL、柠檬酸10.3g和蒸馏水1000mL配制而成，pH值2.4。The chromogenic solution B is prepared from 700 mg of tetramethylbenzidine, 40 mL of DMSO, 10.3 g of citric acid and 1000 mL of distilled water, with a pH value of 2.4.
所述反应终止液为2M的硫酸液。The reaction termination solution is 2M sulfuric acid solution.
实施例1、SARS-CoV-2纳米抗体库的构建Example 1. Construction of SARS-CoV-2 Nanobody Library
取200ug SARS-CoV-2病毒原始株S蛋白和RBD蛋白SARS-CoV-2抗原(北京义翘神州生物有限公司)与等体积完全弗氏佐剂混合，充分乳化后注射至骆驼，以后每隔两周加强免疫一次，其中在加强免疫中使用不完全弗氏佐剂与免疫原的混合液，颈背部皮下多点免疫，共免疫5次。从第三次免疫开始，每次免疫后一周从颈锁静脉采血并检测血清效价。Take 200ug of S protein and RBD protein SARS-CoV-2 antigen of the original strain of SARS-CoV-2 virus (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) and mix it with an equal volume of complete Freund's adjuvant, fully emulsify it and inject it into the camel, and then every other Booster immunization was performed once every two weeks. In the booster immunization, a mixture of incomplete Freund's adjuvant and immunogen was used, and multiple points were immunized subcutaneously on the back of the neck for a total of 5 times. Starting from the third immunization, blood was collected from the jugular vein one week after each immunization and serum titers were measured.
从第5次免疫后的外周血中分离白细胞，提取总RNA，经反转录PCR及巢式PCR(其中，反转录PCR和巢式PCR的体系和参数如下所述)，克隆出VHH基因片段，用限制性内切酶SfiI修饰粘性末端，通过T4连接酶将VHH基因片段连接至噬菌粒pComb3Xss(UC Davis的Bruce DHammock教授实验室惠赠)，高效电转化至大肠杆菌ER2738(实验室保存，也可商购获得，例如购自英国NEB公司)，构建SARS-CoV-2的噬菌体纳米抗体库。经测定，初级库容量达109cfu，加入辅助噬菌体(感染复数为20:1)M13KO7(购自NEB公司，货号：N0315S)进行拯救，得到噬菌体纳米抗体库，库容量为1012pfu/mL，库的多样性较好。Leukocytes were isolated from the peripheral blood after the fifth immunization, total RNA was extracted, and the VHH gene was cloned through reverse transcription PCR and nested PCR (the systems and parameters of reverse transcription PCR and nested PCR are as follows) The fragment was modified with the restriction endonuclease SfiI to modify the sticky end, and the VHH gene fragment was ligated to the phagemid pComb3Xss (a gift from the laboratory of Professor Bruce DHammock at UC Davis) through T4 ligase, and then efficiently electrotransformed into E. coli ER2738 (stored in the laboratory , also available commercially, for example from NEB Company in the UK), to construct a phage nanobody library of SARS-CoV-2. It was determined that the primary library capacity reached 109 cfu. Helper phage (multiplicity of infection 20:1) M13KO7 (purchased from NEB Company, product number: N0315S) was added for rescue, and a phage nanobody library was obtained with a library capacity of 1012 pfu/mL. , the diversity of the library is better.
反转录PCR：Reverse transcription PCR:
反转录试剂盒采用PrimeScriptTM RT-PCR Kit，购自Takara公司，商品编号：AK2701。The reverse transcription kit used PrimeScriptTM RT-PCR Kit, purchased from Takara Company, product number: AK2701.
反转录体系如下：The reverse transcription system is as follows:
65℃，反应5min。取出置于冰上，按以下体系加样，进行cDNA第一链合成。65℃, react for 5 minutes. Take it out and place it on ice, add the sample according to the following system, and perform first-strand cDNA synthesis.
30℃10min；42℃1h；72℃5min。30℃10min; 42℃1h; 72℃5min.
巢式PCR：(购自TAKATA公司，货号：6210A)Nested PCR: (Purchased from TAKATA, Cat. No.: 6210A)
第一轮PCR：First round of PCR:
反应体系如下：The reaction system is as follows:
反应程序如下：The reaction procedure is as follows:
第二轮PCR：Second round of PCR:
反应体系如下：The reaction system is as follows:
反应程序如下：The reaction procedure is as follows:
其中，巢式PCR引物序列如下(5′-3′)：Among them, the nested PCR primer sequence is as follows (5′-3′):
GSP-RT：CGCCATCAATRTACCAGTTGA(SEQ ID NO:27)GSP-RT:CGCCATCAATRTACCAGTTGA(SEQ ID NO:27)
LP-leader：GTGGTCCTGGCTGCTCTW(SEQ ID NO:28)LP-leader：GTGGTCCTGGCTGCTCTW(SEQ ID NO:28)
F：CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC(SEQ ID NO:30)F: CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO:30)
R：CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG(SEQ ID NO:29)R: CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG (SEQ ID NO: 29)
其中，R表示碱基A/G，W表示碱基A/T，K表示碱基G/T。Among them, R represents the base A/G, W represents the base A/T, and K represents the base G/T.
实施例2、SARS-CoV-2纳米抗体的筛选Example 2. Screening of SARS-CoV-2 Nanobodies
在96孔酶标板的第1个孔包被SARS-CoV-2原始株病毒S蛋白抗原，包被浓度为1ug/mL，4℃过夜；次日，倒出包被液，用PBST洗涤3次，将酶标板第1、2两个孔用BSA封闭，室温孵育2h；倒出封闭液，用PBST洗涤3次；将实施例1获得的噬菌体纳米抗体库加入第1个孔，反应2h；倒出液体，在洁净的吸水纸上拍干，用PBST洗涤5次；将100μLSARS-CoV-2病毒原始株S1蛋白添加到第1个孔中，反应1h；吸出第1个孔中的液体，加入第2个孔，反应1h，除去与BSA结合的噬菌体；收集洗脱液，取5μL用于滴度测定，其余用于扩增。Coat the first well of the 96-well microplate with the SARS-CoV-2 original strain virus S protein antigen at a coating concentration of 1ug/mL and leave overnight at 4°C; the next day, pour out the coating solution and wash with PBST for 3 days. times, the first and second wells of the enzyme plate were blocked with BSA and incubated at room temperature for 2 hours; the blocking solution was poured out and washed three times with PBST; the phage Nanobody library obtained in Example 1 was added to the first well and reacted for 2 hours. ; Pour out the liquid, pat dry on clean absorbent paper, and wash 5 times with PBST; Add 100 μL SARS-CoV-2 virus original strain S1 protein to the first well and react for 1 hour; Aspirate the liquid in the first well , add to the second well, react for 1 hour to remove the phage bound to BSA; collect the eluate, take 5 μL for titer determination, and the rest for amplification.
将噬菌体洗脱液加入新鲜的大肠杆菌ER2738(实验室保存，也可商购获得，例如购自NEB公司)菌液，37℃，静置15min；加入羧苄青霉素和SB培养基，37℃，220rpm，培养2h；加入辅助噬菌体M13KO7(感染复数MOI＝20:1)(购自NEB公司，货号：N0315S)和卡那霉素，培养过夜；次日，离心取上清，加入PEG-NaCl溶液沉淀纯化噬菌体。Add the phage eluate to fresh Escherichia coli ER2738 (saved in the laboratory, also available commercially, for example from NEB Company) bacterial liquid, and let stand at 37°C for 15 minutes; add carbenicillin and SB culture medium, and set at 37°C. 220 rpm, culture for 2 hours; add helper phage M13KO7 (multiplicity of infection MOI=20:1) (purchased from NEB Company, product number: N0315S) and kanamycin, culture overnight; the next day, centrifuge the supernatant, and add PEG-NaCl solution Precipitate and purify phage.
将扩增产物进行下一轮筛选，保证每轮筛选的加入量相同，抗原包被浓度及S蛋白竞争洗脱浓度按2倍递减，计算每轮的滴度，挑取单克隆进行扩增及ELISA鉴定。经3轮淘选得到阳性单克隆。The amplified products are subjected to the next round of screening to ensure that the amount added in each round of screening is the same. The antigen coating concentration and S protein competitive elution concentration are reduced by 2 times. Calculate the titer of each round and select single clones for amplification and ELISA identification. After three rounds of panning, positive single clones were obtained.
实施例3、SARS-CoV-2纳米抗体的表达Example 3. Expression of SARS-CoV-2 Nanobodies
提取阳性单克隆质粒，转化至大肠杆菌TOP10F’感受态细胞(购自ThermoFishier)，复苏后涂布于固体培养基过夜培养。次日，挑取单个克隆于SB-羧苄培养基中培养，加入IPTG诱导过夜表达；次日，用高压均浆仪裂解细胞，滤膜过滤后用镍柱纯化，即利用组氨酸标签与镍柱中氯化镍的亲和层析对纳米抗体进行分离纯化，得到高纯度的抗SARS-CoV-2纳米抗体，即抗体A1-A8，经氨基酸测序分析，所得纳米抗体的氨基酸序列如SEQ IDNO:1-8所示。The positive monoclonal plasmid was extracted and transformed into E. coli TOP10F' competent cells (purchased from ThermoFishier). After recovery, the cells were spread on solid medium and cultured overnight. The next day, a single clone was picked and cultured in SB-carbenzyl medium, and IPTG was added to induce overnight expression; the next day, the cells were lysed with a high-pressure homogenizer, filtered through a membrane, and purified with a nickel column. That is, using histidine tag and Nanobodies were separated and purified by affinity chromatography with nickel chloride in a nickel column, and high-purity anti-SARS-CoV-2 nanobodies, namely antibodies A1-A8, were obtained. After amino acid sequencing analysis, the amino acid sequence of the obtained nanobodies was as follows: SEQ IDNO:1-8.
实施例4、纳米抗体与SARS-CoV-2病毒S1蛋白的结合曲线Example 4. Binding curve of Nanobodies and SARS-CoV-2 virus S1 protein
将SARS-CoV-2病毒Wild Type原始株S1蛋白和B.1.1.7突变株S1蛋白(北京义翘神州生物有限公司)分别包被于96孔酶标板上，每个孔包被浓度为1ug/mL，4℃过夜反应；次日，甩出孔中的液体，用含0.05％吐温的PBST洗3次，将酶标板倒置在吸水纸上拍干；加入封闭液，37℃孵育30分钟，甩出孔中的液体，用0.05％PBST洗3次，将酶标板倒置在吸水纸上拍干；分别加入100μL不同稀释倍数的实施例3所得的纳米抗体液，37℃孵育30分钟；甩出孔中的液体，用PBST洗3次，将酶标板倒置在吸水纸上拍干；加入酶标二抗(辣根过氧化物酶标记的抗HA标签抗体，购自Roche公司)，37℃孵育30分钟；甩出孔中的液体，用PBST洗板3次，拍干；取A液和B液等体积混匀，每孔加100μL，避光显色10～15分钟，加入终止液终止反应，酶标仪上测定各孔在波长为450nm处的OD值。根据抗体浓度和对应孔中的OD值绘制纳米抗体和SARS-CoV-2病毒Wild Type原始株S1蛋白和B.1.1.7突变株S1蛋白的结合曲线(参见图1)。实验结果表明这8个纳米抗体与Wild Type原始株S1蛋白和B.1.1.7突变株S1蛋白均有较强的亲和力，说明所述纳米抗体具有一定的广谱性。The S1 protein of the original strain of SARS-CoV-2 Wild Type virus and the S1 protein of the B.1.1.7 mutant strain (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) were coated on a 96-well enzyme plate, and the coating concentration of each well was 1ug/mL, react overnight at 4°C; the next day, shake off the liquid in the well, wash 3 times with PBST containing 0.05% Tween, invert the enzyme plate on absorbent paper and pat dry; add blocking solution, and incubate at 37°C After 30 minutes, shake off the liquid in the wells, wash 3 times with 0.05% PBST, invert the enzyme plate on absorbent paper and pat dry; add 100 μL of the Nanobody solution obtained in Example 3 with different dilution ratios, and incubate at 37°C for 30 minutes; shake off the liquid in the well, wash 3 times with PBST, invert the enzyme plate on absorbent paper and pat dry; add enzyme-labeled secondary antibody (horseradish peroxidase-labeled anti-HA tag antibody, purchased from Roche Company ), incubate at 37°C for 30 minutes; shake off the liquid in the well, wash the plate three times with PBST, and pat dry; take equal volumes of solution A and solution B and mix well, add 100 μL to each well, and develop color in the dark for 10 to 15 minutes. Stop the reaction by adding stop solution, and measure the OD value of each well at a wavelength of 450 nm on a microplate reader. Draw the binding curves of Nanobodies and SARS-CoV-2 virus Wild Type original strain S1 protein and B.1.1.7 mutant strain S1 protein based on the antibody concentration and the OD value in the corresponding well (see Figure 1). Experimental results show that these eight nanobodies have strong affinity to the S1 protein of the Wild Type original strain and the S1 protein of the B.1.1.7 mutant strain, indicating that the nanobodies have a certain broad spectrum.
实施例5、纳米抗体与SARS-CoV-2病毒S1蛋白的亲和力曲线Example 5. Affinity curve of Nanobodies and SARS-CoV-2 virus S1 protein
亲和力检测使用的是亲和素探针，利用Octec red 96仪器进行检测，所述亲和力检测方法是本领域常规技术操作，具体操作如下。向黑色无结合力的96孔板第一列的8个孔中加入0.02％吐温-20的PBST；再向第二列8个孔中加入浓度为15ug/ml生物素标记的SARS-CoV-2病毒Wild Type原始株S1蛋白和B.1.1.7突变株S1蛋白。第三、五、七、九，十一列中加入PBST,第四、六、八，十列中加入倍比稀释的本发明所述的纳米抗体，其中每列的第8个孔加入PBST，第十二列中加入甘氨酸2.0，所述上述液体均200ul每孔。大致程序如下：The affinity detection uses an avidin probe, and the Octec red 96 instrument is used for detection. The affinity detection method is a routine technical operation in this field, and the specific operation is as follows. Add 0.02% Tween-20 in PBST to the 8 wells in the first column of a black non-binding 96-well plate; then add biotin-labeled SARS-CoV-1 at a concentration of 15ug/ml to the 8 wells in the second column. 2 Virus Wild Type original strain S1 protein and B.1.1.7 mutant strain S1 protein. Add PBST to the third, fifth, seventh, ninth, and eleventh columns, add doubly diluted Nanobodies of the present invention to the fourth, sixth, eighth, and tenth columns, wherein PBST is added to the 8th well of each column. Add glycine 2.0 to the twelfth column, and the above liquid is 200ul per well. The general procedure is as follows:
1)先将8根亲和素探针(streptavidin-sensor，购自FORTEBIO，货号：18-5019)浸入到第一列PBST中进行平衡60s；1) First, immerse 8 avidin probes (streptavidin-sensor, purchased from FORTEBIO, product number: 18-5019) into the first column of PBST for 60 seconds;
2)再将亲和素探针浸入到SARS-CoV-2S蛋白稀释液中结合3min；2) Dip the avidin probe into the SARS-CoV-2S protein diluent and bind it for 3 minutes;
3)再回到第一，三列PBST中进行两次平衡；3) Go back to the first step and balance twice among the three columns of PBST;
4)平衡后的探针浸入到第四列纳米抗体稀释液中，进行抗原抗体的特异性结合，结合3min；4) The equilibrated probe is immersed in the fourth column of Nanobody diluent to perform specific binding of antigen and antibody for 3 minutes;
5)再回到第三列PBST中进行解离，解离10min。5) Return to the third column of PBST for dissociation and dissociate for 10 minutes.
6)解离后探针在第十二列甘氨酸2.0中再生5s，将结合的纳米抗体完全洗脱下来；6) After dissociation, the probe is regenerated in column 12 glycine 2.0 for 5 seconds to completely elute the bound Nanobody;
7)再回到第十一列PBST中进行中和5s；7) Go back to the eleventh column of PBST and perform neutralization for 5 seconds;
8)重复6)7)两步；8) Repeat steps 6)7);
9)再将探针浸入第五列PBST中进行平衡；重复4)5)6)7)8)步骤依次检测其他纳米抗体与SARS-CoV-2S蛋白结合能力；9) Then immerse the probe in the fifth column of PBST for equilibrium; repeat steps 4) 5) 6) 7) 8) to detect the binding ability of other nanobodies to the SARS-CoV-2S protein;
10)最后将实验数据导入到excel表中；10) Finally, import the experimental data into the excel table;
结果参见图2和表1，结果显示八个纳米抗体对SARS-CoV-2病毒Wild Type原始株S1蛋白亲和力范围为：1.0-8.2nM；对B.1.1.7突变株株S1蛋白亲和力范围为：0.42-6.5nM。The results are shown in Figure 2 and Table 1. The results show that the affinity range of the eight nanobodies for the S1 protein of the original strain of SARS-CoV-2 Wild Type virus is: 1.0-8.2nM; the affinity range for the S1 protein of the B.1.1.7 mutant strain is :0.42-6.5nM.
表1纳米抗体与SARS-CoV-2病毒Wild Type原始株和B.1.1.7突变株S1蛋白的亲和力常数KD(M)Table 1 Affinity constant KD (M) of nanobodies to the S1 protein of SARS-CoV-2 virus Wild Type original strain and B.1.1.7 mutant strain
实施例6、纳米抗体对SARS-CoV-2假病毒感的中和能力检测Example 6. Testing of Neutralization Ability of Nanobodies against SARS-CoV-2 Pseudovirus Infection
首先用DMEM培养基将本发明所述的8种纳米抗体倍比稀释10个浓度梯度，每个浓度的终体积为50ul，其中，第10个梯度中只含有DMEM培养基且纳米抗体浓度为0，并将其作为对照组，再将3ul能够产生约1x105RLUs(相关荧光素酶活性)的SARS-CoV-2 Wild type原始株假病毒(中国科学院上海巴斯德研究所王海坤研究员惠赠，或者也可购自北京云菱生物技术有限公司)加入纳米抗体稀释液中，混匀后37度孵育60min，再将50ul含有10000个HEK293T-hACE2细胞(中国科学院上海巴斯德研究所王海坤研究员惠赠，或者也可购自南京诺唯赞生物科技股份有限公司)加入到病毒-抗体复合物中，充分混匀再加入到96孔细胞培养板中，每个浓度的抗体设置3个重复孔。将细胞培养板放置于37℃培养箱中，培养48h后，将细胞上清弃去，每孔加入100ul Bright-Glo(Promega)，反应2min，转移到白色96孔板中使用Varioskan Flash多功能读数仪检测萤火虫荧光素酶活性值(SARS-CoV-2病毒B.1.1.7突变株假病毒中和实验与原始株假病毒中和实验步骤一致，仅加入的假病毒不同。假病毒和HEK293T-hACE2细胞均由上海巴斯德所王海坤研究员惠赠，或者也可购自北京云菱生物技术有限公司或南京诺唯赞生物科技股份有限公司)。以加入抗体后SARS-CoV-2S假病毒对HEK293T-hACE2细胞感染率和纳米抗体浓度为横纵坐标绘制中和抑制曲线，最后根据曲线计算EC50值。感染率＝(实验孔RLUs值-本底值)/(对照孔RLUs值-本底值)x100％，本底值为只加100ul Bright-Glo读取的值。结果参见图3，实验结果表明八个纳米抗体都能特异性中和SARS-CoV-2S蛋白假病毒。对Wild Type原始株假病毒的中和EC50范围为：47.1-266.4nM,对B.1.1.7突变株假病毒的中和EC50范围为：32.4-125.7nM。First, 8 kinds of Nanobodies according to the invention are diluted into 10 concentration gradients with DMEM culture medium. The final volume of each concentration is 50ul. Among them, the 10th gradient only contains DMEM culture medium and the Nanobody concentration is 0. , and used it as a control group, and then added 3ul of SARS-CoV-2 Wild type original strain pseudovirus capable of producing about 1x105 RLUs (related luciferase activity) (a gift from researcher Wang Haikun of Shanghai Pasteur Institute, Chinese Academy of Sciences, or It can also be purchased from Beijing Yunling Biotechnology Co., Ltd.) and added to the nanobody diluent, mix and incubate at 37 degrees for 60 minutes, and then add 50ul containing 10,000 HEK293T-hACE2 cells (a gift from researcher Wang Haikun of Shanghai Pasteur Institute, Chinese Academy of Sciences, Alternatively, it can be purchased from Nanjing Novozan Biotechnology Co., Ltd.) and added to the virus-antibody complex, mix thoroughly and then add it to a 96-well cell culture plate. Set up 3 replicate wells for each concentration of antibody. Place the cell culture plate in a 37°C incubator. After 48 hours of culture, discard the cell supernatant, add 100ul Bright-Glo (Promega) to each well, react for 2 minutes, transfer to a white 96-well plate and use Varioskan Flash multi-function reading The instrument detects the firefly luciferase activity value (SARS-CoV-2 virus B.1.1.7 mutant strain pseudovirus neutralization experiment is consistent with the original strain pseudovirus neutralization experiment steps, only the added pseudovirus is different. Pseudovirus and HEK293T- hACE2 cells were donated by researcher Wang Haikun of Shanghai Pasteur Institute, or they can be purchased from Beijing Yunling Biotechnology Co., Ltd. or Nanjing Novozan Biotechnology Co., Ltd.). The neutralization inhibition curve was drawn using the infection rate of HEK293T-hACE2 cells by the SARS-CoV-2S pseudovirus after adding the antibody and the concentration of the nanobody as the horizontal and vertical coordinates. Finally, the EC50 value was calculated based on the curve. Infection rate = (RLUs value of experimental wells - background value) / (RLUs value of control wells - background value) x 100%. The background value is the value read by adding only 100ul Bright-Glo. The results are shown in Figure 3. The experimental results show that eight nanobodies can specifically neutralize the SARS-CoV-2S protein pseudovirus. The neutralization EC50 range for Wild Type original strain pseudovirus is: 47.1-266.4nM, and the neutralization EC50 range for B.1.1.7 mutant pseudovirus is: 32.4-125.7nM.
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be used Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent substitutions, improvements, etc. shall be included in the protection scope of the present invention.
序列表sequence list
A1抗体(SEQ ID NO:1)A1 antibody (SEQ ID NO:1)
QVQLVESGGGSVQAGGSLRLSCSVSEYSSKKCMGWFRQAPGKEREGVAGIFTVGGGTYVADSVKGRFTISQDNSKNTVYLQMNSLKPEDTAKYYCAASGWEQLCTWDARDYNFWGQGTQVTVSSQVQLVESGGGSVQAGGSLRLSCSVSEYSSKKCMGWFRQAPGKEREGVAGIFTVGGGTYVADSVKGRFTISQDNSKNTVYLQMNSLKPEDTAKYYCAASGWEQLCTWDARDYNFWGQGTQVTVSS
A2抗体(SEQ ID NO:2)A2 antibody (SEQ ID NO:2)
QVQLVESGGGSVQAGGSLRLSCSVSAYSSKKCMGWFRWAPGKDRDGVAGIATVGTGTYVADSSKGRFTISQDNSKFTVYLQNNSLKPEQTAKYYCADSWWEQLCTWDAKDYLAWGQGTQVTVSSQVQLVESGGGSVQAGGSLRLSCSVSAYSSKKCMGWFRWAPGKDRDGVAGIATVGTGTYVADSSKGRFTISQDNSKFTVYLQNNSLKPEQTAKYYCADSWWEQLCTWDAKDYLAWGQGTQVTVSS
A3抗体(SEQ ID NO:3)A3 antibody (SEQ ID NO:3)
QVQLVESGGGSVQAGGSLRLSCAVSEYSSKKCVGWFRQAPGSEREAVAGIFTVGGGTYVADPVKGRFTISQDNTKNTVYLQMNSLKPEDTAMYYCAAAGWGQVCTFRAPDYNFWGQGTQVTVSSQVQLVESGGGSVQAGGSLRLSCAVSEYSSKKCVGWFRQAPGSEREAVAGIFTVGGGTYVADPVKGRFTISQDNTKNTVYLQMNSLKPEDTAMYYCAAAGWGQVCTFRAPDYNFWGQGTQVTVSS
A4抗体(SEQ ID NO:4)A4 antibody (SEQ ID NO:4)
QVQLVESGGGSVQAGGSLRLSCAVSEYSSKKCMGWFRQAPGKEREGVAGIFTVGGGTYVADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAASGWGQTCTWDARDYNFWGQGTQVTVSSQVQLVESGGGSVQAGGSLRLSCAVSEYSSKKCMGWFRQAPGKEREGVAGIFTVGGGTYVADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAASGWGQTCTWDARDYNFWGQGTQVTVSS
A5抗体(SEQ ID NO:5)A5 antibody (SEQ ID NO:5)
QVQLVESGGGFVQAGGSLRLSCNVSEYASKKCMGWFKQAPDKEREGVAGIFTVGTGTYVADQVKGRFLISQDNAKNTVYLQMMSLKPEDTAMYYCAAAGWGLTCQWRAWDYNFWGQGTQVIVSSQVQLVESGGGFVQAGGSLRLSCNVSEYASKKCMGWFKQAPDKEREGVAGIFTVGTGTYVADQVKGRFLISQDNAKNTVYLQMMSLKPEDTAMYYCAAAGWGLTCQWRAWDYNFWGQGTQVIVSS
A6抗体(SEQ ID NO:6)A6 antibody (SEQ ID NO:6)
QVQLVESGGGFVQAGGSLRLWCAVSEYASKKCMGWFRQAPGKERYGVAGIFYVGTGTYVADQVKGRFTISDDNAKNHLQMMSLKPVDTAMYYCYAAGWGQTCQWHARDYLFWGQGTQVTVLSQVQLVESGGGFVQAGGSLRLWCAVSEYASKKCMGWFRQAPGKERYGVAGIFYVGTGTYVADQVKGRFTISDDNAKNHLQMMSLKPVDTAMYYCYAAGWGQTCQWHARDYLFWGQGTQVTVLS
A7抗体(SEQ ID NO:7)A7 antibody (SEQ ID NO:7)
QVQLVESEGGSVQAGGSLRLSCAVSAYSSKKCIGWFRQAHGKEREGVAGIFTVGGGTYVADSVKGRFTISQDNAKVHLQMNNLKPEDTAVYYCAASGWGQLCTFHARDYNFWGQGTQVTVSAQVQLVESEGGSVQAGGSLRLSCAVSAYSSKKCIGWFRQAHGKEREGVAGIFTVGGGTYVADSVKGRFTISQDNAKVHLQMNNLKPEDTAVYYCAASGWGQLCTFHARDYNFWGQGTQVTVSA
A8抗体(SEQ ID NO:8)A8 antibody (SEQ ID NO:8)
QVQLVESEGGSVQAGGSLRLSCAVSAWSSDKCIGQFRQAHGKEVEGVAGIFTVGAGTYVADSVDGRFTESQDNAKVHLQNNNLKPEQTAVYYCAADGWGLLCTWHARDLNVWGQGTTVTVSAQVQLVESEGGSVQAGGSLRLSCAVSAWSSDKCIGQFRQAHGKEVEGVAGIFTVGAGTYVADSVDGRFTESQDNAKVHLQNNNLKPEQTAVYYCAADGWGLLCTWHARDLNVWGQGTTVTVSA
CDR1CDR1
SEQ ID NO:9:EYSSKKCSEQ ID NO:9:EYSSKKC
SEQ ID NO:10:AYSSKKCSEQ ID NO:10:AYSSKKC
SEQ ID NO:11:AYSSKKCSEQ ID NO:11:AYSSKKC
SEQ ID NO:12:EYASKKCSEQ ID NO:12:EYASKKC
SEQ ID NO:13:AWSSDKCSEQ ID NO:13:AWSSDKC
CDR2CDR2
SEQ ID NO:14:IFTVGGGTSEQ ID NO:14:IFTVGGGT
SEQ ID NO:15:IATVGTGTSEQ ID NO:15:IATVGTGT
SEQ ID NO:16:IFTVGTGTSEQ ID NO:16:IFTVGTGT
SEQ ID NO:17:IFYVGTGTSEQ ID NO:17:IFYVGTGT
SEQ ID NO:18:IFTVGAGTSEQ ID NO:18:IFTVGAGT
CDR3CDR3
SEQ ID NO:19:AASGWEQLCTWDARDYNFSEQ ID NO:19:AASGWEQLCTWARDYNF
SEQ ID NO:20:AASGWGQTCTWDARDYNFSEQ ID NO:20:AASGWGQTCTWARDYNF
SEQ ID NO:21:AASGWGQLCTFHARDYNFSEQ ID NO:21:AASGWGQLCTFHARDYNF
SEQ ID NO:22:ADSWWEQLCTWDAKDYLASEQ ID NO:22:ADSWWEQLCTWDAKDYLA
SEQ ID NO:23:AAAGWGQVCTFRAPDYNFSEQ ID NO:23:AAAGWGQVCTFRAPDYNF
SEQ ID NO:24:AAAGWGLTCQWRAWDYNFSEQ ID NO:24:AAAGWGLTCQWRAWDYNF
SEQ ID NO:25:YAAGWGQTCQWHARDYLFSEQ ID NO:25:YAAGWGQTCQWHARDYLF
SEQ ID NO:26:AADGWGLLCTWHARDLNVSEQ ID NO:26:AADGGWGLLCTWHARDLNV
GSP-RT：CGCCATCAATRTACCAGTTGA(SEQ ID NO:27)GSP-RT:CGCCATCAATRTACCAGTTGA(SEQ ID NO:27)
LP-leader：GTGGTCCTGGCTGCTCTW(SEQ ID NO:28)LP-leader：GTGGTCCTGGCTGCTCTW(SEQ ID NO:28)
R：CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG(SEQ ID NO:29)R: CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG (SEQ ID NO: 29)
F：CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC(SEQ ID NO:30)F: CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO:30)
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| CN112094342A (en)* | 2020-09-25 | 2020-12-18 | 中国科学技术大学 | Alpaca source nano antibody combined with SARS-CoV-2RBD |
| CN112094343A (en)* | 2020-09-25 | 2020-12-18 | 中国科学技术大学 | Alpaca source nano antibody combined with SARS-CoV-2RBD |
| Title |
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| Development of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity and avidity;Jianbo Dong et al;Emerging Micorbes ＆ Infections;第9卷(第1期);1-3* |
| SARS-CoV-2 nanobodies 2.0;Fabien Labroussaa et al;Signal Transduction and Targeted Therapy;第6卷(第202期);1-3* |
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| CN114539395A (en) | 2022-05-27 |
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