Llama-derived nanobody s140 and applications thereof

Abstract

The present application relates to a llama-derived nanobody and its application, in particular to a llama-derived nanobody S140 or an antigen-binding fragment thereof capable of binding to SARS-CoV-2 S2 and its application, wherein the antibody comprises a heavy chain variable region, and the heavy chain variable region comprises the following CDRs: a CDR1 with an amino acid sequence as shown in SEQ ID NO:1, a CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:3. The nanobody S140 of the present application can effectively inhibit the infection of SARS-CoV-2 original strain and its variant strains, and has great potential for clinical application.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an alpaca-derived nanobody and its application, and more specifically, to an alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 S2, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a method for its preparation, transformed cells, and a pharmaceutical composition containing the above, and their application in the preparation of drugs for the prevention or treatment of COVID-19. Background Technology

[0002] Since the end of 2019, the epidemic caused by the novel coronavirus (also known as SARS-CoV-2 or COVID-19) belonging to the family Coronaviridae has continued to spread globally. In addition, other coronaviruses belonging to the same family, such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), are also major pathogens affecting the human respiratory system. They are mainly transmitted through droplets, aerosols, and contact, and are highly contagious, easily causing public panic. Therefore, these viruses that cause respiratory diseases seriously endanger public health and safety. Especially in recent years, the frequent occurrence of respiratory infectious diseases and the continuous mutation of viruses have posed a great threat to the health and safety of the people, national economic development, and social stability.

[0003] Neutralizing antibody drugs mainly work by binding to antigens on the surface of pathogenic microorganisms, preventing specific molecules expressed by the pathogenic microorganisms from binding to cell surface receptors, thus achieving a "neutralizing" effect.

[0004] Both SARS-CoV and SARS-CoV-2 viruses possess a glycosylated spike protein (S) on their surface. This S protein interacts with the host cell receptor protein ACE2 and triggers membrane fusion. Therefore, blocking the binding of the S protein to ACE2 is an effective approach to treating SARS-CoV-2 infection. The S protein consists of two subunits, S1 and S2. The S2 subunit is involved in the fusion of the viral membrane and cell membrane. Antibodies targeting the S2 subunit can exert antiviral effects through a neutralization mechanism. Especially against RNA viruses like SARS-CoV-2, which are prone to mutation and immune escape, single-specific antibodies are insufficient to meet long-term therapeutic needs. Therefore, the isolation and identification of neutralizing antibodies targeting different epitopes is urgently needed.

[0005] Nanobodies, also known as single-domain antibodies (VHH), have some unique advantages compared to traditional mAbs (~150 kDa): small molecular weight (~15 kDa), low immunogenicity, better solubility and stability, and longer CDR3 regions. With these properties, nanobodies can be used as single domains or modular units to build more complex molecules, such as multivalent antibodies against different antigens to expand the spectrum. Importantly, nanobodies can be easily aerosolized and delivered directly to the lungs by inhalers, making them potential drugs for treating respiratory diseases. Therefore, isolating and identifying nanobodies with cross-reactivity is to provide potential drug reserves for the current COVID-19 pandemic and possible future coronavirus infections. SUMMARY

[0006] Invention objectives

[0007] The present application aims to provide a llama-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 S2, a polynucleotide encoding the same, a nucleic acid construct comprising the same, an expression vector comprising the same, a method for preparing the same, a transformed cell, and a pharmaceutical composition comprising the same, as well as their use in the preparation of a drug for preventing or treating COVID-19. The llama-derived nanobody or antigen-binding fragment thereof of the present application is a nanobody with high neutralizing activity and strong binding ability to SARS-CoV-2 S2 protein, which can effectively inhibit the infection of SARS-CoV-2 original strain and a series of variant strains. The nanobody has the advantages of small molecular weight (~15 kDa), low immunogenicity, better solubility and stability, and longer CDR3 region, can be aerosolized for administration, can directly reach the lungs, and has a faster onset, providing a potential treatment strategy for COVID-19 or other coronavirus infections.

[0008] Solution

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] In a first aspect, the present application provides a llama-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 S2, wherein the antibody comprises a heavy chain variable region comprising the following CDRs:

[0011] a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 (i.e., GFTFSMYA),

[0012] a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2 (i.e., IGASDTVY),

[0013] and CDR3 is shown as amino acid sequence of SEQ ID NO: 3 (i.e. RRVQFVRSEY).

[0014] In a particular embodiment, the heavy chain variable region further comprises four framework regions FR1-4, which are staggered in order with the CDR1, CDR2 and CDR3.

[0015] In a preferred embodiment, the amino acid sequences of the FR1-4 are shown as SEQ ID NO: 4 (i.e. QVQLQESGGGLVQPGGSLRLSCGAS), SEQ ID NO: 5 (i.e. MSWYRQAPGKERELVAA), SEQ ID NO: 6 (i.e. TDSVKGRFTISRDNAKNMAYLQMNSLKPEDTAVYYCHA) and SEQ ID NO: 7 (i.e. WGLGTQVTVSS), respectively.

[0016] In a preferred embodiment, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 8:

[0017] wherein the underlined parts are framework regions FR1-4, and the blacked parts are CDR1, CDR2 and CDR3 of the heavy chain variable region, respectively.

[0018] In a second aspect, the present application provides a polynucleotide encoding the llama-derived nanobody or antigen-binding fragment thereof according to the first aspect described above.

[0019] Further, the polynucleotide is DNA or mRNA.

[0020] Further, the polynucleotide has a nucleotide sequence shown as SEQ ID NO: 9:

[0021] CAGGTGCAGCTGCAGGAGAGCGGAGGAGGGCTGGTGCAGCCCGGAGGAAGCCTGAGACTGAGCTGCGGCGCCTCAGGATTCACCTTTAGCATGTATGCCATGAGCTGGTACAGGCAGGCCCCTGGCAAAGAAAGAGAGCTGGTGGCAGCCATTGGAGCCAGCGACACAGTGTACACCGACTCCGTGAAAGGCAGGTTCACAATCAGCAGAGACAACGCCAAGAACATGGCATACCTGCAAATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCCACGCCAGAAGAGTGCAGTTCGTGAGAAGCGAGTACTGGGGACTGGGCACACAAGTGACAGTGTCCAGC.

[0022] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide according to the second aspect as described above.

[0023] Further preferably, the nucleic acid construct further comprises at least one expression regulatory element operably linked to the polynucleotide. For example, a histidine tag, a stop codon, etc.

[0024] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid construct according to the third aspect as described above.

[0025] In a fifth aspect, the present application provides a transformed cell comprising the polynucleotide according to the second aspect as described above, the nucleic acid construct according to the third aspect as described above, or the expression vector according to the fourth aspect as described above.

[0026] In a sixth aspect, the present application provides a pharmaceutical composition comprising the llama-derived nanobody or antigen-binding fragment thereof binding to SARS-CoV-2 RBD according to the first aspect as described above, the polynucleotide according to the second aspect as described above, the nucleic acid construct according to the third aspect as described above, the expression vector according to the fourth aspect as described above, or the transformed cell according to the fifth aspect as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0027] Further preferably, the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation.

[0028] Further preferably, the nasal spray is selected from the group consisting of an aerosol, a spray, and a powder spray.

[0029] Further preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, granules, granulates, soft / hard capsules, film-coated tablets, pellets, sublingual tablets and pastes.

[0030] Further preferably, the parenteral formulation is a transdermal formulation, an ointment, a plaster, a liquid for external use, an injectable or a bolus formulation.

[0031] In a seventh aspect, the present application provides use of a llama-derived nanobody or an antigen-binding fragment thereof binding to SARS-CoV-2 S2 as defined in the first aspect above, a polynucleotide as defined in the second aspect above, a nucleic acid construct as defined in the third aspect above, an expression vector as defined in the fourth aspect above, or a transformed cell as defined in the fifth aspect above, or a pharmaceutical composition as defined in the sixth aspect above, in the preparation of a medicament for preventing, treating or detecting a coronavirus infection.

[0032] Preferably, the coronavirus is a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

[0033] Further preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and / or Delta (B.1.617.2) variant strain of SARS-CoV-2.

[0034] In an eighth aspect, the present application provides a method for preventing or treating a coronavirus, comprising: administering to a subject in need thereof a prophylactically or therapeutically effective amount of a llama-derived nanobody or an antigen-binding fragment thereof binding to SARS-CoV-2 S2 as defined in the first aspect above, a polynucleotide as defined in the second aspect above, a nucleic acid construct as defined in the third aspect above, an expression vector as defined in the fourth aspect above, or a transformed cell as defined in the fifth aspect above, or a pharmaceutical composition as defined in the sixth aspect above.

[0035] In a ninth aspect, the present application provides a method for detecting a coronavirus, comprising using a llama-derived nanobody or an antigen-binding fragment thereof binding to SARS-CoV-2 S2 as defined in the first aspect above.

[0036] Preferably, the coronavirus is a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

[0037] Further preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and / or Delta (B.1.617.2) variant strain of SARS-CoV-2.

[0038] The amount of the effective component of the pharmaceutical composition of the present application to be administered varies depending on the subject to be administered, the organ of the subject, the symptoms, the method of administration, etc., and can be determined according to the kind of the dosage form, the method of administration, the age and body weight of the patient, the symptoms of the patient, etc., based on the judgment of a medical doctor.

[0039] Beneficial effects

[0040] The present application is directed to the development of nanobody drugs against SARS-CoV-2 by immunizing alpacas with SARS-CoV-2 S protein, constructing antibody library, screening specific nanobodies using phage display technology, etc. Nanobodies that bind to SARS-CoV-2 S2 were screened, which are designated as nanobody S140 in this paper. It was confirmed by surface plasmon resonance technology that the nanobody S140 of the present application specifically binds to SARS-CoV-2 S2 with high affinity. In addition, it was confirmed by antibody neutralization test that the nanobody S140 of the present application can neutralize SARS-CoV-2 original strain and its series of variant strains with high neutralization activity.

[0041] The present application provides potential nanobody new drugs for the clinical prevention, treatment and detection of SARS-CoV-2 original strain and its variant strains. BRIEF DESCRIPTION OF DRAWINGS

[0042] One or more embodiments are illustrated by way of example in the figures that constitute a part of this patent document. This exemplary illustration is not intended to limit the embodiments in any way. The word "exemplary," as used herein means "serving as an example, instance, or illustration.” Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0043] Figure 1 is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 S-his protein described in Example 1 of the present application;

[0044] Figure 2 is a schematic diagram of the molecular sieve chromatography and Western-blot identification results of the SARS-CoV-2 S2-his protein described in Example 1 of the present application;

[0045] Figure 3is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the nanobody S140 described in Example 4 of the present application;

[0046] Figure 4 is a graph of the affinity identification results of the nanobody S140 and SARS-CoV-2 S2 protein described in Example 5 of the present application;

[0047] Figure 5 is a schematic diagram of the effect of the nanobody S140 in neutralizing SARS-CoV-2 pseudovirus infection determined in Example 7 of the present application. DETAILED DESCRIPTION

[0048] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application. Unless otherwise explicitly indicated, in the entire specification and claims, the term “comprise” or its variants such as “contain” or “include” etc. will be understood as including the stated elements or components, and not excluding other elements or components.

[0049] In addition, in order to better illustrate the present application, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means, etc. which are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present application.

[0050] The present application will be described in detail below.

[0051] Definitions

[0052] “Nanobody”, i.e. “heavy-chain single-domain antibody”, this kind of antibody only contains one heavy chain variable region (VHH, variable domain of heavy chain of heavy-chain antibody), compared with other antibodies, the light chain is naturally missing.

[0053] Due to the biophysical advantages of nanobodies themselves, they can be easily atomized and directly delivered to the lungs through inhalers, thereby treating infections caused by respiratory viruses, and are considered to be very potential antibody drugs.

[0054] When referring to ligand / receptor, antibody / antigen or other binding pairs, "specific" binding refers to the determination of the presence or absence of a binding reaction between a protein, e.g., a Nanobody of the present application, and a SARS-CoV-2 RBD protein in a heterogeneous population of proteins and / or other biological agents. Thus, under designated conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0055] The chemical materials used in the following examples of the present application, such as reagents, enzymes, culture media, antibiotics and milk, are commercially available, for example, TRIzol is purchased from Invitrogen, Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.

[0056] Some commonly used biological materials, such as competent cells, vectors, helper phages, cells to be transformed, etc., are also commercially available, for example, pCAGGS vector is purchased from MiaoLing Plasmid, 293F cells, HEK293T cells, etc. are purchased from ATCC; electrocompetent E. coli TG1 cells are purchased from Lucigen, VCSM13 helper phage is purchased from StrataGene, plasmid pMES4 is purchased from Addgene; protein A chip is purchased from GE Healthcare; Vero cells are purchased from ATCC CCL81.

[0057] Some synthetic biological materials, such as primers, sequences, etc., which are materials that need to be synthesized artificially, are all entrusted to a synthesis company to complete, for example, the primers (SED ID NO: 14-19) in the present application are synthesized by Beijing Chengke Biological Technology Co., Ltd.

[0058] Example 1: Expression and purification of SARS-CoV-2 S-his, SARS-CoV-2 S2-his proteins

[0059] A signal peptide (as shown in SEQ ID NO: 11) is connected to the 5' end of the SARS-CoV-2 S protein coding sequence (as shown in SEQ ID NO: 10), and a coding sequence of a hexa-His-tag and a translation termination codon (TGA) are connected to the 3' end of the signal peptide. Through restriction enzyme sites EcoRI and XhoI, it is constructed into a pCAGGS vector, transfected into 293F cells, and the expression of SARS-CoV-2 S-his protein is carried out. The cell culture fluid containing the target protein is subjected to nickel ion affinity chromatography (HisTrap TM excel(GE)) and gel filtration chromatography (Superose TM6Increase 10 / 300GL(GE)) purification, the relatively pure target protein SARS-CoV-2 S-his can be obtained. The SDS-PAGE identification size of SARS-CoV-2 S-his protein is about 200KD, and the results are shown in Figure 1

[0060] Similarly, the coding sequence of 6 histidine tag (hexa-His-tag) and the translation termination codon (TGA) at the 3' end of the SARS-CoV-2 S2 protein coding sequence (as shown in SEQ ID NO: 12) is constructed into the pFastBacl vector (the sequence is shown in SEQ ID NO: 13) through the restriction enzyme sites EcoRI and Xhol. The exogenous gene constructed on the pFastBac plasmid is transposed into the bacmid in E. coli DH10Bac by using site-specific Tn7 transposon to generate recombinant bacmid. The bacmid is transfected into sf9 cells to generate recombinant baculovirus that can express the target gene. The virus titer is confirmed by plaque method, and the virus with confirmed titer is used to infect sf9 cells to amplify the virus. The P3 virus can be directly used for protein expression. The virus is used to infect Hi5 cells to express SARS-CoV-2 S2-his protein. After the cell culture solution containing the target protein is purified by nickel ion affinity chromatography (HisTrap TM excel((GE Healthcare)) and gel filtration chromatography (SuperdexTM200Increase 10 / 300GL column(GE Healthcare)), the relatively pure target protein can be obtained. The Western-blot identification size of SARS-CoV-2 S2-his protein is about 60KD, and the results are shown in Figure 2 .

[0061] Example 2: Llama immunization and antibody library construction

[0062] ​The SARS-CoV-2 S protein with 6 histidine tags prepared in Example 1, 200 μg, was diluted with PBS to a final volume of 1 mL, 1 ml of complete Freund's adjuvant was emulsified for 5 min, and immunization was performed by subcutaneous multiple point injection. After that, immunization was performed once every two weeks, and MF59 water-soluble adjuvant was used to emulsify the S protein. On the 12th day after the fifth immunization, 50-60 mL of blood was collected, and PBMCs (peripheral blood mononuclear cells) were separated. The separated PBMCs were added to 1 mL of TRIzol, and total RNA was extracted according to the procedure of the instruction manual. Using the extracted total RNA as a template, the Superscript II First-Strand Synthesis System for RT-PCR kit was used to synthesize cDNA with random primer oligo-dT 12-18 Primers were synthesized to synthesize cDNA. Using specific primers CALL001 and CALL002 (primer sequences are shown in Table 1), PCR was performed using cDNA as a template, and a 700 bp band was cut and recovered. The purified DNA was used as a template, and nest PCR was performed using nest primers VHH-BACK and PMCF to amplify the nanobody (VHH) sequence, and the VHH sequence with a size of about 400 bp was recovered and purified.

[0063] Using double enzyme digestion, the VHH fragment was ligated into the plasmid pMES4 through the restriction enzyme sites Pst I and BstE II. The purified cloning vector and electrocompetent E. coli TG1 cells were mixed, and the cloning vector was transformed into the electrocompetent E. coli TG1 cells using an electroporator (BIO-RAD electroporator MicroPulser). All were plated on selective medium containing ampicillin, and after overnight culture at 37°C, all colonies were collected in LB medium, centrifuged and the supernatant was discarded, and the cells were resuspended in LB, which was an antibody library.

[0064] Table 1. Reaction primers

[0065]

[0066] Example 3: Screening of specific nanobodies by phage display technology

[0067] The E. coli TG1 transfected with the recombinant plasmid of Example 2 was taken, and VCSM13 helper phage was added at a multiplicity of infection (MOI) of about 20. After overnight culture, centrifugation was performed at 4000 rpm, and the supernatant was taken and filtered through a 0.22 μm membrane. PEG6000 / NaCl was added at a volume ratio of 1:4, and after mixing, the mixture was placed at 4°C for at least 1 hour. Centrifugation was performed at 8000 x g for 30 min, the supernatant was discarded, and the precipitate was resuspended with PBS. The collected phage particles were obtained, and the phage titer was determined.

[0068] 2 x 10 11 The collected phage was mixed with an equal volume of 5% (w / v) skimmed milk, and added to a 96-well plate coated with SARS-CoV-2 S-his antigen. After incubation at room temperature for 1 h, specific phage was eluted with 0.2 M glycine, and the eluted phage was neutralized with Tris-HCl (pH 9.1). Then, the phage was used to infect E. coli TG1 cells, and the phage was amplified. A 96-well plate coated with SARS-CoV-2 S-his antigen was prepared again, and the second round of panning was performed to enrich phage expressing specific nanobodies. A total of 3 rounds of panning were performed. After each round of panning, different single colonies were randomly picked from the agar plate with colonies, cultured in a 37°C shaker, and then VCSM13 helper phage was added for overnight expansion. The next day, the culture solution was centrifuged, and the phage supernatant was taken for ELISA experiments (using SARS-CoV-2 S-his protein as the coating antigen). When the OD 450nM >0.2, it was determined to be a positive reaction. The corresponding clone was taken, and specific primers MP57 and GIII were used for sequencing of the plasmid (primer sequences are shown in Table 2). The sequence encoding VHHs in the plasmid was obtained. Through sequence determination, the core coding sequence of S140 was obtained.

[0069] Table 2. Reaction primers

[0070]

[0071] Example 4: Expression and purification of nanobody S140

[0072] To make the heavy chain variable region of S140 more complete, the coding sequence of QVQLQ (CAGGTGCAGCTGCAG) was added to the 5' end of the core coding sequence of S140 obtained in Example 3, and the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT) was added to the 3' end, to obtain a nucleotide sequence as in SEQ ID NO: 9, which is the coding sequence of the nanobody S140 of the present application, then a signal peptide (SEQ ID NO: 20) was added in front of it, and the coding sequence of a hexa-His-tag and a translation termination codon TGA were added after it, which was constructed into a pCAGGS vector through restriction enzyme cutting sites EcoRI and XhoI, and transfected into 293F cells. After 5 days of culture, the supernatant was collected, centrifuged at 5000 rpm for 30 min, filtered through a 0.22 μm filter, and then purified by nickel ion affinity chromatography (HisTrap TM excel (GE Healthcare) and gel filtration chromatography (Superdex TM 75Increase 10 / 300 GL column (GE Healthcare) to obtain relatively pure target protein. The target peak was determined by SDS-PAGE, and the results are shown in Figure 3 , to obtain purified nanobody S140.

[0073] Example 5: Detection of the binding ability of antibodies to SARS-CoV-2 S2 by surface plasmon resonance technology

[0074] Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0075] The SA chip (purchased from GE Healthcare) was selected, and the SARS-CoV-2 S2-biotin protein obtained in Example 1 was immobilized on the chip through the affinity of the SA chip and biotin. The S140 antibody protein was diluted with PBST buffer (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.05% Tween) by a factor of two, and was loaded from low concentration to high concentration one by one. The kinetic curve of the binding of nanobody S140 to SARS-CoV-2 S2 protein is shown in Figure 4 The kinetic association constant (ka), dissociation constant (kd) and equilibrium dissociation constant (K D) The calculation of these parameters was performed using the BIAevaluation software 8K (Biacore, Inc.) software as shown in Table 3. The results of Table 3 show that the Nanobody S140 is able to bind to SARS-CoV-2 S2 with a higher affinity.

[0076] Table 3 Binding constants (ka), dissociation constants (kd) and equilibrium dissociation constants (Kd) of antibodies binding to SARS-CoV-2 S2 protein D )

[0077]

[0078]

[0079] Example 6: Packaging of SARS-CoV-2 wild type pseudovirus

[0080] 1) The gene encoding the 18 amino acids after the S protein of SARS-CoV-2 wild type (WT) was removed, and the remaining sequence of the S protein was synthesized (synthesis service was provided by Suzhou Jinyuzhi), obtaining the nucleotide sequence of SARS-CoV-2-WT-S-del18 gene, the sequence of which is shown in SEQ ID NO: 21.

[0081] 2) The protein gene obtained in 1) was cloned into the pCAGGS vector to obtain the expression plasmid pCAGGS-SARS-CoV-2-WT-S-del18.

[0082] The packaging steps of SARS-CoV-2 wild type and variant pseudovirus are as follows:

[0083] a. Cell preparation: HEK293T cells were plated in a 10 cm cell culture dish, and the cell confluence density was about 80% the next day. The culture medium was DMEM medium containing 10% FBS.

[0084] b. Transfection: 30 μg of the expression plasmid of the S protein in step 2) above was transfected with PEI per 10 cm cell culture dish, and the target plasmid was mixed with PEI at a ratio of 1:3 before transfection. The culture medium (DMEM medium containing 10% FBS) was changed 4-6 h after transfection, and the cells were cultured at 37°C for 24 h.

[0085] c. Virus addition: The pseudovirus packaging backbone virus G*VSV-delG (purchased from Wuhan Pivot Biosciences Technology Co., Ltd.) was added to the HEK293T cells after transfection above, and incubated at 37°C for 2 h. The culture medium (DMEM medium containing 10% FBS) was changed, and VSV-G antibody (hybridoma cells expressing the antibody were purchased from ATCC cell bank) was added, and the cells were further cultured in the incubator for 30 h.

[0086] d. Collecting toxicity: collect the supernatant at 3000 rpm for 10 min, filter through a 0.45 μm sterile filter in a clean bench to remove cell debris, aliquot, and store in a -80°C refrigerator.

[0087] A pseudovirus of SARS-CoV-2 wild type (SARS-CoV-2 WT) was obtained.

[0088] Example 7: Detection of neutralization of SARS-CoV-2 pseudovirus infection by nanobody S140

[0089] The purified nanobody S140 obtained in Example 4 was diluted 5 times from 250 μg / mL to the 9th gradient (0.6 ng / mL), and the diluted nanobody S140 was mixed with 1.6 x 10 4 TCID 50 The pseudovirus of SARS-CoV-2 wild type obtained in Example 6 was mixed and incubated at 37°C for 1 h, and then added to a 96-well plate pre-seeded with Vero cells (purchased from ATCC CCL81). After 18-20 hours of incubation, detection was performed by CQ1 Confocal Quantitative Image Cytometer (Yokogawa). According to the number of cells with GFP fluorescence, the neutralization ability of the antibody to the above-mentioned SARS-CoV-2 wild type pseudovirus was calculated, and the results are shown in Figure 5 , and the IC50 data are not shown.

[0090] In summary, nanobody S140 can be used as a high neutralization activity llama-derived nanobody against SARS-CoV-2 wild type and its mutant strains.

[0091] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> A llama-derived nanobody S140 and its application <130> 1087-210324F <160> 21 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1 of the heavy chain variable region of the Nanobody of the application S140 <220> <221> DOMAIN <222> (1)..(8) <400> 1 Gly Phe Thr Phe Ser Met Tyr Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR2 of the heavy chain variable region of the Nanobody of the application S140 <220> <221> DOMAIN <222> (1)..(8) <400> 2 Ile Gly Ala Ser Asp Thr Val Tyr 1 5 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR3 of the heavy chain variable region of the Nanobody of the application S140 <220> <221> DOMAIN <222> (1)..(10) <400> 3 Arg Arg Val Gln Phe Val Arg Ser Glu Tyr 1 5 10 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of framework region FR1 of Nanobody S140 of the invention <220> <221> DOMAIN <222> (1)..(25) <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Gly Ala Ser 20 25 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of framework region FR2 of Nanobody S140 of the invention <220> <221> DOMAIN <222> (1)..(17) <400> 5 Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 15 Ala <210> 6 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of framework region FR3 of Nanobody S140 of the invention <220> <221> DOMAIN <222> (1)..(38) <400> 6 Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 1 5 10 15 Asn Met Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 20 25 30 Val Tyr Tyr Cys His Ala 35 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the framework region FR4 of the Nanobody of the application S140 <220> <221> DOMAIN <222> (1)..(11) <400> 7 Trp Gly Leu Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain variable region of the Nanobody of the application S140 <220> <221> DOMAIN <222> (1)..(117) <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Gly Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Ala Met Ser Trp Tyr Arg Gin Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ala lie Gly Ala Ser Asp Thr Val Tyr Thr Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Met Ala Tyr Leu Gin 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ala 85 90 95 Arg Arg Val Gin Phe Val Arg Ser Glu Tyr Trp Gly Leu Gly Thr Gin 100 105 110 Val Thr Val Ser Ser 115 <210> 9 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of Nanobody S140 of the application <220> <221> misc_feature <222> (1)..(351) <400> 9 caggtgcagc tgcaggagag cggaggaggg ctggtgcagc ccggaggaag cctgagactg 60 agctgcggcg cctcaggatt cacctttagc atgtatgcca tgagctggta caggcaggcc 120 cctggcaaag aaagagagct ggtggcagcc attggagcca gcgacacagt gtacaccgac 180 tccgtgaaag gcaggttcac aatcagcaga gacaacgcca agaacatggc atacctgcaa 240 atgaacagcc tgaagcccga ggacaccgcc gtgtactact gccacgccag aagagtgcag 300 ttcgtgagaa gcgagtactg gggactgggc acacaagtga cagtgtccag c 351 <210> 10 <211> 3603 <212> DNA <213> Artificial Sequence <220> <223> SARS-CoV-2 S protein coding sequence <220> <221> misc_feature <222> (1)..(3603) <400> 10 agccagtgcg tgaacctgac cacacggacc cagctccctc ccgcctacac aaactctttc 60 acccggggcg tgtactaccc cgacaaggtg ttccggtcta gcgtgctcca ctctacacag 120 gacctgttcc tccctttctt cagcaacgtg acatggttcc acgccatcca cgtgtctggc 180 acaaacggca caaagcggtt cgacaacccc gtgctccctt tcaacgacgg cgtgtacttc 240 gccagcaccg agaagtctaa cattatccgg ggctggattt tcggcaccac actcgactct 300 aagacacagt ccctcctgat tgtgaacaac gccacaaacg tggtgattaa ggtgtgcgag 360 ttccagttct gcaacgaccc tttcctgggc gtgtactacc acaagaacaa caagtcttgg 420 atggagtctg agttcagagt gtactctagc gccaacaact gcaccttcga gtacgtgtcc 480 cagcctttcc tcatggacct ggagggcaag cagggcaact tcaagaacct gagagagttc 540 gtgttcaaga acattgacgg ctacttcaag atttactcta agcacacccc aattaacctc 600 gtgagggacc tccctcaggg cttctccgcc ttagaaccac tggtggacct ccctattggc 660 attaacatca cacgcttcca gacactgctc gccctccacc ggtcttacct gaccccaggc 720 gactctagct ctggctggac agccggcgcc gccgcctact acgtgggcta cctgcagcct 780 aggaccttcc tcctgaagta caacgagaac ggcacaatta ccgacgccgt ggactgcgcc 840 ctggacccac tgtccgagac aaagtgcaca ctgaagtcct tcacagtgga gaagggcatt 900 taccagacat ctaacttccg ggtgcagcct acagagtcta ttgtgcggtt cccaaacatc 960 acaaacctgt gccctttcgg cgaggtgttc aacgccaccc ggttcgcctc tgtgtacgcc 1020 TGGAACC GGA AGCGGATCTC TAAC TGC TGGCCGACTACT CC GTGCTGTACAAC TCCGCC 1080 TCTTTCTCTA CATTCAAGTG CTACGGCGTG TCCCCTACAA AGCTGAACGA CCTGTGCTTC 1140 ACCAACGTGT ACGCCGACTC TTTCTGTATT AGAGGCGACG AGGTGAGGCA GATTGCCCCC 1200 GGCCAGACAG GCAAGATCGC CGACTACAAC TACAAGCTGC CCGACGACTT CACAGGCTGC 1260 GTGATCGCCT GGAAC TCTAACCAACCTGGACTCTAAGGTGGGC GGCACTA CAAC TACCTG 1320 TACAGACTGT TCCGGAAGTC TAACCTGAAG CCATTCGAGA GGGACAT TAGC ACCGAGATT 1380 TACCAGGCCG GCTCTACCCC ATGCAACGGC GTGGAGGGCT TCAACTGCTA CTTCCCAC T G 1440 CAGTCCTACG GCTTCCAGCC TACAAACGGC GTGGGCTACC AGCCTTACCG GGTGGTG GTG 1500 CTGTCTTTCG AGCTGCTCCA CGCCCCCGCC ACAGTGTGCG GCCCAAAGAA GAGCACAAAC 1560 CTCGTGAAGA ACAAGTGCGT GAAC T TCAAC T TCAACGGCCTC ACAGGCACAG GC GTGCTC 1620 ACC GAGTCTAACAAGAAGTT CCTCCCTTTCC AGCAGTTCGG CC GCA CAT T GCC GAC ACC 1680 ACC GACGCCGTGC GGGACCC T CAGAC ACTGGAAATTC TCGAC ATCACCCCTTGC AGCTTC 1740 GGCGGCGTGT CC GT GATCAC CCC AGGCA CA AAC ACATCTA ACC AGGTGGC CGTGCTGTAC 1800 CAGGACGTGA CTGCACCGAG GTCC AGTGGCC ATCCACGCCG ACC AGCTCACCCCA ACA 1860 TGGAGGGTGT ACAGCACAGG CTCTAACGTG TTCC AGACCC GGGCCGGCTG CCTCATTGGC 1920 GCCGAGCACG TGAACAAC TC TTACGAGTGC GACATCCCTA TTGGCGCCGG C ATTTGC GCC 1980 TCTTACCAGA CCC AGAC AAAC TCTCC ATCT AGCGCCTCCT CTGTGGCCTC TC AGAGCATT 2040 ATTGCCTAC AC CATGTCTCT GGGCGCCGAG AACTCTGTGG CCTACTCTAA CAAC TCTATT 2100 GCC ATCCCTA CAAAC T TCA CA TTTC TGTG ACC ACCGAGATTCTCC CAGT GTCT ATGACC 2160 AAGAC ATCTG TGGACTGC ACC ATGTAC ATTTGC GGC GACT CC ACCGAGTG CTCT AACCTC 2220 CTGCTCCAGT AC GGCTCTTT CTGC ACCC AGCTC ACCGC GCC TGAC AGG CATCGCCGTG 2280 GAGC AGGA CA AGAAC ACCC AGGAGGTGTTC GCCC AGGTG AAGC AGATTT ACA AGACCCCC 2340 CCAATTAAGG ACTTCGGCGG CTTCAACTTC TCTC AGATTCTCCCCGACCC ATCCAAGCCT 2400 AGCAAGCGGT CCTTC ATTAGGACCTCCTG TTCA ACAAGGT AC ACTGGCCGAC GCCGGC 2460 ttcattaagc agtacggcga ctgcctgggc gacattgccg cccgggacct gatttgcgcc 2520 cagaagttca acggcctcac agtgctcccc ccactgctca ccgacgagat gattgcccag 2580 tacacatctg ccctcctggc cggcacaatt acatctggct ggaccttcgg cgccggcgcc 2640 gccctgcaga tccctttcgc catgcagatg gcctaccgct tcaacggcat cggcgtgaca 2700 cagaacgtgc tgtacgagaa ccagaagctg atcgccaacc agttcaacag cgccattggc 2760 aagattcagg actctctgag cagcacagcc agcgccctgg gcaagctgca ggacgtggtg 2820 aaccagaacg cccaggccct gaacacactg gtgaagcagc tgtcttctaa cttcggcgcc 2880 atttctagcg tgctgaacga cattctgtcg cggctggacc ctccagaggc cgaggtgcag 2940 attgacaggc tcatcacagg cagactgcag tctctgcaga catacgtgac ccagcagctg 3000 attagagccg ccgagattag agcctccgcc aacctggccg ccaccaagat gagcgagtgc 3060 gtgctcggcc agtctaagcg ggtggacttc tgcggcaagg gctaccacct catgtctttc 3120 cctcagtccg cccctcacgg cgtggtgttc ctccacgtga catacgtgcc cgcccaggag 3180 aagaacttca ccacagcccc cgccatttgc cacgacggca aggcccactt ccctagggag 3240 ggcgtgttcg tgtctaacgg cacccactgg ttcgtgaccc agcggaactt ctacgagcct 3300 cagattatta ccacagacaa cacattcgtg agcggcaact gcgacgtggt gattggcatt 3360 gtgaacaaca cagtgtacga cccactgcag cctgagttgg actctttcaa ggaggaactc 3420 gacaagtact tcaagaacca cacatctcct gacgtggacc tgggcgacat tagcggcatt 3480 aacgcctctg tggtgaacat tcagaaggag attgacagac tgaacgaggt ggccaagaac 3540 ctgaacgagt ctctcattga cctgcaggag ctgggcaagt acgagcagta cattaagtgg 3600 cct 3603 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Signal peptide sequence for expression of SARS-CoV-2 S protein <220> <221> misc_signal <222> (1)..(60) <400> 11 gccaccatgc acagctcagc actgctctgt tgcctggtcc tcctgactgg ggtgagggcc 60 <210> 12 <211> 1571 <212> DNA <213> Artificial Sequence <220> <223> SARS-CoV-2 S2 protein coding sequence <220> <221> misc_feature <222> (1)..(1571) <400> 12 agagcgtggc ttcccagtcc atcatcgctt acaccatgtc cctcggtgct gagaacagcg 60 tggcttacag caacaacagc atcgctatcc ctaccaactt caccatctcc gtgaccaccg 120 agatcctgcc tgtgagcatg accaagacct ccgtggactg caccatgtac atctgcggcg 180 acagcaccga gtgcagcaac ctcttgttgc agtacggtag cttctgcacc cagttgaaca 240 gggctttgac cggaatcgct gtggagcagg acaagaacac ccaggaggtg ttcgctcagg 300 tgaagcagat ttacaagacc ccccctatca aggacttcgg cggattcaac ttctcccaaa 360 ttttgcccga ccctagcaag ccaagcaagc gtagcttcat cgaggacctg ctgttcaaca 420 aggtcacctt ggccgacgcc ggtttcatca agcagtacgg cgactgcctg ggtgacatcg 480 ccgctaggga cttgatctgc gctcagaagt tcaacggttt gaccgtcttg cccccattgt 540 TGACC GACGA GATGATCGCT CAGTACACCC TCCGCTCTGT TGGCTGGTAC CATCACCTCCG 600 GATGGACCTT CGGTGCTGGC GCCGCTTTGC AGATCCCTTT CGCCATGCAG ATGGCTTACA 660 GATTCAACGG AATCGGAGTG ACCCAGAACG TGTTGTACGA GAACCAGAAG CTGATCGCTA 720 ACCAGTTCAA CAGCGCTATC GGAAAGATCC AGGACAGCCT GTCCTCCACC GCAAGCGCTT 780 TGGGTAAGTT GCAGGACGTC GTCAACCAAG ACGCTCAGGC TCTGAACACC TTGGTGAAGC 840 AGTTGTCCAG CAACTTCGGC GCTATCTCCT CCgtTTGAA CGACATCCTG TCCCgtTTGG 900 ACAAGGTCGA GGCTGAGGTG CAGATCGACA GGTtgATCAC CggCCGTTTG CAGTCCTTGC 960 AGACCTACGT GACCCAGCAG TTGATCAGGG CCGCCGAGAT CAGGGCATCT GCTAACCTGG 1020 CCGCTACCAA GATGTCCGAG TGCgtCTTGG GTCAGTCCAA GAGAGTGGAC TTCTGCGGAA 1080 AGGGATACCA CTTGATGTCC TTCCCACAGA GCGCTCCACA CGGAGTGGTG TTCTTGCACG 1140 TCACCTACGT GCCCGCTCAG GAGAAGAACT TCACCACCgc CCCGCTATC TGCCACGACG 1200 GTAAGGCTCA CTTCCCACGC GAGGgtGTGT TCgtCAGCAA CGGCACCCAC TGgtTCgtCA 1260 CCCAGCGTAA CTTCTACGAG CCTCAGATCA TCACCACCGA CAACACCTTC GTGTCCGGTA 1320 ACTGCGACGT GGTCA TCGGA ATCGTGAACA ACACC GTGTACGACCCTTTGCAGCCTGAGC 1380 TGGACTCCTT CAAGGAGGAA TTAGACAAGT ACTTCAAGAACCACACCTCCCCTGACGTGG 1440 ACTTGGGCAT CATCAGCGGT ATCAACGCCT CTGTCGTGAAC ATCCAGAAGG AGATCGACA 1500 GGCTGAACGAGGTGGCTAAGAACCCTGAACGAGTCCCTCATCGACTTGCAGGAGTTGGGT A 1560 AGTACGAGCA G 1571 <210> 13 <211> 4884 <212> DNA <213> Artificial Sequence <220> <223> pFastBac vector sequence <220> <221> misc_feature <222> (1)..(4884) <400> 13 GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG TGGTTACGCG CAGCGTGACC 60 GCTACACTTG CCAGCGCCCT AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC 120 ACGTTCGCCG GCTTTCCCCG TCAAGCTCTA AATCGGGGCT CCCTTTAGG GTTCCGATTT 180 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420 aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 GAGCCTGTGG ATGCTGTGTT ATGTCTCCTC GAGGAGAAGT GGGGGAGGTT TCCCCTGGC TGC 46 TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAACCA CCGCTACCA 1680 GCAGCGGTGG GCTGAACGGG GGCTTCGTGC ACACAGCCCC AGCTTGGAGC GAACGACCT 1980 AGCAGAGCGC AGATACCAAA TACTGTCCTT CTTAGTGTAG CCCTAGTTAG GCCACCACCT 1800 AAGAAGTCTG TAGCACCGCC TACATACTTC GCTCTGCTAT CTTGTACCAG TGGCTGCT 1860 GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG 1920 GCAGCGGTGG GCTGAACGGG GGCTTCGTGC ACACAGCCCC AGCTTGGAGC GAACGACCT 1980 TACACCGAAC TGAGATACCT ACAGCGTGAG CATTGAGAAA GCGCCACGCT TCCC GAAGGG 2040 AGAAAGGCAG ACAGGTATCC GGTAAGCGGC AGGTCGGAAA GGAGAGCGCA CGAGGGAG 2100 CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTCGCACA CCTCTGACT 2160 GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA CGCCAGCAAC 2220 GCAGCGGTGG GCTGAACGGG GGCTTCGTGC ACACAGCCCC AGCTTGGAGC GAACGACCT 1980 TTATCCCCTG ATTCTGTGGA TAACCCTATT ACCGCCTTTG AGTGAGCTGA TACCCTCGC 2340 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 CGGTATT TTC TCCTTACGC ATCTGTGCGGT ATTTACACC GCAGACCAGC CCGTAACCT 2460 GGCAAAATCG GTTACGGTTG AGTAATAAAT GGATGCCCTG CGTAAGCGGT GTGGGCGGA 2520 CAATAAAGTC TTA AACTGAA CAA AATAGAT CTA AACTATG ACAATAAAGT CT A AACTAG 2580 ACAGAATAGT TGTA AACTGAA ATCAGTCCAG TTATGCTGT GAAAAAGCAT ACTGGACTTT 2640 TGTATGGCTA AAGCAAACTT TCATTTTC TGAAGTGCAA TTGCCC GTC GTATTAAGA 2700 GGGGC GTCGGCC AAGGGCATGGTAAAGACTA TATTCGCGGC GTTGTGACAA TTTACC GAAC 2760 A ACTCCGCGGC GGG AAGCC GATCTC GGC TTG AACGAATT GTTAGGTGGC GGTACTTGGG 2820 TCGATATCAA AGTG CATCATTCTTCCC GT ATGCCCAAC TTTGTATAGA GAGCCACTGC 2880 GGATCGTCAC CGTAATCTGC TTGCACGTAG ATCACATAAG CACCAAGCGC GTTGGCCTCA 2940 TGCTTGAGGA GATTGATGAG CGCGGTGGCA ATGCCCTGCCT CCGGTGCTCG CC GGAGACT 3000 GCGAGATCAT AGATATAGAT CTC ACTACGC GGCTGCTCAAACCTGGGCAG AACGTAAGCC 3060 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180. 3240. ccgaactcac ccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300. ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420. acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat cagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600. cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720 cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 120 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 180 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 240 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 300 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 360 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 420 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 480 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 540 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 600 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 660 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 720 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 780 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 840tctggatctg atcactgctt gagcctagga gatccgaacc agataagtga aatctagttc 4560 caaactattt tgtcattttt aattttcgta ttagcttacg acgctacacc cagttcccat 4620 ctattttgtc actcttccct aaataatcct taaaaactcc atttccaccc ctcccagttc 4680 ccaactattt tgtccgccca cagcggggca tttttcttcc tgttatgttt ttaatcaaac 4740 atcctgccaa ctccatgtga caaaccgtca tcttcggcta ctttttctct gtcacagaat 4800 gaaaattttt ctgtcatctc ttcgttatta atgtttgtaa ttgactgaat atcaacgctt 4860 atttgcagcc tgaatggcga atgg 4884 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer CALL001 <220> <221> misc_feature <222> (1)..(23) <400> 14 gtcctggctg ctcttctaca agg 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer CALL002 <220> <221> misc_feature <222> (1)..(23) <400> 15 ggtacgtgct gttgaactgt tcc 23 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer VHH-BA <220> <221> misc_feature <222> (1)..(29) <400> 16 gatgtgcagc tgcaggagtc tggrggagg 29 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer PMCF <220> <221> misc_feature <222> (1)..(34) <400> 17 ctagtgcggc cgctgaggag acggtgacct gggt 34 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer MP57 <220> <221> misc_feature <222> (1)..(20) <400> 18 ttatgcttcc ggctcgtatg 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer G III <220> <221> misc_feature <222> (1)..(19) <400> 19 ccacagacag ccctcatag 19 <210> 20 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Signal peptide sequence for expression of Nanobody S140 of the application <220> <221> misc_signal <222> (1)..(60) <400> 20 gccaccatgc acagcagcgc cctgctgtgc tgcctggttc tgctgaccgg agtgagggcc 60 <210> 21 <211> 3786 <212> DNA <213> Artificial Sequence <220> <223> Coding nucleotide sequence of SARS-CoV-2-WT-S-del18 <220> <221> misc_feature <222> (1)..(3786) <400> 21 atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cctgaccaca 60 agaacacagc tgccccccgc ctacaccaac agcttcacaa gaggcgtgta ctaccccgac 120 aaggtgttca gaagcagcgt cctccacagc acccaagacc tgttcctgcc cttcttcagc 180 aacgtgacct ggttccacgc catcagcggc accaacggca ccaagagatt cgacaacccc 240 gtgctgccct tcaacgacgg cgtgtacttc gctagcaccg agaagagcaa catcatcaga 300 ggctggatct tcggcaccac cctggacagc aaaacacaga gcctgctgat cgtgaacaac 360 gccacaaacg tggtgatcaa ggtgtgcgag tttcagttct gcaacgaccc cttcctgggc 420 gtgtaccaca agaacaacaa gagctggatg gagagcgagt tccgggtgta cagcagcgcc 480 aacaactgca ccttcgagta cgtgagccaa cccttcctga tggacctgga gggcaagcaa 540 ggcaatttta agaacctgag agagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600 tacagcaagc acacccccat caacctggtg agagacctgc cccaaggctt cagcgccctg 660 gagcccctgg tggacctgcc catcggcatc aacatcacaa gatttcagac cctgctggcc 720 ctgcacagaa gctatctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 GAGAAGAGTGC ATGCTGGAGA AGCCTGGGAC ATGAGCTACA GAGCGGCTGG 60 ggctatcagc cctacagagt ggtcgtgctg agcttcgagc tgctgcacgc ccccgccacc 1560 gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttcaacttc 1620 aacggcctca ccgggaccgg cgtgctgacc gagagcaaca agaagttcct gcctttccaa 1680 cagttcggca gagacatcga cgacaccacc gacgccgtca gagaccctca gaccctggag 1740 atcctggaca tcacaccctg cagcttcggc ggcgtgagcg tgatcacccc cggcaccaac 1800 acaagcaacc aagtggccgt gctgtaccaa ggcgtgaact gcaccgaggt gcccgtggcc 1860 atccacgccg atcagctgac ccccacctgg agagtgtaca gcaccggcag caacgtgttt 1920 cagacaagag ccggctgcct gatcggcgcc gagcacgtga acaacagcta cgagtgcgac 1980 atccccatcg gcgccggcat ctgcgctagc tatcagacac agaccaacag ccacagaaga 2040 gctagaagcg tggctagcca aagcatcatc gcctacacca tgagcctggg cgccgagaac 2100 agcgtggcct acagcaacaa cagcatcgcc atccccacca acttcaccat cagcgtgacc 2160 accgaaatcc tgcctgtgag catgaccaag acaagcgtgg actgcaccat gtacatctgc 2220 GGCGACAGCACCGAGTGCAGCAACCTGCTCCTGCAGTACGGCAGCTTCTGC ATTCAGCTG 2280 AACAGAGCCCTGACCggcatcgccgtggagcaagacaagAacacccaagaggtgttcgcc 2340 CAAGTGAAGCAGATCTACAAgaccccccccAtcaaggacttcggcggcttcaacttcagc 2400 CAAAATCCTGCCTGACCCTAGCAAGCCTAGCAAGAGAAGCTTcatcgaggacctgctgttc 2460 AACAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTGGGCGAC 2520 ATCGCCGCTAGAGACCTGATCTGCGCTCAGAAGTTCAACGGCCTGACCCTGCCCCCC 2580 CTGCTGACCgACGAGATGATCGCTCAGTACAAgCGCCCtGCTCGCTGGCACCAtCACA 2640 AGCGGGTGGA CCTTCGGCGCCGGGGCCGCCCTGCAGATCCCCTTCGCCATGCAGATGGCC 2700 TACAGATTC AACGGCATCGGCgtGA CACAGAACGTGCTGTACGAGAATCAGAAGCTGATC 2760 GCCAATCAGTTCAACAGCGCCATCggCAAGAtCcaAGACAgCCTGAGCAGCACCgCTAGC 2820 GCCCTGGGCAAGCTGCAAGACGTGgtGAATCAGAACGCCAAGCCCTGAACACCCTGgtG 2880 AAGCAGCTGAGCAGCAACttCggCGCCAtCAGCAGCgtGCTGAACGACAtCCTGGCTAGA 2940 ctggacagg tggaggccga ggtgcagatc gadogactga tcaccggcag actgcagagc 3000 ctgcagacct acgtgacaca gcagctgatc agagccgccg agatcagagc tagcgccac 3060 ctggccgcca ccagatgag cgagtgcgtg ctggggcaga gcaagagt ggactctgc 3120 ggcaagggct accacctgat gagctccct cagagcgccc cccacggcgt ggtgttcctg 3180 cacgtgacct acgtgcccgc ccagagaag aacttcacca ccgcccccgc catctgccac 3240 gacggcaagg cccactccc tagagaggggc gtgttcgtga gcaacggcac ccactggttc 3300 gtgacacaga gaaacttcta cgagcctcag atcatcacca cccacacac cttcgtgagc 3360 ggcaactgcg acgtggtgat cggcatcgtg aaaacaccg tgtacgaccc tctgcagccc 3420 gagctggaca gcttcagga ggagctggac aagtacttca agaccacac aagccccgac 3480 gtggacctgg gcgacatcag cgggatcac gctagcgtgg tgacattca gaaggaatc 3540 gagagactga atgaggtggc caagaacctg aacgagagcc tgatcgacct gcaagagctg 3600 ggcaagtacg agcagtacat caagtggccc tgtacatct ggctggggctt catcgccggc 3660 CTGATCGCCATCGTGATGGTGACC ATCATGCTGTGCTG CATGACAAGCTGCTGCTCCTGT 3720 CTGAAGGGGTGCTGCAGCTGC GGCAGCTGCTGCAAGGACTACAAGGACGATGACGACAAG 3780 GGCCCC 3786

Claims

1. An alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 S2, comprising a heavy chain variable region. The heavy chain variable region comprises the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO: 1, CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and CDR3 with the amino acid sequence shown in SEQ ID NO:

3.

2. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 S2 according to claim 1, characterized in that, The heavy chain variable region also includes four frame regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

3. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 S2 according to claim 2, characterized in that, The amino acid sequences of FR1-4 are shown in SEQ ID NO:4, 5, 6, and 7, respectively.

4. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 S2 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

8.

5. A polynucleotide encoding an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 S2 as described in any one of claims 1 to 4.

6. The polynucleotide according to claim 5, characterized in that, The polynucleotide is DNA or mRNA.

7. The polynucleotide according to claim 6, characterized in that, The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

9.

8. A nucleic acid construct comprising the polynucleotide as described in any one of claims 5 to 7.

9. The nucleic acid construct according to claim 8, characterized in that, It also includes at least one expression regulatory element operatively linked to the polynucleotide.

10. An expression vector comprising the nucleic acid construct of claim 8 or 9.

11. A transformed cell comprising the polynucleotide of any one of claims 5 to 7, the nucleic acid construct of claim 8 or 9, or the expression vector of claim 10.

12. A pharmaceutical composition comprising an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 S2 as described in any one of claims 1 to 4, a polynucleotide as described in any one of claims 5 to 7, a nucleic acid construct as described in claim 8 or 9, an expression vector as described in claim 10 or a transformed cell as described in claim 11, and a pharmaceutically acceptable carrier and / or excipient.

13. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is in the form of a nasal spray, oral preparation, suppository, transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

14. The pharmaceutical composition according to claim 13, characterized in that, The nasal spray is selected from aerosols, sprays, and powders; The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, and film-coated formulations; The injectable formulation is a push-in formulation.

15. The pharmaceutical composition according to claim 14, characterized in that, The tablets are sublingual tablets; The powder is a granule; The pills are small pills; The granules are fine granules.

16. The use of an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 S2 as described in any one of claims 1 to 4, a polynucleotide as described in any one of claims 5 to 7, a nucleic acid construct as described in claim 8 or 9, an expression vector as described in claim 10, a transformed cell as described in claim 11, or a pharmaceutical composition as described in any one of claims 12 to 15 in the preparation of a medicament for the prevention, treatment, or detection of infection with the original strain of SARS-CoV-2.

17. A method for detecting a raw SARS-CoV-2 strain for non-diagnostic purposes, comprising using an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 S2 as described in any one of claims 1 to 4.

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