Constructs of nanobody s43 and uses thereof

Abstract

The application provides a construct based on a nanobody S43 specifically binding to SARS-CoV-2 RBD (including a multivalent nanobody and a nanobody fusion protein), a related product thereof and application thereof; the construct based on the nanobody S43 specifically binding to SARS-CoV-2 RBD (including the multivalent nanobody and the nanobody fusion protein) can effectively inhibit SARS-CoV-2 infection and infection of a variant strain thereof, can be administered in a nebulization mode, can directly reach the lung, has a relatively fast effect and has a long half-life, and provides a more effective treatment strategy for preventing or treating a new coronavirus and a variant strain thereof.

Description

[0001] This application is a divisional application of the invention patent application filed on March 21, 2022, with application number 202210278937.X and invention title "Construction of Nanoantibody S43 and its Application". Technical Field

[0002] This invention relates to the field of biomedicine, specifically to constructs of nanobody S43 and their applications, and more specifically, to multivalent nanobodies based on nanobody S43 that specifically binds to SARS-CoV-2 RBD, nanobody fusion proteins, polynucleotides encoding the same, nucleic acid constructs containing the polynucleotides, expression vectors containing the nucleic acid constructs, cells transformed containing the aforementioned polynucleotides, nucleic acid constructs or expression vectors, and pharmaceutical compositions containing any of the above products, and their applications in the preparation of medicaments for the prevention or treatment of novel coronavirus, and in the preparation of reagents or kits for the detection of novel coronavirus or the diagnosis of novel coronavirus infection. Background Technology

[0003] Since December 2019, the COVID-19 pandemic caused by the novel coronavirus (SARS-CoV-2), 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 primarily 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 significant threat to the health and safety of the people, as well as national economic development and social stability.

[0004] The current COVID-19 outbreak has spurred the development of various vaccines and antiviral drugs. Vaccination is an effective way to prevent serious infectious diseases; however, vaccines are only suitable for uninfected individuals, and their development cycle is long and clinical trials are complex. For confirmed patients, treatment relies on antiviral drugs, one type of which is therapeutic antibody drugs, primarily neutralizing antibodies. Neutralizing antibody drugs work by binding to antigens on the surface of pathogens, preventing specific molecules expressed by the pathogens from binding to cell surface receptors, thus achieving a "neutralizing" effect. The SARS-CoV-2 virus has a glycosylated spike protein (S) on its surface. This S protein can interact with the host cell receptor protein ACE2 and trigger membrane fusion. Therefore, blocking the binding of the S protein to ACE2 is an effective way to treat COVID-19 infection.

[0005] Conventional monoclonal antibodies are generally administered intravenously. However, the concentration of monoclonal antibodies delivered intravenously into the lungs is very low, significantly reducing the antiviral efficacy of the neutralizing antibodies and failing to effectively reduce viral load in the lungs. Since the novel coronavirus initially infects the upper respiratory tract, its first interaction with the immune system primarily occurs on the respiratory mucosa. Therefore, for SARS-CoV-2 infections transmitted through the respiratory tract, in addition to focusing on serum antibodies, it is necessary to consider, design, and develop suitable antibody drugs from the perspective of mucosal immunity. For example, nebulized administration can achieve higher local concentrations of antibody drugs in the respiratory tract, more effectively blocking viral infection upon viral invasion.

[0006] Nanobodies have attracted considerable attention as therapeutic drugs; for example, the nanobodies Caplacizumab (Cablivi) developed by Ablynx... TM One example is ALX-0171, a trivalent nanobody drug used to treat acquired thrombotic thrombocytopenic purpura, which is the first nanobody drug to be approved for marketing. Another example is ALX-0171, a nanobody candidate drug that is used to treat respiratory syncytial virus (RSV) infection in children. It is administered via nebulization and has entered Phase II clinical trials (https: / / clinicaltrials.gov). These all suggest that nanobody drugs are safe and feasible.

[0007] Therefore, developing nanobody drugs that target the novel coronavirus and are suitable for respiratory mucosal immunity has potential clinical application value and prospects. Summary of the Invention

[0008] Purpose of the invention

[0009] The present invention aims to provide a construct (including multivalent nanobodies and nanobodies fusion proteins) of a nanobody S43 that specifically binds to SARS-CoV-2 RBD, a polynucleotide encoding the polynucleotide, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, cells transformed containing the polynucleotide, the nucleic acid construct or the expression vector, and a pharmaceutical composition containing any of the above products, and their use in the preparation of a medicament for the prevention or treatment of novel coronavirus, and in the preparation of a reagent or kit for the detection of novel coronavirus or the diagnosis of novel coronavirus infection.

[0010] The present invention provides a construct of nanobody S43 based on specific binding to SARS-CoV-2 RBD (including multivalent nanobody and nanobody fusion protein), which can effectively inhibit SARS-CoV-2 infection and its variants. It can be administered by nebulization, can reach the lungs directly, has a rapid onset of action and a long half-life, and provides a more effective treatment strategy for SARS-CoV-2 and its variants.

[0011] Solution

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] In a first aspect, the present invention provides a multivalent nanobody comprising two or more nanobodies that specifically bind to SARS-CoV-2 RBD, wherein the nanobodies specifically binding to SARS-CoV-2 RBD include the following CDRs:

[0014] The amino acid sequence is CDR1 as shown in SEQ ID NO:1 (i.e., GFTLDYYAIG).

[0015] The amino acid sequence is CDR2 as shown in SEQ ID NO:2 (i.e., CISSNNSTYYADSVKG), and

[0016] The amino acid sequence is CDR3 as shown in SEQ ID NO:3 (i.e., EPDYSGVYYYTCGWTDFGS).

[0017] In a specific implementation, the nanobody that specifically binds to SARS-CoV-2RBD further includes four framework regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence;

[0018] Preferably, the amino acid sequences of FR1-4 are as shown in SEQ ID NO:4 (i.e., QVQLQESGGGLVQPGGSLRLTCAPS), SEQ ID NO:5 (i.e., WFRQAPGKEREGVS), SEQ ID NO:6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA), and SEQ ID NO:7 (i.e., WGQGTQVTVSS), respectively.

[0019] In a preferred embodiment, the nanobody that specifically binds to SARS-CoV-2RBD has an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8; preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO:8 below:

[0020] QVQLQESGGGLVQPGGSLRLTCAPS GFTLDYYAIG WFRQAPGKEREGVS CISSNNSTYYADSVKG RFT ISRDNAKNTVYLQMNSLKPEDTAVYYCAA EPDYSGVYYYTCGWTDFGS WGQGTQVTVSS The underlined parts represent the frame regions FR1-4, and the bolded parts represent the heavy chain variable regions CDR1, CDR2, and CDR3.

[0021] In a preferred embodiment I, the multivalent nanobody is composed of two or more, preferably three, nanobodies that specifically bind to SARS-CoV-2RBD, linked together by a linker;

[0022] The Linker is (GGGGS)n, where n = 1, 2, 3, or 4, preferably n = 2 or 3.

[0023] As a further preferred embodiment of implementation I, the multivalent nanobody is a trivalent nanobody having the amino acid sequence shown in SEQ ID NO:9:

[0024] QVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYT CGWTDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLTC APSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQGTQVTVSS(SEQ ID NO:9).

[0025] In a preferred embodiment II, the multivalent nanobody is an IgM pentamer formed from the following fusion protein, the structure of which from the N-terminus to the C-terminus is shown in formula (I):

[0026] ALB (I)

[0027] in,

[0028] A is a single nanobody that specifically binds to SARS-CoV-2RBD, or a multivalent nanobody as described in the preferred embodiment I above;

[0029] B is the Fc fragment of human IgM; preferably, the Fc fragment of human IgM has the amino acid sequence shown in SEQ ID NO:10 (i.e., VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY), or the same as SEQ ID NO:10. The amino acid sequence shown in NO:10 has at least 95%, 96%, 97%, 98%, or 99% sequence identity.

[0030] L is (GGGGS)m, where m = 0, 1, 2, 3, or 4.

[0031] As a preferred embodiment of the above-mentioned fusion protein, it has the amino acid sequence shown in SEQ ID NO:11:

[0032] QVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQG TQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPD QDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPA REQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO:11).

[0033] Secondly, the present invention provides a nano-antibody fusion protein, the structure of which from the N-terminus to the C-terminus is shown in formula (I):

[0034] ALB (I)

[0035] in,

[0036] A is a single nanobody that specifically binds to SARS-CoV-2 RBD, as defined in the first aspect above; or, A is a multivalent nanobody according to the preferred embodiment I of the first aspect above, i.e., a multivalent nanobody composed of two or more, preferably three, nanobodies that specifically bind to SARS-CoV-2 RBD linked by a linker, wherein the linker is (GGGGS)n, where n = 1, 2, 3, or 4, preferably n = 2 or 3;

[0037] B is the Fc fragment of human IgM; preferably, the Fc fragment of human IgM has an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:10.

[0038] L is (GGGGS)m, where m = 0, 1, 2, 3, or 4.

[0039] As a preferred embodiment of the above-mentioned fusion protein, it has an amino acid sequence as shown in SEQ ID NO:11.

[0040] Thirdly, the present invention provides a polynucleotide that encodes a multivalent nanobody as described in the first aspect above, or a nanobody fusion protein as described in the second aspect above.

[0041] In a specific implementation, the polynucleotide is DNA or mRNA;

[0042]

[0043]

[0044] Fourthly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the third aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide. Examples include histidine tags, stop codons, etc.

[0045] Fifthly, the present invention provides an expression vector comprising the nucleic acid construct as described in the fourth aspect above.

[0046] In a sixth aspect, the present invention provides a transformed cell comprising the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, or the expression vector as described in the fifth aspect above.

[0047] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a multivalent nanobody as described in the first aspect above, a nanobody fusion protein as described in the second aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, or transformed cells as described in the sixth aspect above, and a pharmaceutically acceptable carrier and / or excipient.

[0048] In specific implementations, the pharmaceutical composition may be in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0049] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0050] Preferably, the oral preparation is selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments;

[0051] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

[0052] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0053] Eighthly, the present invention provides the use of the multivalent nanobody as described in the first aspect above, the nanobody fusion protein as described in the second aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the expression vector as described in the fifth aspect above, the transformed cell as described in the sixth aspect above, or the pharmaceutical composition as described in the seventh aspect above in the preparation of a medicament for the prevention and / or treatment of novel coronavirus infection.

[0054] In a specific implementation plan, the novel coronavirus may be the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain;

[0055] Preferably, the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain, or Omicron (B.1.1.529) subtype BA.2 strain; more preferably, the SARS-CoV-2 variant strain is a Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain, or Omicron (B.1.1.529) subtype BA.2 strain.

[0056] In a ninth aspect, the present invention provides the use of the multivalent nanobody as described in the first aspect, the nanobody fusion protein as described in the second aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, or the transformed cell as described in the sixth aspect in the preparation of reagents or kits for detecting the novel coronavirus or for diagnosing novel coronavirus infection.

[0057] In a specific implementation plan, the novel coronavirus may be the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain;

[0058] Preferably, the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain, or Omicron (B.1.1.529) subtype BA.2 strain; more preferably, the SARS-CoV-2 variant strain is a Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain, or Omicron (B.1.1.529) subtype BA.2 strain.

[0059] In a tenth aspect, the present invention provides a novel coronavirus detection kit, comprising a multivalent nanobody as described in the first aspect, a nanobody fusion protein as described in the second aspect, a polynucleotide as described in the third aspect, a nucleic acid construct as described in the fourth aspect, an expression vector as described in the fifth aspect, or transformed cells as described in the sixth aspect.

[0060] Beneficial effects

[0061] This invention relates to drug development using nanobody constructs targeting the novel coronavirus. The S43-based nanobody constructs of this invention can bind to SARS-CoV-2 RBD with high affinity and can neutralize pseudoviruses and live viruses of the SARS-CoV-2 prototype strain and a series of variant strains with high neutralizing activity. These findings indicate that the S43-based nanobody constructs are novel coronavirus (SARS-CoV-2) nanobodies that can bind to SARS-CoV-2 RBD with high affinity and possess high neutralizing activity.

[0062] In particular, the inventors have demonstrated through a series of experiments that the trivalent nanobody (TS43) and IgM pentamer (MS43) based on nanobody S43 of the present invention have significantly improved neutralizing activity and significantly prolonged half-life compared with their monomer (i.e., nanobody S43). They achieve mucosal immunity, can limit viral replication and further cross the mucosal barrier, control the mucosal transmission of the virus, provide a potential new antibody drug that can be administered by aerosolization for the clinical prevention and treatment of novel coronavirus, and can achieve sensitive and reliable detection of novel coronavirus. Attached Figure Description

[0063] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0064] Figure 1 This is a schematic diagram of the structure of the nanobody constructs TS43 and MS43 constructed in Example 1 of the present invention;

[0065] Figure 2 This is a graph showing the S43 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of this invention;

[0066] Figure 3 This is a graph showing the results of molecular sieve chromatography and SDS-PAGE identification of TS43 protein as described in Example 1 of this invention;

[0067] Figure 4 This is a graph showing the MS43 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of this invention;

[0068] Figure 5 This is an SDS-PAGE identification result diagram of the SARS-CoV-2 RBD-his protein (A), the RBD-his protein (B) of the variant strain Omicron (B.1.1.529) subtype BA.1, and the RBD-his protein (C) of the Omicron (B.1.1.529) subtype BA.2 as described in Example 2 of this invention.

[0069] Figure 6 This is a schematic diagram illustrating the neutralization effect of the three antibodies on the SARS-CoV-2 prototype strain pseudovirus infection as measured in Example 5 of this invention.

[0070] Figure 7 This is a schematic diagram illustrating the effect of the three antibodies on neutralizing the pseudovirus infection of the SARS-CoV-2 variant strain Delta (B.1.617.2) as measured in Example 5 of this invention.

[0071] Figure 8 This is a schematic diagram illustrating the effect of the three antibodies measured in Example 5 of the present invention on the neutralization of SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 pseudovirus infection.

[0072] Figure 9 This is a schematic diagram illustrating the effect of the three antibodies measured in Example 5 of the present invention on the neutralization of SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.2 pseudovirus infection.

[0073] Figure 10 These are the neutralizing activities of the three antibodies against pseudoviruses before and after nebulization as measured in Example 7 of this invention; wherein, A is the result of the neutralizing activity of nanobody S43 against SARS-CoV-2 prototype strain pseudoviruses before and after nebulization, B is the result of the neutralizing activity of nanobody construct TS43 against SARS-CoV-2 prototype strain pseudoviruses, and C is the result of the neutralizing activity of nanobody construct MS43 against SARS-CoV-2 variant strain Delta (B.1.617.2) pseudoviruses. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0075] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0076] The present invention will now be described in detail.

[0077] definition

[0078] "Nanobodies," also known as "heavy chain single-domain antibodies," contain only one variable domain of heavy chain (VHH) and, unlike other antibodies, naturally lack the light chain.

[0079] Due to their inherent biophysical advantages, nanobodies can be easily atomized and delivered directly to the lungs via inhalers to treat viral respiratory infections, making them a highly promising antibody drug.

[0080] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction of the nanobody of the present invention to the SARS-CoV-2 RBD protein, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0081] The reagents, enzymes, culture media, antibiotics, and milk used in the following examples of the present invention are all commercially available products. For example, TRIzol was purchased from Invitrogen, and the Superscript II First-Strand Synthesis System for RT-PCR kit was purchased from Invitrogen.

[0082] Some commonly used biological materials, such as competent cells, vectors, helper phages, and cells to be transformed, are also commercially available products. For example, the pCAGGS vector was purchased from MiaoLingPlasmid; 293F cells and HEK293T cells were purchased from ATCC; the Series Sensor Chip SA chip was purchased from GE Healthcare; and Vero cells were purchased from ATCC CCL81.

[0083] Some synthetic biological materials, such as primers and sequences, which require artificial synthesis, are outsourced to synthetic companies. For example, the coding sequence of TS43 in this invention was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0084] Example 1: Construction, expression, and purification of antibodies based on the trivalent form (TS43) and IgM pentamer form (MS43) of nanobody S43.

[0085] The structural schematic diagrams of the monovalent nanobody and its trivalent and IgM pentamer forms in this embodiment are shown below. Figure 1 As shown.

[0086] The basic nanobody S43 used was obtained in our laboratory by immunizing alpacas with SARS-CoV-2 S protein, constructing an antibody library, and screening using phage display technology. The amino acid sequence of the monovalent nanobody S43 is shown in SEQ ID NO:8. It can bind to SARS-CoV-2 RBD with high affinity and specificity (binding constant is 1.2E-10±1.4E-11M), and in the pseudovirus neutralization experiment, it can neutralize SARS-CoV-2 pseudovirus with high neutralization activity. These results indicate that the S43 nanobody is a novel coronavirus (SARS-CoV-2) alpaca-derived nanobody that can bind to SARS-CoV-2 RBD with high affinity and has high neutralization activity.

[0087] The coding sequence of the monovalent nanobody S43 (as shown in SEQ ID NO:14) was linked to a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15) at the 5' end, a six-histidine tag (hexa-His-tag) at the 3' end, and the translation stop codon TGA. Using restriction endonuclease sites EcoRI and XhoI, the nanobody was constructed into the pCAGGS vector (purchased from Invitrogen). The resulting recombinant vector was then transfected into 293F cells (purchased from Invitrogen) for S43-his protein expression. Cell culture medium containing the target protein was subjected to nickel ion affinity chromatography (HisTrap). TMExcel (GE Healthcare) and gel filtration chromatography (Superdex) TM After purification (using a 75% increase in 10 / 300 GL column (GE Healthcare)), a relatively pure target protein can be obtained. The SDS-PAGE analysis of the S43-his protein showed a size of approximately 15 kDa, as shown in the results. Figure 2 As shown.

[0088] Using two (GGGGS)3 sequences, the coding sequences of three nanobodies S43 (as shown in SEQ ID NO:14) were tandemly linked (directly synthesized by Beijing Qingke Biotechnology Co., Ltd.). A signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15) was attached to the 5' end, and the coding sequence of a six-histidine tag (hexa-His-tag) and the translation stop codon TGA were attached to the 3' end. The recombinant vector was constructed into a pCAGGS vector (purchased from Invitrogen) using restriction endonuclease sites EcoRI and XhoI. The resulting recombinant vector was then transfected into 293F cells (purchased from Invitrogen) for TS43-his protein expression. Cell culture medium containing the target protein was subjected to nickel ion affinity chromatography (HisTrap). TM Excel (GE Healthcare) and gel filtration chromatography (Superdex) TM After purification using a 200 Increase 10 / 300 GL column (GE Healthcare), a relatively pure target protein can be obtained. SDS-PAGE analysis of the TS43-his protein revealed a size of approximately 50 kDa, as shown in the results. Figure 3 As shown.

[0089] The coding sequence of nanobody S43 (as shown in SEQ ID NO:14) was linked to the coding sequence of the Fc of human IgM antibody (as shown in SEQ ID NO:16) via homologous recombination. A signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15) was attached to the 5' end, and a translation stop codon TGA was attached to the 3' end. The pCAGGS vector (purchased from Invitrogen) was constructed using restriction endonuclease sites EcoRI and XhoI to obtain the pCAGGS-S43-IgM Fc recombinant expression vector. The coding sequence of the J chain (as shown in SEQ ID NO:17) was linked to the 3' end with the translation stop codon TGA. The pCAGGS-J chain recombinant expression vector was constructed using restriction endonuclease sites EcoRI and XhoI. The two recombinant expression vectors pCAGGS-S43-IgM Fc and pCAGGS-J chain were co-transfected into 293F cells (purchased from Invitrogen) to express the S43-IgM Fc fusion protein and the J chain. The two then self-assembled to form the IgM form of MS43 protein. The resulting MS43 protein was analyzed using HiTrap. TM IgM Purification HP (GE Healthcare) and Superose TM 6. Increase 10 / 300 GL (GE Healthcare) purification, followed by SDS-PAGE identification. The SDS-PAGE identification of MS43 protein showed a size of approximately 70 kDa, as shown in the results. Figure 4 As shown.

[0090] Example 2: Expression and purification of SARS-CoV-2 and its variant strain RBD

[0091] The coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA were added to the 3' end of the coding sequence of the RBD protein (the amino acid sequence of which is shown in SEQ ID NO:18) of the original SARS-CoV-2 strain. The recombinant vector was constructed into the pCAGGS vector (purchased from Invitrogen) by using the restriction endonuclease sites EcoRI and XhoI. The resulting recombinant vector was then transfected into 293F cells (purchased from Invitrogen) for the expression of SARS-CoV-2 RBD-his protein.

[0092] The coding sequences of the RBD protein (amino acid sequence shown in SEQ ID NO:19) of the SARS-CoV-2 variant strain Omicron(B.1.1.529) subtype BA.1 and the coding sequences of the RBD protein (amino acid sequence shown in SEQ ID NO:20) of the Omicron(B.1.1.529) subtype BA.2 were respectively linked to the 3' end of a six-histidine tag (hexa-His-tag) and a translation stop codon TGA. Expression was then performed using the bac-to-bac baculovirus expression system (Invitrogen). The pFastbac1 plasmid containing the target gene was transformed into DH10Bac competent cells to generate recombinant bacmids. The recombinant bacmids were transfected into Sf9 cells for viral amplification, and protein expression was performed in Hi5 cells. After 48 h of expression, the supernatant from Hi5 cells was collected and processed using HisTrap. TM Excel (GE Healthcare) purifies soluble proteins using nickel affinity chromatography.

[0093] Cell culture medium containing the target protein was subjected to HisTrap nickel affinity chromatography. TM Excel (GE Healthcare) and Superdex gel filtration chromatography TM After purification using a 200 Increase 10 / 300 GL column (GE Healthcare), a relatively pure target protein can be obtained. SDS-PAGE analysis of the SARS-CoV-2 RBD-his protein, the RBD-his protein of the variant strain Omicron (B.1.1.529) subtype BA.1, and the RBD-his protein of Omicron (B.1.1.529) subtype BA.2 showed a size of approximately 30 kDa, as shown below. Figure 5 As shown in A to C.

[0094] Example 3: Detection of the binding ability of various antibodies to the RBD protein of the original SARS-CoV-2 strain and its variants using surface plasmon resonance technology.

[0095] Surface plasmon resonance analysis was performed using a Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0096] The monovalent nanobody S43 and its constructs TS43 and MS43 prepared in the above examples were biotinylated and then immobilized onto a Series Sensor Chip SA chip (Cytiva Life Sciences). The RBD proteins of the original SARS-CoV-2 strain and its variants prepared in the above examples were serially diluted with PBST buffer (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.05% Tween), and loaded onto the chip one by one from low to high concentration. The binding kinetic constants were calculated using BIAevaluation software 8K (Biacore, Inc.). The equilibrium dissociation constants (K0) between each antibody and each RBD were calculated. D As shown in Table 1, the results indicate that monovalent nanobody S43 and its constructs TS43 and MS43 can bind to the RBD proteins of the original SARS-CoV-2 strain and the variant strains Omicron subtype BA.1 and Omicron subtype BA.2 with high affinity. Table 1: Affinity results between monovalent nanobody S43 and its constructs TS43 and MS43 and the RBD proteins of the original and variant SARS-CoV-2 strains.

[0097]

[0098] Example 4: Packaging of original SARS-CoV-2 strains and variant pseudoviruses

[0099] 1) The last 18 amino acids of the S protein encoding the original SARS-CoV-2 strain (WT) and the variant strains Delta (B.1.617.2), Omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529) subtype BA.2 were removed, and the remaining S protein sequences were synthesized (synthesis services were provided by Suzhou Genewiz), resulting in the nucleotide sequences of the genes SARS-CoV-2-WT-S-del18, B.1.617.2-S-del18, B.1.1.529-BA.1-S-del18 and B.1.1.529-BA.2-S-del18, as shown in SEQ ID NO:21~24, respectively.

[0100] 2) The protein gene obtained in 1) was cloned into the pCAGGS vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18, pCAGGS-B.1.617.2-S-del18, pCAGGS-B.1.1.529-BA.1-S-del18 and pCAGGS-B.1.1.529-BA.2-S-del18.

[0101] The packaging steps for the original SARS-CoV-2 strain and its variant pseudoviruses are as follows:

[0102] a. Cell preparation: Seed HEK293T cells (purchased from ATCC CRL-3216) in 10cm cell culture dishes and allow the cell confluence to reach approximately 80% by the second day. The culture medium is DMEM containing 10% FBS.

[0103] b. Transfection: Take the expression plasmids of each S protein from step 2) above, and transfect 30 μg of plasmid / 10 cm cell culture dish with PEI. Mix the target plasmid and PEI at a ratio of 1:3 before transfection. Change the culture medium (DMEM medium containing 10% FBS) after 4-6 hours and incubate at 37℃ for 24 hours.

[0104] c. Virus addition: The pseudovirus packaging backbone virus G*VSV-delG (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) was added to the above-transfected HEK293T cells and incubated at 37°C for 2 hours. The culture medium was changed (DMEM medium containing 10% FBS), and VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank) was added. The cells were then cultured in an incubator for another 30 hours.

[0105] d. Collection of the virus: Collect the supernatant, centrifuge at 3000 rpm for 10 min, filter through a 0.45 μm sterile filter in a laminar flow hood to remove cell debris, aliquot, and freeze at -80℃.

[0106] Pseudoviruses were obtained from the SARS-CoV-2 prototype strain (SARS-CoV-2WT) and the variant strains Delta (B.1.617.2), Omicron (B.1.1.529) subtype BA.1, and Omicron (B.1.1.529) subtype BA.2.

[0107] Example 5: Detection of antibody neutralization of pseudovirus infection

[0108] The purified monovalent nanobody S43 and its constructs TS43 and MS43 (prepared in Example 1) were serially diluted 5-fold to the 9th gradient (2.56 pg / mL). The diluent was mixed with 1.6 × 10⁻⁶ ppm. 4 TCID 50In Example 4, a series of SARS-CoV-2 original strains and variant pseudoviruses were mixed and incubated at 37°C for 1 hour, then added to 96-well plates pre-inoculated with Vero cells (purchased from ATCC CCL81). After incubation for 18–20 hours, the results were detected using a CQ1 Confocal Quantitative Image Cytometer (Yokogawa). Based on the number of cells displaying GFP fluorescence, the neutralizing capacity of the antibody against the aforementioned series of SARS-CoV-2 prototype strains and variant strains Delta, BA.1, and BA.2 pseudoviruses was calculated, and the results are shown below. Figures 6-9 As shown in Table 2, the results show that the pseudovirus neutralization effect of the constructs TS43 and MS43 was improved compared with that of the monovalent nanobody S43.

[0109] Table 2. Neutralizing ability of monovalent nanobody S43 and its constructs TS43 and MS43 against pseudoviruses of the original and variant strains of SARS-CoV-2.

[0110]

[0111] Note: IC 50 (μg / mL) represents the half-maximum inhibitory concentration (WMC) of the antibody. * indicates that the highest concentration of 10 mg / mL was used to measure the inhibition rate, but 100% inhibition was not achieved.

[0112] Example 6: Detection of antibody neutralization of live virus infection

[0113] In this embodiment, the neutralizing effect of each antibody against live SARS-CoV-2 virus was determined using a live virus neutralization assay based on cytopathic effect (CPE). The specific steps are as follows:

[0114] The purified monovalent nanobody S43, as well as TS43 and MS43 (prepared in Example 1), were serially diluted 2-fold to the 11th gradient, with 4 replicate wells for each gradient and 50 μL per well. The diluent was mixed with an equal volume of 100 TCID50. 50 The original SARS-CoV-2 strain or its variant Delta and Omicron subtype BA.1 were incubated at 37°C. After 1 hour, the mixture was added to suspended Vero cells and incubated at 37°C for another 3 days. Cytopathic effects were observed and recorded. The IC50 of the nanobodies and their constructs was calculated using GraphPadPrism 7.0. 50 All experiments were conducted in the Biosafety Level 3 (BSL3) laboratory of the Chinese Center for Disease Control and Prevention.

[0115] The neutralizing effects of monovalent nanobody S43 and its TS43 and MS43 on the live virus of the original strain and its variant strain of SARS-CoV-2 are shown in Table 3. The results in Table 3 show that TS43 and MS43 have good inhibitory effects on the live virus of both the original strain and its variant strain of SARS-CoV-2.

[0116] Table 3. Neutralizing ability of monovalent nanobody S43 and its constructs TS43 and MS43 against live SARS-CoV-2 original strains and variant strains.

[0117]

[0118] Note: IC 50 (μg / mL) is the half-inhibitory concentration of the antibody.

[0119] Example 7: Detection of antibody stability before and after nebulization

[0120] Using an Aerogen Solo (Aerogen Inc., Chicago, USA) nebulizer, monovalent nanobody S43 and its constructs TS43 and MS43 were nebulized, respectively. The nebulized antibodies were then collected using an all-glass SKC (Eighty Four, PA, USA) containing 20 mL of PBS, and a pseudovirus neutralization test was performed as described in Example 5. The results are as follows... Figure 10 As shown, A represents the neutralizing activity of nanobody S43 against SARS-CoV-2 prototype strain pseudovirus before and after nebulization; B represents the neutralizing activity of nanobody construct TS43 against SARS-CoV-2 prototype strain pseudovirus; and C represents the neutralizing activity of nanobody construct MS43 against SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus. Figure 10 The results showed that the nanobody constructs TS43 and MS43 of the present invention maintained stable neutralizing activity against pseudoviruses of the SARS-CoV-2 prototype strain or its variant strains before and after nebulization, suggesting that they are both suitable for administration via nebulization.

[0121] The above results indicate that the S43-based nanobody constructs TS43 and MS43 of the present invention have the potential to be developed into high- and medium-activity antibody drugs, particularly nebulized drugs, for the treatment of novel coronavirus and its variant strains.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and 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. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> Construction and application of nanobody S43 <130> 1087-220048F-D2 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR1 sequence of the VHH chain of nanobody S43 <400> 1 Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly 1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR2 sequence of the VHH chain of nanobody S43 <400> 2 Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 3 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR3 sequence of the VHH chain of nanobody S43 <400> 3 Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr Asp 1 5 10 15 Phe Gly Ser <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR1 sequence of the VHH chain of nanobody S43 <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 Thr Cys Ala Pro Ser 20 25 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR2 sequence of the VHH chain of nanobody S43 <400> 5 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 <210> 6 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR3 sequence of the VHH chain of nanobody S43 <400> 6 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR4 sequence of the VHH chain of nanobody S43 <400> 7 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of the VHH chain of nanobody S43 <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 Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 100 105 110 Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 9 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of a trivalent nanobody <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 100 105 110 Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly 145 150 155 160 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Thr Cys Ala Pro 165 170 175 Ser Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala 180 185 190 Pro Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Ser Ser Asn Asn Ser 195 200 205 Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 210 215 220 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Pro Asp Tyr Ser Gly Val 245 250 255 Tyr Tyr Tyr Thr Cys Gly Trp Thr Asp Phe Gly Ser Trp Gly Gln Gly 260 265 270 Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 275 280 285 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 290 295 300 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 305 310 315 320 Ser Leu Arg Leu Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 325 330 335 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 340 345 350 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys 355 360 365 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 370 375 380 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 385 390 395 400 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 405 410 415 Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 420 425 430 <210> 10 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Fc fragment of human IgM <400> 10 Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg 1 5 10 15 Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala 20 25 30 Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly 35 40 45 Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala 50 55 60 Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile 65 70 75 80 Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp 85 995<! His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro 100 105 110 Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala 115 120 125 Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp 130 135 140 Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly 145 150 155 160 Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala 165 170 175 Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn 180 185 190 Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser 195 200 205 Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala Leu His Arg 210 215 220 Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg 225 230 235 240 Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp 245 250 255 Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys 260 265 270 Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr 275 280 285 Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly 290 295 300 Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val 305 310 315 320 Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn 325 330 335 Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 340 345 <210> 11 <211> 476 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of a nanobody fusion protein <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 100 105 110 Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Val 115 120 125 Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp 130 135 140 Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr 145 150 155 160 Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys 165 170 175 Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys 180 185 190 Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys 195 200 205 Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His 210 215 220 Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp 225 230 235 240 Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala Ser 245 250 255 Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp Leu 260 265 270 Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly Glu 275 280 285 Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala Thr 290 295 300 Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn Ser 305 310 315 320 Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser Pro 325 330 335 Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala Leu His Arg Pro 340 345 350 Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu 355 360 365 Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val 370 375 380 Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr 385 390 395 400 Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr Phe 405 410 415 Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly Glu 420 425 430 Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val Thr 435 440 445 Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn Val 450 455 460 Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 465 470 475 <210> 12 <211> 1293 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence of a trivalent nanobody <400> 12 caggtccaac tccaagagag cggcggcggc ctcgtccaac ccggaggatc actcagactc 60 acatgcgccc caagcggctt cacactcgac tactacgcca tcggctggtt cagacaagcc 120 cccggcaaag agagagaagg agtgtcttgc attagcagca acaacagcac ctactacgcc 180 gacagtgtca aaggaagatt caccatcagc agggacaacg ctaagaacac cgtgtatctc 240 cagatgaact cactgaagcc cgaggacacc gccgtgtact actgcgccgc cgagcccgac 300 tacagcggcg tttactacta cacctgcgga tggaccgact tcggcagctg gggccaagga 360 acccaagtca ccgtgagcag cggaggcgga ggaagcggcg gtggaggaag tggcggaggc 420 ggatctgggg ggggaggatc aggcggagga ggaagccagg tgcagctgca ggagagcgga 480 ggaggactgg tgcagccagg aggaagcctg agactgacat gcgcaccaag cggattcaca 540 ctggactatt atgctatcgg atggttcaga caggcccctg gaaaagagag agagggggtg 600 agctgcatca gcagcaataa ctccacatac tacgccgata gcgtcaaggg gaggttcact 660 attagcaggg acatgcaa gaacacagtg tacctgcaga tgacagcct gaagcccgaa 720 gabaccgccg tctactg cgcagccgag cccgattaca gcggcgtgta ctactacaca 780 tgcggatgga cagactcgg ctcctggggc caagcaccc aagtgaccgt gtcaagcgga 840 ggcgggggg gcggaggagg tggagtgga ggggggggat ctggcggggg aggaagtgga 900 ggaggaggat cacaggtgca gctccaggag agcgggggag gactggtcca gccaggagggg 960 agcctgagac tcacatgtgc acccagcgga tttacactgg attacgc catcggatgg 1020 tttaggcagg cacccgggaa aggagagag ggcgtgagct gcattagcag taatacagc 1080 acctattacg cggactcagt gaggggcgg ttcaccata gcaggtaa cgccagaac 1140 accgtctacc tgcagatgaa tagcctgaaa cccgaagaca cagccgtgta ctattgcgcc 1200 gccgaacccg actactctgg agtgtactac ataccctgcg gctggaccga ctttggcagc 1260 tggggcaag gcaccaggt gaccgtgagc agt 1293 <210> 13 <211> 1428 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence of a nanobody fusion protein <400> 13 caggtgcagc tgcaggagag cggaggaggg ctggtgcagc ccggaggaag cctgagactg 60 acctgcgccc ccagcggatt caccctggat tattatgcta ttggctggtt taggcaggct 120 cccggcaaag agagagaggg ggtgtcatgc attagcagca ataactcaac ctactacgcc 180 gacagcgtca agggacgctt caccatttcc agggacaacg ctaagaacac cgtgtatctc 240 cagatgaata gcctgaagcc cgaggacacc gcagtgtact actgcgccgc cgagcccgac 300 tacagcggtg tgtattacta cacctgcgga tggaccgact tcggcagctg gggccaggga 360 acccaggtga cagtgagcag cgtgatcgcc gagctgcccc ccaaggtgag cgtgttcgtg 420 ccccctagag acggcttctt cggcaaccct agaaagagca agctgatctg ccaagccacc 480 ggcttctccc ctagacagat ccaagtgagc tggctgagag agggcaagca agtgggcagc 540 ggcgtcacaa cagaccaagt gcaagccgag gccaagga gcggccccac cacctacaag 600 gtgacaagca ccctgaccat caaggagagc gactggctgg ggcagagcat gttcacctgc 660 agagtggacc agaggcct gacctttcag cagaacgcta gcagcatgtg cgtgcccgac 720 caagacaccg ccatcagagt gttcgccatc ccccctagct tcgctagcat cttcctgacc 780 aagagcacca agctgacctg cctcgtgacc gatctgacca cctacgacag cgtgaccatc 840 agctggacaa gacagaacgg cgaggccgtg aagacccaca ccaacatcag cgagagccac 900 cccaacgcca ccttcagcgc cgtgggcgag gctagcatct gcgaggacga ctggaacagc 960 ggcgagagat tcacctgcac cgtgacccac accgacctgc ctagccccct gaagcagacc 1020 atcagcagac ccaagggcgt ggccctgcac agacccgacg tgtacctgct gccccccgct 1080 agagagcagc tgaacctgag agagagcgcc accatcacct gcctggtgac cggctttagc 1140 cccgctgacg tgttcgtgca gtggatgcag agaggcagc ccctgagccc cgagaagtac 1200 gtgacaagcg cccccatgcc cgagccccaa gccccccggca gatacttcgc ccacagcatc 1260 ctgaccgtga gcgaggaaga gtggaacacc ggcgagacct acacctgcgt ggtggcccac 1320 gaggccctgc ccaacagagt gaccgagaga accgtggaca agagcaccgg caagcccacc 1380 ctgtacaacg tgagcctggt gatgagcgac accgccggca cctgctac 1428 <210> 14 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence of nanobody S43 VHH chain <400> 14 caggtgcagc tgcaggagag cggaggaggg ctggtgcagc ccggaggaag cctgagactg 60 acctgcgccc ccagcggatt caccctggat tattatgcta ttggctggtt taggcaggct 120 cccggcaaag agagagaggg ggtgtcatgc attagcagca ataactcaac ctactacgcc 180 gacagcgtca agggacgctt caccatttcc agggacaacg ctaagaacac cgtgtatctc 240 cagatgaata gcctgaagcc cgaggacacc gcagtgtact actgcgccgc cgagcccgac 300 tacagcggtg tgtattacta cacctgcgga tggaccgact tcggcagctg gggccaggga 360 acccaggtga cagtgagcag c 381 <210> 15 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Signal peptide sequence <400> 15 Ala Thr Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr 1 5 10 15 Gly Val Arg Ala 20 <210> 16 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> The coding sequence of the Fc of human IgM antibody <400> 16 gtgatcgccg agctgccccc caaggtgagc gtgttcgtgc cccctagaga cggcttcttc 60 ggcaacccta gaaagagcaa gctgatctgc caagccaccg gcttctcccc tagacagatc 120 caagtgagct ggctgagaga gggcaagcaa gtgggcagcg gcgtcacaac agaccaagtg 180 caagccgagg ccaaggagag cggccccacc acctacaagg tgacaagcac cctgaccatc 240 aaggagagcg actggctggg gcagagcatg ttcacctgca gagtggacca cagaggcctg 300 acctttcagc agaacgctag cagcatgtgc gtgcccgacc aagacaccgc catcagagtg 360 ttcgccatcc cccctagctt cgctagcatc ttcctgacca agagcaccaa gctgacctgc 420 ctcgtgaccg atctgaccac ctacgacagc gtgaccatca gctggcaag agaacggc 480 gaggccgtga agacccacac caacatcagc gagagccacc ccaacgccac cttcagcgcc 540 gtgggcgagg ctagcatctg cgaggacgac tggaacagcg gcgagagatt cacctgcacc 600 gtgacccaca ccgacctgcc tagccccctg aagcagacca tcagcagacc caagggcgtg 660 gccctgcaca gacccgacgt gtacctgctg ccccccgcta gagagcagct gaacctgaga 720 gagagcgcca ccatcacctg cctggtgacc ggctttagcc ccgctgacgt gttcgtgcag 780 tggatgcaga gagggcagcc cctgagcccc gagaggatcg tgacaagcgc ccccatgccc 840 gagccccaag cccccggcag atacttcgcc cacagcatcc tgaccgtgag cgaggaagag 900 tggaacaccg gcgagaccta cacctgcgtg gtggcccacg aggccctgcc caacagagtg 960 accgagagaa ccgtggacaa gagcaccggc aagcccaccc tgtacaacgt gagcctggtg 1020 atgagcgaca ccgccggcac ctgctac 1047 <210> 17 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence of J chain <400> 17 atgaagaacc acctgctgtt ctggggcgtg ctggccgtgt tcatcaaggc cgtgcacgtg 60 aaggcccaag aggacgagag aatcgtgctg gtggacaaca agtgcaagtg cgctagaatc 120 acaagcagaa tcatcagaag cagcgaggac cccaacgagg acatcgtgga gagaaacatc 180 agaatcatcg tgcccctgaa caacagagag aacatcagcg accccacaag ccccctgaga 240 acaagattcg tgtaccacct gagcgacctg tgcaagaagt gcgaccccac cgaggtggag 300 ctggacaatc agatcgtgac cgccacacag agcaacatct gcgacgagga cagcgccacc 360 gagacctgct acacctacga cagaaacaag tgctacaccg ccgtggtgcc cctggtgtac 420 ggcggcgaga ccagatggt ggagaccgcc ctgaccccg acgcctgcta ccccgac 477 <210> 18 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURES <223> See the SARS‐CoV‐2 update page <400> 18 Met Phe Will Be Leo Will Be Leo Pro Leo Will Be Gln Cys Arg 1 5 10 15 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 20 25 30 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 35 40 45 Only Trp Asn Arg Lys Arg Ile Serves Asn Cys Only Asp Tyr Serves 50 55 60 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 65 70 75 80 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 85 90 95 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 100 105 110 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 115 120 125 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 130 135 140 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 145 150 155 160 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 165 170 175 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 180 185 190 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 195 200 205 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 210 215 220 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 225 230 235 <210> 19 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of the RBD protein of subtype BA.1 of the mutant strain Omicron (B.1.1.529) <400> 19 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 Ala His Ser Ala Phe Ala Arg Val Gln Pro Thr Glu Ser Ile Val Arg 35 40 45 Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala 50 55 60 Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn 65 70 75 80 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr 85 90 95 Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe 100 105 110 Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg 115 120 125 Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys 130 135 140 Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys 145 150 155 160 Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe 165 170 175 Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile 180 185 190 Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys 195 200 205 Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly 210 215 220 His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala 225 230 235 240 Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn 245 250 255 Lys Cys Val Asn Phe 260 <210> 20 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of the RBD protein of the variant strain Omicron (B.1.1.529) subtype BA.2 <400> 20 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 Ala His Ser Ala Phe Ala Arg Val Gln Pro Thr Glu Ser Ile Val Arg 35 40 45 Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala 50 55 60 Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn 65 70 75 80 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Phe Ala Pro Phe Phe Ala 85 90 95 Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe 100 105 110 Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asn Glu Val Ser 115 120 125 Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys 130 135 140 Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys 145 150 155 160 Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe 165 170 175 Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile 180 185 190 Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys 195 200 205 Tyr Phe Pro Leu Arg Ser Tyr Gly Phe Arg Pro Thr Tyr Gly Val Gly 210 215 220 His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala 225 230 235 240 Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn 245 250 255 Lys Cys Val Asn Phe 260 <210> 21 <211> 3789 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Nucleotide sequence of SARS-CoV-2-WT-S-del18 gene <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 gctactacg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggc 840 acaatcaccg acgccgtcga ctgccccctg gaccccctga gccagaccaa gtgcaccctg 900 aagagcttca ccgtggagaa gggcatctat cagacaagca acttcagagt gcagcccacc 960 gagagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1020 gccacaagat tcgctagcgt gtacgcctgg aacagaaaga gaatcagcaa ctgcgtggcc 1080 gactacagcg tgctgtacaa cagcgctagc ttcagcacct tcaagtgcta cggcgtcagc 1140 cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcaga 1200 ggcgacgagg tgagacagat cgcccccggg cagaccggca agatcgccga ctacaactac 1260 aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggactcc 1320 aaggtgggcg gcaactacaa ctacctgtac agactgttca gaaagagcaa cctgaagccc 1380 ttcgagagag acatcagcac cgagatctac caagccggca gcaccccctg caacggcgtg 1440 gagggcttca actgctactt ccccctgcag agctacggct ttcagcccac ctacggcgtg 1500 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 ggcgacagca ccgagtgcag caacctgctc ctgcagtacg gcagcttctg cattcagctg 2280 aacagagccc tgaccggcat cgccgtggag caagacaaga acacccaaga ggtgttcgcc 2340 caagtgaagc agatctacaa gacccccccc atcaaggact tcggcggctt caacttcagc 2400 caaatcctgc ctgaccctag caagcctagc aagagaagct tcatcgagga cctgctgttc 2460 aacaaggtga ccctggccga cgccggcttc atcaagcagt acggcgactg cctgggcgac 2520 atcgccgcta gagacctgat ctgcgctcag aagttcaacg gcctgaccgt gctgcccccc 2580 ctgctgaccg acgagatgat cgctcagtac acaagcgccc tgctcgctgg caccatcaca 2640 agcgggtgga ccttcggcgc cggggccgcc ctgcagatcc ccttcgccat gcagatggcc 2700 tacagattca acggcatcgg cgtgacacag aacgtgctgt acgagaatca gaagctgatc 2760 gccaatcagt tcaacagcgc catcggcaag atccaagaca gcctgagcag caccgctagc 2820 gccctgggca agctgcaaga cgtggtgaat cagaacgccc aagccctgaa caccctggtg 2880 aagcagctga gcagcaactt cggcgccatc agcagcgtgc tgaacgacat cctggctaga 2940 ctggacaagg tggaggccga ggtgcagatc gatagactga tcaccggcag actgcagagc 3000 ctgcagacct acgtgacaca gcagctgatc agagccgccg agatcagagc tagcgccaac 3060 ctggccgcca ccaagatgag cgagtgcgtg ctggggcaga gcaagagat ggacttctgc 3120 ggcaagggct accacctgat gagcttccct cagagcgcc cccacggcgt ggtgttcctg 3180 cacgtgacct acgtgcccgc ccaagagaag aacttcacca ccgcccccgc catctgccac 3240 gacggcaagg cccacttccc tagagagggc gtgttcgtga gcaacggcac ccactggttc 3300 gtgacacaga gaaacttcta cgagcctcag atcatcacca cccacaacac cttcgtgagc 3360 ggcaactgcg acgtggtgat cggcatcgtg aacaacaccg tgtacgaccc tctgcagccc 3420 gagctggaca gcttcaagga ggagctggac aagtacttca agaaccacac aagccccgac 3480 gtggacctgg gcgacatcag cgggatcaac gctagcgtgg tgaacattca gaaggaaatc 3540 gacagactga atgaggtggc caagaacctg aacgagagcc tgatcgacct gcaagagctg 3600 ggcaagtacg agcagtacat caagtggccc tggtacatct ggctgggctt catcgccggc 3660 ctgatcgcca tcgtgatggt gaccatcatg ctgtgctgca tgacaagctg ctgctcctgt 3720 ctgaaggggt gctgcagctg cggcagctgc tgcaaggact acaaggacga tgacgacaag 3780 ggcccctga 3789 <210> 22 <211> 3792 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Nucleotide sequence of B.1.617.2-S-del18 gene <400> 22 atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgagaaca 60 cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120 aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180 240. aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 420. aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc ctggacgtgt actaccacaa gaacaacaag tcttggatgg agtctggcgt gtactctagc gccaacaact gcaccttcga gtacgtgtcc cagcctttcc tcatggacct ggagggcaag 540 cagggcaact tcaagaacct gagagagttc gtgttcaaga acattgacgg ctacttcaag atttactcta agcacacccc aattacctc gtgagggacc tccctcaggg cttctccgtg ttagaaccac tggtggacct ccctattggc attack cacgcttcca gacactgctc gccctccacc ggtcttacct gaccccaggc gactctagct ctggctggac agccggcgcc 780 gccgcctact acgtgggcta cctgcagcct aggaccttcc tcctgaagta caacgagaac 840 ggcacaatta ccgacgccgt ggactgcgcc ctggacccac tgtccgagac aaagtgcaca ctgaagtcct tcacagtgga gaagggcatt taccagacat ctaacttccg ggtgcagcct 960 acagagtcta ttgtgcggtt cccaaacatc acaaacctgt gccctttcgg cgaggtgttc 1020 aacgccaccc ggttcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 1080 gccgactact ccgtgctgta caactccgcc tctttctcta cattcaagtg ctacggcgtg 1140 tcccctacaa agctgaacga cctgtgcttc accaacgtgt acgccgactc tttcgtgatt 1200 agaggcgacg aggtgaggca gattgccccc ggccagacag gcaagatcgc cgactacaac 1260 tacaagctgc ccgacgactt cacaggctgc gtgatcgcct ggaactctaa caacctggac 1320 tctaaggtgg gcggcaacta caactacaga tacagactgt tccggaagtc taacctgaag 1380 ccattcgaga gggacattag caccgagatt taccaggccg gctctaagcc atgcaacggc 1440 gtggagggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 1500 gtgggctacc agccttaccg ggtggtggtg ctgtctttcg agctgctcca cgcccccgcc 1560 acagtgtgcg gcccaaagaa gagcacaaac ctcgtgaaga acaagtgcgt gaacttcaac 1620 ttcaacggcc tcacaggcac aggcgtgctc accgagtcta acaagaagtt cctccctttc 1680 cagcagttcg gccgcgacat tgccgacacc accgacgccg tgcgggaccc tcagacactg 1740 gaaattctcg acatcacccc ttgcagcttc ggcggcgtgt ccgtgatcac cccaggcaca 1800 aacacatcta accaggtggc cgtgctgtac cagggcgtga actgcaccga ggtgccagtg 1860 gccatccacg ccgaccagct caccccaaca tggagggtgt acagcacagg ctctaacgtg 1920 ttccagaccc gggccggctg cctcattggc gccgagcacg tgaacaactc ttacgagtgc 1980 gacatcccta ttggcgccgg catttgcgcc tcttaccaga cccagacaaa ctctagacgg 2040 agagcccggt ctgtggcctc tcagagcatt attgcctaca ccatgtctct gggcgccgag 2100 aactctgtgg cctactctaa caactctatt gccatcccta caaacttcac aatttctgtg 2160 accaccgaga ttctcccagt gtctatgacc aagacatctg tggactgcac catgtacatt 2220 tgcggcgact ccaccgagtg ctctaacctc ctgctccagt acggctcttt ctgcacccag 2280 ctcaaccgcg ccctgacagg catcgccgtg gagcaggaca agaacaccca ggaggtgttc 2340 gcccaggtga agcagattta caagaccccc ccaattaagg acttcggcgg cttcaacttc 2400 tctcagattc tccccgaccc atccaagcct agcaagcggt ccttcattga ggacctcctg 2460 ttcaacaagg tgacactggc cgacgccggc ttcattaagc agtacggcga ctgcctgggc 2520 gacattgccg cccgggacct gatttgcgcc cagaagttca acggcctcac agtgctcccc 2580 ccactgctca ccgacgagat gattgcccag tacacatctg ccctcctggc cggcacaatt 2640 acatctggct ggaccttcgg cgccggcgcc gccctgcaga tccctttcgc catgcagatg 2700 gcctaccgct tcaacggcat cggcgtgaca cagaacgtgc tgtacgagaa ccagaagctg 2760 atcgccaacc agttcaacag cgccattggc aagattcagg actctctgag cagcacagcc 2820 agcgccctgg gcaagctgca gaacgtggtg aaccagaacg cccaggccct gaacacactg 2880 gtgaagcagc tgtcttctaa cttcggcgcc atttctagcg tgctgaacga cattctgtcg 2940 cggctggaca aggtggaggc cgaggtgcag attgacaggc tcatcacagg cagactgcag 3000 tctctgcaga catacgtgac ccagcagctg attagagccg ccgagattag agcctccgcc 3060 aacctggccg ccaccaagat gagcgagtgc gtgctcggcc agtctaagcg ggtggacttc 3120 tgcggcaagg gctaccacct catgtctttc cctcagtccg cccctcacgg cgtggtgttc 3180 ctccacgtga catacgtgcc cgcccaggag aagaacttca ccacagcccc cgccatttgc 3240 cacgacggca aggcccactt ccctagggag ggcgtgttcg tgtctaacgg cacccactgg 3300 ttcgtgaccc agcggaactt ctacgagcct cagattatta ccacagacaa cacattcgtg 3360 agcggcaact gcgacgtggt gattggcatt gtgaacaaca cagtgtacga cccactgcag 3420 cctgagttgg actctttcaa ggaggaactc gacaagtact tcaagaacca cacatctcct 3480 gacgtggacc tgggcgacat tagcggcatt aacgcctctg tggtgaacat tcagaaggag 3540 attgacagac tgaacgaggt ggccaagaac ctgaacgagt ctctcattga cctgcaggag 3600 ctgggcaagt acgagcagta cattaagtgg ccttggtaca tttggctggg cttcattgcc 3660 ggcctgatcg ccattgtgat ggtgaccatc atgctgtgct gcatgacatc ttgctgcagc 3720 tgcctgaagg gctgctgctc ttgcggctct tgctgcaagg actacaagga cgacgatgac 3780 aagggacctt aa 3792 <210> 23 <211> 3795 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Nucleotide sequence of gene B.1.1.529‑BA.1‑S‑del18 <400> 23 atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60 cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120 aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180 aacgtgacat ggttccacgt gatctctggc acaaacggca caaagcggtt cgacaacccc 240 gtgctccctt tcaacgacgg cgtgtacttc gccagcattg agaagtctaa cattatccgg 300 ggctggattt tcggcaccac actcgactct aagacacagt ccctcctgat tgtgaacaac 360 gccacaaacg tggtgattaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420 cacaagaaca acaagtcttg gatggagtct gagttcagag tgtactctag cgccaacaac 480 tgcaccttcg agtacgtgtc ccagcctttc ctcatggacc tggagggcaa gcagggcaac 540 ttcaagaacc tgagagagtt cgtgttcaag aacattgacg gctacttcaa gatttactct 600 aagcacaccc caattatgt gagggaacca gaagacctcc ctcagggctt ctccgcctta 660 gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720 ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780 gcctactacg tgggctacct gcagcctagg accttctcc tgaagtacaa cgagaacggc 840 acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900 aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960 gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020 gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080 gactactccg tgctgtacaa cctggcccct ttcttcacat tcaagtgcta cggcgtgtcc 1140 cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200 ggcgacgagg tgaggcagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260 aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320 aaggtgtctg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380 ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440 gccggcttca actgctactt cccactgcgc tcctactcct tccggcctac atacggcgtg 1500 ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560 gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620 aacggcctca agggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680 cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740 attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800 acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860 atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920 caaacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980 atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040 gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100 tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160 accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220 ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280 aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340 caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400 cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460 aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520 attgccgccc gggacctgat ttgcgcccag aagttcaagg gcctcacagt gctcccccca 2580 ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640 tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700 taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760 gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820 gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880 aagcagctgt cttctaagtt cggcgccatt tctagcgtgc tgaacgacat tttctcgcgg 2940 ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000 ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060 ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120 ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180 cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240 gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300 gtgacccagc ggaacttcta cgagcctcag attattacca cagacaacac attcgtgagc 3360 ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420 gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480 gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540 gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600 ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660 ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720 ctgaagggct gctgctcttg cggctcttgc tgcaaggact acaaggacga cgatgacaag 3780 ggaccttaac tcgag 3795 <210> 24 <211> 2913 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Nucleotide sequence of B.1.1.529 - BA.2 - S - del18 gene <400> 24 atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgatcaca 60 cggacccaga gctacacaaa ctctttcacc cggggcgtgt actaccccga caaggtgttc cggtctagcg tgctccactc tacacaggac ctgttcctcc ctttcttcag caacgtgaca tggttccacg ccatccacgt gtctggcaca aacggcacaa agcggttcga caaccccgtg ctccctttca acgacggcgt gtacttcgcc agcaccgaga agtctaacat tatccggggc 300 tggattttcg gcaccacact cgactctaag acacagtccc tcctgattgt gaacaacgcc 360 acaaacgtgg tgattaaggt gtgcgagttc cagttctgca acgacccttt cctggacgtg 420 tactaccaca agaacaacaa gtcttggatg gagtctgagt tcagagtgta ctctagcgcc aacaactgca ccttcgagta cgtgtcccag cctttcctca tggacctgga gggcaagcag 540 ggcaacttca agaacctgag aggttcgtg ttcaagaca ttgacggcta cttcaagatt tactctaagc acaccccaat taacctcggc agggacctcc ctcagggctt ctccgcctta 720. gaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780 gcctactacg tgggctacct gcagcctagg accttctcc tgaagtacaa cgagaacggc 840 acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900 aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960 gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020 gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080 gactactccg tgctctacaa cttcgcccc ttcttcgcct tcaagtgcta cggcgtgtcc 1140 cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200 ggcaacgagg tgagccagat tgccccggc cagacaggca acatcgccga ctacaactac 1260 aagctgcccg aggacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320 aaggtgggcg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380 ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440 gccggcttca actgctactt cccactgcgg tcctacggct tccggcctac atacggcgtg 1500 ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560 gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620 aacggcctca caggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680 cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740 attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800 acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860 atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920 cagacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980 atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040 gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100 tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160 accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220 ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280 aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340 caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400 cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460 aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520 attgccgccc gggacctgat ttgcgcccag aagttcaacg gcctcacagt gctcccccca 2580 ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640 tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700 taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760 gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820 gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880 aagcagctgt cttctaagtt cggcgccatt agc 2913

Claims

1. The application of a multivalent nanobody in the preparation of a drug for the prevention or treatment of SARS-CoV-2 infection, characterized in that, The multivalent nanobody is composed of three nanobodies that specifically bind to SARS-CoV-2 RBD linked together by a linker; The nanobody specifically binding to SARS-CoV-2 RBD includes 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; and, The Linker is (GGGGS)n, where n = 1, 2, 3, or 4.

2. The application according to claim 1, characterized in that, n = 2 or 3.

3. The application according to claim 1, characterized in that, The nanobody that specifically binds to SARS-CoV-2 RBD also includes four framework regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

4. The application according to claim 3, characterized in that, The amino acid sequences of FR1-4 are shown in SEQ ID NO:4, 5, 6, and 7, respectively.

5. The application according to claim 1, characterized in that, The nanobody that specifically binds to SARS-CoV-2 RBD has an amino acid sequence as shown in SEQ ID NO:

8.

6. The application according to any one of claims 1-5, characterized in that, The SARS-CoV-2 mentioned refers to the original SARS-CoV-2 strain and / or a variant of SARS-CoV-2.

7. The application according to claim 6, characterized in that, The SARS-CoV-2 variant strains mentioned are Alpha, Beta, Gamma, Kappa, Delta strains, Omicron subtype BA.1 strain and / or Omicron subtype BA.2 strain.

8. The application according to any one of claims 1-5, characterized in that, The drug is in the form of nasal spray, oral preparation, suppository, transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

9. The application according to claim 8, 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.

10. The application according to claim 9, characterized in that, The tablets are sublingual tablets; the powder is a granule; the pills are small pills; and the granules are fine granules.

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