A nanobody that specifically binds to AAV2 and its application

Abstract

This application belongs to the field of nanobody technology, and more specifically, relates to a nanobody that specifically binds to AAV2 and its applications. The nanobody provided in this application can specifically bind to AAV2 adeno-associated virus (AAV2) without binding to other AAV serotypes such as AAV5, AAV8, and AAV9, achieving highly sensitive and specific detection of AAV2 AAV. In practical applications, using the nanobody coupled with a solid-phase carrier provided in this application as an affinity packing material can effectively capture AAV2 AAV and gently elute it, effectively improving virus purification and recovery, and is suitable for high-purity recovery of AAV2 AAV.

Description

Technical Field

[0001] This application belongs to the field of nanobody technology, and more specifically, relates to a nanobody that specifically binds to AAV2 and its application. Background Technology

[0002] Adeno-associated virus (AAV) belongs to the Parvoviridae family. It is a non-replicating, non-enveloped, single-stranded DNA virus that is infectious to both dividing and non-dividing cells. AAV viruses can infect a wide variety of tissues and cells, exhibiting little or no pathogenicity and immunogenicity during host cell proliferation. They can also stably express their carried genes over long periods, making them suitable as vectors for in vivo gene transfer. Currently, several human AAV serotypes have been identified, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV2, AAV10, AAV11, and AAV12, as well as their mutants. Different AAV serotypes have different capsid protein spatial structures, sequences, and tissue specificities, and their recognition and binding to cell surface receptors differ significantly. Therefore, the tissue types, cell types, and infection efficiencies of different AAV serotypes vary.

[0003] AAV2, one of the most widely used serotypes in gene therapy, can infect various cell types, including dividing and non-dividing cells. It can effectively transduce multiple cell types both in vivo and in vitro, making it an ideal vector for gene therapy. The AAV2 capsid protein has high affinity for specific cell surface receptors (such as heparan sulfate proteoglycan and fibroblast growth factor receptor 1), enabling it to naturally target the retina, skeletal muscle, liver, and central nervous system. Furthermore, AAV2 vectors can maintain high levels of gene expression in vivo for several years, making them widely used in the field of gene-engineered drugs and significant for the treatment of genetic diseases. Currently, the FDA-approved drug Luxturna (voretigeneneparvovec) uses an AAV2 vector to deliver the RPE65 gene, successfully treating Leber congenital amaurosis type 2, resulting in significant improvement in the patient's visual function. Developing an antibody with high affinity, good stability, and the ability to specifically bind to AAV2, and preparing it as an AAV2 affinity packing material, is of great significance for the enrichment, purification, detection, and quality control of AAV2 gene therapy vectors. Currently available broad-spectrum antibodies that can simultaneously recognize multiple AAV serotypes have low affinity for specific AAV2 serotypes, poor purification efficiency, and complex preparation processes with long preparation cycles, making them unsuitable for mass production, thus limiting the clinical application of AAV2. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide a nanobody that specifically binds to AAV2 and its application, aiming to solve the problems of low affinity of existing nanobodies for AAV2 serotype, poor purification effect, and complex preparation process, long preparation cycle and high cost of nanobodies.

[0005] To achieve the above objectives, in a first aspect, this application provides a nanobody that specifically binds to AAV2, wherein the heavy chain variable region of the nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequence of the complementarity-determining region of the nanobody is selected from any group of (1)-(11):

[0006] (1): The amino acid sequences are as shown in SEQ ID NO.1 (CDR1), SEQ ID NO.2 (CDR2), and SEQ ID NO.3 (CDR3);

[0007] (2): The amino acid sequences are as shown in SEQ ID NO.8 (CDR1), SEQ ID NO.9 (CDR2), and SEQ ID NO.10 (CDR3);

[0008] (3): The amino acid sequences are as shown in SEQ ID NO.14 (CDR1), SEQ ID NO.15 (CDR2), and SEQ ID NO.16 (CDR3);

[0009] (4): The amino acid sequences are as shown in SEQ ID NO.20 (CDR1), SEQ ID NO.21 (CDR2), and SEQ ID NO.22 (CDR3);

[0010] (5): The amino acid sequences are as shown in SEQ ID NO.26 CDR1, SEQ ID NO.27 CDR2 and SEQ ID NO.28 CDR3;

[0011] (6): The amino acid sequences are as shown in SEQ ID NO.31 (CDR1), SEQ ID NO.32 (CDR2), and SEQ ID NO.33 (CDR3);

[0012] (7): The amino acid sequences are as shown in SEQ ID NO.35 CDR1, SEQ ID NO.36 CDR2 and SEQ ID NO.37 CDR3;

[0013] (8): The amino acid sequences are as shown in SEQ ID NO.40 (CDR1), SEQ ID NO.41 (CDR2), and SEQ ID NO.42 (CDR3);

[0014] (9): An amino acid sequence that has at least 90% sequence homology with (1), (2), (3), (4), (5), (6), (7) or (8);

[0015] (10): The amino acid sequences shown in (1), (2), (3), (4), (5), (6), (7) or (8) are functionally conserved variants with one or more amino acid substitutions, deletions, or insertions;

[0016] (11): The amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8) is a chemically modified sequence.

[0017] Preferably, the heavy chain variable region of the above-mentioned nanobody further includes framework regions FR1, FR2, FR3, and FR4.

[0018] Preferably, the amino acid sequence of the framework region of the above-mentioned nanobody is selected from any group of (a)-(k):

[0019] (a): Amino acid sequences as shown in SEQ ID NO.4 FR1, SEQ ID NO.5 FR2, SEQ ID NO.6 FR3 and SEQ ID NO.7 FR4;

[0020] (b): Amino acid sequences as shown in SEQ ID NO.11 FR1, SEQ ID NO.12 FR2, SEQ ID NO.13 FR3 and SEQ ID NO.7 FR4;

[0021] (c): Amino acid sequences as shown in SEQ ID NO.17 FR1, SEQ ID NO.18 FR2, SEQ ID NO.19 FR3 and SEQ ID NO.7 FR4;

[0022] (d): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.24 FR2, SEQ ID NO.25 FR3 and SEQ ID NO.7 FR4;

[0023] (e): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.30 FR3 and SEQ ID NO.7 FR4;

[0024] (f): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.34 FR3 and SEQ ID NO.7 FR4;

[0025] (g): Amino acid sequences as shown in SEQ ID NO.38 FR1, SEQ ID NO.24 FR2, SEQ ID NO.39 FR3 and SEQ ID NO.7 FR4;

[0026] (h): Amino acid sequences as shown in SEQ ID NO.43 FR1, SEQ ID NO.44 FR2, SEQ ID NO.45 FR3 and SEQ ID NO.7 FR4;

[0027] (i): An amino acid sequence that has at least 90% sequence homology with (a), (b), (c), (d), (e), (f), (g), or (h);

[0028] (j): Functionally conserved variants of the amino acid sequences shown in (a), (b), (c), (d), (e), (f), (g), or (h) with one or more substitutions, deletions, or insertions of one or more amino acids;

[0029] (k): The amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8) is a chemically modified sequence.

[0030] More preferably, the amino acid sequence of the heavy chain variable region of the above-mentioned nanobody is selected from any one of (I)-(IV):

[0031] (I): Any one of the amino acid sequences in SEQ ID NO.46 to SEQ ID NO.53;

[0032] (II): An amino acid sequence that has at least 90% sequence homology with (I);

[0033] (III): Functionally conserved variants of the amino acid sequence shown in (I) by one or more substitutions, deletions, or insertions of one or more amino acids;

[0034] (Ⅳ): The amino acid sequence shown in (I) is a chemically modified sequence.

[0035] Secondly, this application provides a multivalent construct comprising at least one of the above-mentioned nanobodies, or a combination thereof.

[0036] Thirdly, this application provides a fusion protein comprising one or more antigen-binding functional portions; wherein the antigen-binding functional portions include the aforementioned nanobody.

[0037] Preferably, the fusion protein further includes a detection tag or a purification tag.

[0038] Fourthly, this application provides a solid-phase carrier for the isolation and / or purification of AAV2 adeno-associated virus, wherein the aforementioned nanobody is coupled to the surface of the solid-phase carrier.

[0039] Fifthly, this application provides a nucleic acid comprising a nucleic acid sequence encoding the aforementioned nanobody.

[0040] Sixthly, this application provides an expression vector containing the aforementioned nucleic acid.

[0041] Seventhly, this application provides a host cell comprising the aforementioned nucleic acid or the aforementioned expression vector.

[0042] Eighthly, this application provides the application of the above-mentioned nanobody, the above-mentioned multivalent construct, the above-mentioned fusion protein or the above-mentioned solid-phase carrier in the immunodetection, enrichment or purification of AAV2 adeno-associated virus.

[0043] Ninthly, this application provides an AAV2 adeno-associated virus detection kit, which includes the above-mentioned nanobody, the above-mentioned multivalent construct, the above-mentioned fusion protein or the above-mentioned solid-phase carrier.

[0044] In a tenth aspect, this application provides an AAV2 adeno-associated virus purification kit, which includes the above-mentioned nanobody, the above-mentioned multivalent construct, the above-mentioned fusion protein or the above-mentioned solid-phase carrier.

[0045] In summary, the technical solutions conceived in this application have the following main technical advantages compared with the prior art:

[0046] (1) The nanobody provided in this application can specifically bind to AAV2 adeno-associated virus without binding to other AAV serotypes such as AAV5, AAV8, and AAV9, thus achieving highly sensitive and specific detection of AAV2 adeno-associated virus. In practical applications, using the nanobody-conjugated solid-phase carrier provided in this application as an affinity packing material can effectively capture AAV2 adeno-associated virus and gently elute it, effectively improving the virus purification and recovery effect, and is suitable for the high-purity recovery of AAV2 adeno-associated virus.

[0047] (2) The nanobody preparation process provided in this application is short, with the incubation binding time between the phage library containing nanobodies and the antigen (AAV2 adeno-associated virus) being only 10 minutes, which is significantly shorter than the conventional screening process (the incubation binding time between the phage library containing nanobodies and the antigen is 30 minutes). In actual production, it can significantly shorten the production cycle of nanobodies, greatly reduce production costs, and facilitate mass production. Attached Figure Description

[0048] Figure 1 The results are obtained by SDS-PAGE silver staining of the affinity packing material prepared in this application, which is conjugated with eight nanobodies respectively, to purify AAV2 virus. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0050] In the description of the embodiments in this application, the words "exemplary" or "for example" are used to indicate that they are examples, illustrations, or descriptions. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design options. Specifically, the use of the words "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0051] In the description of the embodiments in this application, unless otherwise stated, "multiple" means two or more.

[0052] The term "antibody" as used in this application is used in its broadest sense to include immunoglobulins or other types of molecules containing one or more antigen-binding domains that specifically bind to antigens, and proteins or peptides that exhibit binding specificity to a particular antigen. Specific examples of antibodies may include intact antibodies (e.g., classic tetrachain antibody molecules), single-chain antibodies, nanobodies, multispecific antibodies, etc. Classic antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains linked together by disulfide bonds. Based on the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind antigens, while the constant region (such as the Fc fragment) is used to initiate downstream effects, such as antibody-dependent cell-mediated cytotoxicity (DCC). Within the variable regions of the heavy and light chains, there are three local regions with a higher degree of variability in amino acid composition and sequence, which are key sites for antibody-antigen binding and are therefore also called complementarity-determining regions (CDRs). The amino acid sequence of the CDRs can be determined using a numbering scheme generally accepted in the art, such as Kabat, Chothia, IMGT, AbM, or Contact. The three complementarity-determining regions of the heavy chain are designated HCDR1, HCDR2, and HCDR3, and the three complementarity-determining regions of the light chain are designated LCDR1, LCDR2, and LCDR3. Each variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be composed of three CDRs and four FRs, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0053] The term "nanobody" is also known as "single domain antibody (sdAb)" or "V". H "H antibody" refers to an antibody molecule that has antigen-binding ability, includes a heavy chain variable region but lacks a light chain.

[0054] The term "specific binding" refers to the fact that nanobodies react more frequently, faster, longer for longer, and / or have greater affinity for a specific target antigen than they react with other target antigens.

[0055] The term "sequence identity," also known as "sequence homology," refers to the degree of similarity between amino acid sequences or nucleotide sequences, and is usually expressed as a percentage.

[0056] In this application, "functionally conserved variant" or "variant" refers to an amino acid sequence in which one or more amino acids are substituted, deleted, or inserted without altering the desired properties (such as antigen affinity and / or antigen specificity). It is understood that, based on the specific nanobody sequence provided in this application, corresponding variants of the nanobody provided in this application can be obtained by substituting, deleting, or adding a few amino acids and verifying or screening the binding ability or biological activity of the resulting product with the corresponding antigen. These variants should also be included within the scope of this application.

[0057] The term "multivalent construct" refers to a construct that has at least two antigen-binding sites / binding units. Multivalent constructs can be, but are not limited to, bivalent, trivalent, or tetravalent constructs.

[0058] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and refer to nucleotide polymers. These nucleotide polymers can contain natural and / or non-natural nucleotides. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained within a nucleic acid molecule or polynucleotide.

[0059] The term "fusion protein" refers to an artificially generated protein molecule (e.g., through genetic engineering) composed of at least two distinct peptides. These peptides do not exist in nature or are not present in the same protein molecule. Common examples of fusion proteins, including antibody fragments, include, but are not limited to, multispecific antibodies and enzyme-labeled antibodies used for immunoassay.

[0060] The term "vector" refers to a nucleic acid molecule (e.g., nucleic acid, plasmid, or virus) that can be engineered to contain a target polynucleotide (e.g., the coding sequence of a target polypeptide) or that can replicate in a host cell. A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences (such as promoters and / or enhancers) that regulate the expression of the target polynucleotide, and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric analysis, such as β-galactose). The term "expression vector" refers to a vector used to express a target polypeptide in a host cell.

[0061] This application provides a nanobody that specifically binds to AAV2. The heavy chain variable region of the nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequences of the complementarity-determining regions of the nanobody are selected from any group of (1)-(11):

[0062] (1): The amino acid sequences are as shown in SEQ ID NO.1 (CDR1), SEQ ID NO.2 (CDR2), and SEQ ID NO.3 (CDR3);

[0063] (2): The amino acid sequences are as shown in SEQ ID NO.8 (CDR1), SEQ ID NO.9 (CDR2), and SEQ ID NO.10 (CDR3);

[0064] (3): The amino acid sequences are as shown in SEQ ID NO.14 (CDR1), SEQ ID NO.15 (CDR2), and SEQ ID NO.16 (CDR3);

[0065] (4): The amino acid sequences are as shown in SEQ ID NO.20 (CDR1), SEQ ID NO.21 (CDR2), and SEQ ID NO.22 (CDR3);

[0066] (5): The amino acid sequences are as shown in SEQ ID NO.26 CDR1, SEQ ID NO.27 CDR2 and SEQ ID NO.28 CDR3;

[0067] (6): The amino acid sequences are as shown in SEQ ID NO.31 (CDR1), SEQ ID NO.32 (CDR2), and SEQ ID NO.33 (CDR3);

[0068] (7): The amino acid sequences are as shown in SEQ ID NO.35 CDR1, SEQ ID NO.36 CDR2 and SEQ ID NO.37 CDR3;

[0069] (8): The amino acid sequences are as shown in SEQ ID NO.40 (CDR1), SEQ ID NO.41 (CDR2), and SEQ ID NO.42 (CDR3);

[0070] (9): An amino acid sequence that has at least 90% sequence homology with (1), (2), (3), (4), (5), (6), (7) or (8);

[0071] (10): The amino acid sequences shown in (1), (2), (3), (4), (5), (6), (7) or (8) are functionally conserved variants with one or more amino acid substitutions, deletions, or insertions;

[0072] (11): The amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8) is a chemically modified sequence.

[0073] The nanobody provided in this application can specifically bind to AAV2 adeno-associated virus without binding to other AAV serotypes such as AAV5, AAV8, and AAV9. It can be used to purify AAV2 virus. During the purification process, AAV2 virus can be captured and eluted very gently, and the recovered AAV2 virus has high purity.

[0074] In some embodiments, the heavy chain variable region of the above-mentioned nanobody also includes framework regions FR1, FR2, FR3, and FR4.

[0075] In some embodiments, the amino acid sequence of the framework region of the above-mentioned nanobody is selected from any group of (a)-(k):

[0076] (a): Amino acid sequences as shown in SEQ ID NO.4 FR1, SEQ ID NO.5 FR2, SEQ ID NO.6 FR3 and SEQ ID NO.7 FR4;

[0077] (b): Amino acid sequences as shown in SEQ ID NO.11 FR1, SEQ ID NO.12 FR2, SEQ ID NO.13 FR3 and SEQ ID NO.7 FR4;

[0078] (c): Amino acid sequences as shown in SEQ ID NO.17 FR1, SEQ ID NO.18 FR2, SEQ ID NO.19 FR3 and SEQ ID NO.7 FR4;

[0079] (d): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.24 FR2, SEQ ID NO.25 FR3 and SEQ ID NO.7 FR4;

[0080] (e): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.30 FR3 and SEQ ID NO.7 FR4;

[0081] (f): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.34 FR3 and SEQ ID NO.7 FR4;

[0082] (g): Amino acid sequences as shown in SEQ ID NO.38 FR1, SEQ ID NO.24 FR2, SEQ ID NO.39 FR3 and SEQ ID NO.7 FR4;

[0083] (h): Amino acid sequences as shown in SEQ ID NO.43 FR1, SEQ ID NO.44 FR2, SEQ ID NO.45 FR3 and SEQ ID NO.7 FR4;

[0084] (i): An amino acid sequence that has at least 90% sequence homology with (a), (b), (c), (d), (e), (f), (g), or (h);

[0085] (j): Functionally conserved variants of the amino acid sequences shown in (a), (b), (c), (d), (e), (f), (g), or (h) with one or more substitutions, deletions, or insertions of one or more amino acids;

[0086] (k): The amino acid sequence shown in (a), (b), (c), (d), (e), (f), (g) or (h) is a chemically modified sequence.

[0087] In some embodiments, the amino acid sequence of the heavy chain variable region of the above-mentioned nanobody is selected from any one of (I)-(IV):

[0088] (I): Any one of the amino acid sequences in SEQ ID NO.46 to SEQ ID NO.53;

[0089] (II): An amino acid sequence that has at least 90% sequence identity with (I);

[0090] (III): Functionally conserved variants of the amino acid sequence shown in (I) by one or more substitutions, deletions, or insertions of one or more amino acids;

[0091] (Ⅳ): The amino acid sequence shown in (I) is a chemically modified sequence.

[0092] It should be noted that if an amino acid sequence has at least 90% sequence identity with any one of SEQ ID NO.46 to SEQ ID NO.53 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and the amino acid sequence also has excellent antigen (AAV2 adeno-associated virus) affinity and antigen specificity, then it is also within the scope of protection of this application.

[0093] In some embodiments, the above chemical modifications include one or more of cyclization, N-methylation, acetylation, phosphorylation, succinylation, cardiomylation, palmitoylation, glycosylation, isopreneation, ubiquitination, biotinylation, polyethylene glycol modification, and fluorescent labeling.

[0094] Nanobodies exhibit high chemical stability, solubility, and rapid tissue penetration due to their size and compact folding. Furthermore, nanobodies are readily convertible into multivalent modes, such as bivalent, trivalent, tetravalent, or other multispecific constructs. Based on this, this application provides a multivalent construct comprising at least one of the aforementioned nanobodies, or a combination thereof.

[0095] In some embodiments, the multivalent construct is a bivalent construct or a trivalent construct.

[0096] This application provides a fusion protein comprising one or more antigen-binding functional portions, wherein the antigen-binding functional portions include the aforementioned nanobody.

[0097] In some embodiments, the fusion protein further includes a detection tag or a purification tag. The detection tag facilitates the detection of nanobodies or the immune complexes formed between the nanobodies and their corresponding antigens (AAV or its VP protein), and can be used to indicate the presence or content of AAV or its VP protein in the sample. This application does not specifically limit the detection tag, and includes, but is not limited to, enzymes commonly used in immunoassays, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP); and fluorescent proteins, such as GFP. Purification tags include, but are not limited to, His6 tags, Flag tags, MBP tags, GST tags, and SUMO tags, which facilitate the separation of the aforementioned nanobodies from the sample.

[0098] This application also provides a solid-phase carrier for the isolation and / or purification of AAV2 adeno-associated virus, wherein the aforementioned nanobodies are coupled to the surface of the solid-phase carrier. In some embodiments, the nanobodies are immobilized on the surface of the solid-phase carrier by binding to functional groups such as hydroxyl, carboxyl, and amino groups on the surface of the solid-phase carrier through a coupling agent. The aforementioned solid-phase carrier includes, but is not limited to, polymer microspheres, agarose gels, dextran, cellulose, polycarbonate, digested fiber, silica, or magnetic microspheres.

[0099] In practical applications, when the above-mentioned solid-phase carrier is used for the isolation and / or purification of AAV2 adeno-associated virus, the solid-phase carrier with nanoantibodies coupled to its surface can be used to prepare an affinity packing material. Then, the affinity packing material is contacted with the sample solution to adsorb AAV2 virus particles in the solution. Subsequently, the solid-phase carrier is separated from the sample and the adsorbed AAV2 adeno-associated virus is eluted, thereby achieving the isolation or purification of AAV2 adeno-associated virus.

[0100] This application also provides a nucleic acid comprising a nucleic acid sequence encoding the aforementioned nanobody.

[0101] It is well known to those skilled in the art that nucleic acid sequences encoding nanobodies can be operatively linked to vectors. Based on this, this application provides an expression vector comprising the aforementioned nucleic acid molecules. It is understood that the aforementioned vector refers to any genetic element, including but not limited to plasmids, bacteriophages, transposons, kinases, chromosomes, viruses (such as retroviruses and lentiviruses), viral particles, etc., which, when bound to appropriate control elements, can replicate and transfer gene sequences into cells.

[0102] In some embodiments, the expression vector may also contain other genes, such as marker genes that allow selection of the expression vector in appropriate host cells and under appropriate conditions. Furthermore, the expression vector may also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. These expression control elements include, but are not limited to, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation.

[0103] This application also provides a host cell comprising the aforementioned nucleic acid molecules or expression vectors, capable of being used to prepare the aforementioned nanobodies. The host cell can be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells include mammalian cells (such as primate or non-primate cells), fungal cells (such as yeast), plant cells, and insect cells. It also includes, but is not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and their derived cells, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells.

[0104] The nanobodies, multivalent constructs, fusion proteins, or solid-phase carriers provided in this application can be used for the isolation and / or purification of AAV2 adeno-associated virus.

[0105] This application also provides the application of the above-mentioned nanobodies, multivalent constructs, fusion proteins, or solid-phase carriers in the preparation of AAV2 adeno-associated virus detection or purification kits. In this field, methods for antigen detection using antibodies include, but are not limited to, antigen-antibody precipitation reactions, immunodiffusion experiments, immunoturbidimetry, immunoelectrophoresis (including Western blotting), immunofluorescence techniques, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA), among which ELISA methods can be further classified into direct ELISA, indirect ELISA, sandwich ELISA, etc. All of these detection methods can utilize the nanobodies provided in this application for AAV2 adeno-associated virus detection.

[0106] On the other hand, this application also provides an AAV2 adeno-associated virus detection kit, which includes the above-mentioned nanobody, the above-mentioned multivalent construct, and the above-mentioned fusion protein. It is understood that when using the fusion protein for immunoassay, various enzymes (such as HRP) or fluorescent protein (GFP) can be used to facilitate the detection of AAV2 adeno-associated virus.

[0107] This application also provides an AAV2 adeno-associated virus purification kit, which includes the above-mentioned nanobody, the above-mentioned multivalent construct or the above-mentioned fusion protein, and also includes the above-mentioned solid-phase carrier.

[0108] It should be understood that materials of the same or similar type, model, quality, properties, or function as the reagents and instruments used in the following embodiments can be used to implement this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0109] The main reagents and materials used in the following examples are from the following sources:

[0110] Lymphocyte separation medium was purchased from Solarbio Science, TRIzol™ Reagent kit from Thermo Fisher Scientific, Taq enzyme from TAKARA Biotech, rabbit anti-goat secondary antibody from Genscript Biotech, NHS-activated 4FF affinity packing material and SC-Ni NTA FF affinity column from Shangchun Biotechnology (Wuhan) Co., Ltd., iTaq Universal SYBR Green Supermix from Bio-Rad, and AVIpure AAV2 affinity resin from REPLIGEN.

[0111] The following is an example:

[0112] Example 1: Screening of anti-AAV2 adeno-associated virus nanobodies

[0113] 1.1 Preparation of alpaca immune library

[0114] An adult healthy alpaca was immunized with purified AAV2 adeno-associated virus at a dose of 1×10⁻⁶. 11 One virus particle per alpaca was administered, with a total of three immunizations given at two-week intervals. After immunization, 50 mL of peripheral blood was collected from the alpaca, and B lymphocytes were isolated using lymphocyte separation medium (catalog number P8900).

[0115] 1.2 Nested PCR amplification

[0116] Total RNA was extracted from B lymphocytes using the TRIzol™ Reagent kit (catalog number 15596018) and reverse transcribed into cDNA. The VHH gene was then amplified by nested PCR using this cDNA as a template. The DNA polymerase used for nested PCR amplification was Taq polymerase (catalog number R001A), and the amplification program was set according to the Taq instructions. The primers used for nested PCR amplification are shown in Table 1.

[0117] Table 1 Primers used for nested PCR amplification

[0118]

[0119] 1.3 Construction of a nanobody phage display library

[0120] The amplified VHH gene fragment and the phage display vector pComb 3XSS were digested with Sfil restriction endonuclease. The digested VHH gene fragment and the linearized vector pComb 3XSS were then ligated using T4 DNA ligase. The ligation product was recovered and electroporated into *E. coli* TG1 competent cells to construct a nanobody phage display library. The library size was determined to be 1.83 × 10⁻⁶ using the plate serial dilution method. 9 The cloning efficiency of the nanobody gene insertion vector was 98%.

[0121] 1.4 Screening for anti-AAV2 adeno-associated virus nanobodies

[0122] To meet the requirements of immunoaffinity purification, we aim to obtain nanobodies with a "fast up-and-down" binding characteristic. Unlike conventional screening strategies (where the phage display library and antigen incubation time for nanobodies is 30 minutes), this application significantly shortens the library-antigen incubation time while employing a gentle elution method to screen for anti-AAV2 adeno-associated virus nanobodies. The specific screening steps are as follows:

[0123] First round of screening: AAV2 adeno-associated virus was coated onto microplates at a dose of 100 μL / well, with a virus coating dose of 1 × 10⁻⁶. 11The microplate was coated overnight at 4°C with vp / mL, blocked the next day with 1% bovine serum albumin (BSA), and washed with PBST. 100 μL of the constructed nanobody phage display library was added, and the microplate was incubated at room temperature for 10 min. The microplate was then washed with PBST and PBS, respectively. 100 μL of citrate buffer (50 mM, pH 3.0) was added to the microplate, and the plate was gently shaken for 2 min. The eluent was collected. The eluent was used to infect E. coli TG1 cells in the logarithmic growth phase. A small amount of infected E. coli TG1 cells was used to determine the titer of the eluent using a plate gradient dilution method. The remainder was amplified to obtain an anti-AAV2 specific phage enrichment library.

[0124] Second round of screening: The same procedures as in the first round of screening were used, except that 1% ovalbumin (OVA) was used to block the microplate, the library was incubated for 5 min, and 100 μL of the enriched library obtained in the first round of screening was combined with a microplate coated with AAV2 adeno-associated virus. After elution and library expansion, an anti-AAV2 specific phage enriched library was obtained.

[0125] The third round of screening: The same procedures as in the first round of screening were used, except that the library incubation time was 5 min and 100 μL of the enriched library obtained in the second round of screening was used to bind to a microplate coated with AAV2 adeno-associated virus.

[0126] The fourth round of screening used the same procedures as the first round of screening, except that the microplate was blocked with 1% ovalbumin (OVA), the library was incubated for 5 minutes, and 100 μL of the enriched library obtained in the third round of screening was used to bind the microplate coated with AAV2 adeno-associated virus.

[0127] The phage eluent obtained from the fourth round of screening was used to infect E. coli TG1. Single colonies were randomly picked from the culture dishes used for titer determination and inoculated into 2 mL deep-well plates, and incubated at 30°C until OD500. 600 Approximately 0.5 μL of IPTG was used to induce VHH expression at a final concentration of 4 mM. The bacterial culture supernatant containing the nanobody was collected by centrifugation. The specific response of the nanobody to AAV2 adeno-associated virus was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: Coating with 1×10⁻⁶... 11AAV2 adeno-associated virus (AAV2) at vp / mL was blocked with 5% skim milk, followed by the addition of appropriately diluted bacterial culture supernatant containing nanobodies. The mixture was incubated at 37°C for 1 h, washed three times with PBST to remove unbound antibodies, and then HRP-conjugated rabbit anti-goat secondary antibody (1:5000 dilution) was added. After incubation at 37°C for 1 h, the mixture was washed three times with PBST, followed by TMB (3,3′,5,5′-tetramethylbenzidine) substrate solution for 15 min of color development. The color development was terminated with 2M H2SO4. The absorbance of each well was read at 450 nm. Negative controls (bacterial culture supernatant containing only blocking agent, without nanobodies) and blank wells (buffered buffer) were included. The OD values ​​of the test wells were recorded. 450 A value more than three times that of the negative control is considered positive. The results of positive clone detection are shown in Table 2.

[0128] Table 2. Detection results of positive clones screened in this application.

[0129]

[0130] As shown in Table 2, eight positive clones obtained in this application have affinity for AAV2 adeno-associated virus.

[0131] Example 2: Preparation and large-scale production of anti-AAV2 adeno-associated virus nanobodies

[0132] 2.1 Preparation of anti-AAV2 adeno-associated virus nanobodies

[0133] After preserving the clones corresponding to the positive wells in Example 1, the vectors were extracted and sequenced. The antibody sequencing was performed by Suzhou Genewiz Company. The VHH coding gene sequence was obtained. After translating the DNA sequence into an amino acid sequence, the sequence was compared. Clones with identical CDR1, CDR2, and CDR3 sequences were considered as the same antibody strain, while clones with different CDR sequences were considered as different antibody strains.

[0134] Sequencing yielded anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8. The complementarity-determining regions (CDRs) (CDR1 to CDR3) and framework regions (FRs) (FR1 to FR4) in the heavy chain variable region are shown in Tables 3 to 10. The amino acid sequences of the heavy chain variable region of anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8 are shown in Table 11. The amino acid sequences were divided based on the IMGT numbering rules.

[0135] Table 3. Sequence information of the complementarity-determining regions CDR1~CDR3 and the framework regions FR1~FR4 of nanobody A2-1

[0136]

[0137] Table 4. Sequence information of the complementarity-determining regions CDR1-CDR3 and the framework regions FR1-FR4 of nanobody A2-2.

[0138]

[0139] Table 5. Sequence information of the complementarity-determining regions CDR1-CDR3 and the framework regions FR1-FR4 of nanobody A2-3.

[0140]

[0141] Table 6. Sequence information of the complementarity-determining regions CDR1-CDR3 and the framework regions FR1-FR4 of nanobody A2-4

[0142]

[0143] Table 7. Sequence information of the complementarity-determining regions CDR1-CDR3 and the framework regions FR1-FR4 of nanobody A2-5.

[0144]

[0145] Table 8. Sequence information of the complementarity-determining regions CDR1-CDR3 and the framework regions FR1-FR4 of nanobody A2-6.

[0146]

[0147] Table 9. Sequence information of the complementarity-determining regions CDR1-CDR3 and the framework regions FR1-FR4 of nanobody A2-7.

[0148]

[0149] Table 10 Sequence information of the complementarity-determining regions CDR1~CDR3 and the framework regions FR1~FR4 of nanobody A2-8

[0150]

[0151] Table 11 Amino acid sequence information of the heavy chain variable regions of nanobodies A2-1 to A2-8

[0152]

[0153] 2.2 Large-scale production of anti-AAV2 adeno-associated virus nanobodies

[0154] A VHH encoding gene fusion sequence with a 6×His tag at the N-terminus was synthesized from the whole genome. The VHH encoding gene could be any one of the nanobody A2-1 to A2-8. The fusion gene was then inserted between the NcoI and NheI restriction sites of the PET25b vector. The resulting expression vector PET25b was transformed into *E. coli* Rosetta (DE3) competent cells to obtain a VHH-expressing recombinant strain. The recombinant strain was inoculated at a 1% inoculum into LB medium containing 50 μg / mL ampicillin (Amp) and cultured at 37°C with shaking until OD... 600 The concentration was set to 0.8, and 0.5 mM IPTG was added to the solution. Induction was performed at 22°C and 200 rpm for 16 h. After centrifugation and discarding the supernatant, the bacterial cells were resuspended in 20 mL of PBS (20 mM, pH 7.4). The supernatant was discarded again after centrifugation. The washed bacterial cells were resuspended in 20 mL of PBS and sonicated for 3-5 min until the solution became clear. The supernatant was then separated by centrifugation at 10,000 rpm for 20 min. The supernatant was purified using a nickel column (SC-Ni NTA FF affinity column, catalog number SCB0E005B). The cells were washed with 10 column volumes (CV) of washing buffer (PBS + 10 mM imidazole) and eluted with 2 CV of elution buffer (PBS + 250 mM imidazole). The eluted sample was dialyzed in PBS buffer at 4°C for 16 h, and then concentrated using a 3 kDa ultrafiltration tube to obtain the purified nanobody. Protein concentration was determined using the BCA method, and purity was detected by SDS-PAGE.

[0155] SDS-PAGE analysis showed that the purity of anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8 was greater than 95%.

[0156] Example 3: Specificity of anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8

[0157] 1×10 11 AAV2, AAV5, AAV8, and AAV9 adeno-associated viruses (vp / mL) were blocked with 5% skim milk, and different nanobody solutions (nanobody A2-1 to A2-8) were added. After binding at 37°C for 1 h, the cells were washed three times with PBST to remove unbound antibodies. Then, HRP-conjugated rabbit anti-goat secondary antibody (1:5000 dilution) was added, and after binding at 37°C for 1 h, the cells were washed three times with PBST. Finally, TMB (3,3',5,5'-tetramethylbenzidine) substrate solution was added for color development for 15 min, and the color development was stopped with 2M H2SO4. The absorbance of each well was read at 450 nm. The specificity detection results are shown in Table 12.

[0158] Table 12 Specificity of Nanobodies A2-1 to A2-8

[0159]

[0160] As shown in Table 12, it can be seen that the nanobodies A2-1 to A2-8 provided in this application only bind to AAV2, but do not bind to other serotypes of viruses such as AAV5, AAV8, and AAV9, indicating that the nanobodies A2-1 to A2-8 provided in this application can specifically bind to AAV2 adeno-associated virus.

[0161] Example 4: Preparation of nanobody-conjugated affinity packing material and its use for purification of AAV2 adeno-associated virus.

[0162] 4.1 Pre-activated affinity filler

[0163] Take 2g of NHS-activated 4FF affinity packing material (product number SCB0E009), and wash it three times with pre-cooled 1mM hydrochloric acid (0°C~4°C) by vacuum filtration, each time for 10min.

[0164] 4.2 Preparation of Nanobody Solution

[0165] The purified nanobody (nanobody A2-1 to A2-8) solution was replaced with a 3 kDa ultrafiltration membrane and placed in coupling buffer (sodium bicarbonate buffer, pH 8.5), and the protein concentration was determined by BCA protein quantification method.

[0166] 4.3 Nanoparticle antibody-conjugated affinity packing material

[0167] 2g of NHS-activated 4FF affinity filler was added to 6mL of nanobody solution. The mass ratio of affinity filler to nanobody was 1g:10mg. The reaction was carried out at 28°C and 250rpm for 3.5h. After the reaction was completed, the nanobody-conjugated affinity filler was blocked overnight with 1M ethanolamine (pH 8.0) to obtain the affinity filler.

[0168] 4.4 Preparation of AAV virus loading solution

[0169] The AAV virus (AAV2, AAV5, AAV8, AAV9 adeno-associated virus) harvested from fermentation was lysed using Tween 20 and incubated with nuclease. The fermentation broth after incubation was filtered through a 0.22 μm filter to obtain the AAV supernatant. The AAV supernatant was then concentrated and replaced with an ultrafiltration membrane to ensure that the AAV sample after the replacement was consistent with the affinity packing material equilibration solution. The sample after the replacement was then filtered through a 0.22 μm filter, and the filtrate was collected as the AAV virus loading solution for the affinity packing material.

[0170] 4.5 Affinity packing material for conjugating nanobodies for the purification of AAV adeno-associated virus

[0171] 1 mL of the affinity packing material for the conjugated nanobody prepared in step 4.3 was loaded into a chromatography column, and the AAV2 adeno-associated virus loading solution prepared in step 4.4 was used for loading to detect the purification effect. Before purification, the chromatography column was equilibrated with PBS buffer. After loading, the column was eluted with citric acid (pH 2.5), phosphate (pH 1.9), and 4M guanidine hydrochloride, respectively. The viral genome titer in the eluted sample was detected using qPCR. Specifically, the sample was diluted 10–100 times with purified water, and 200 μL of the diluted sample was treated with a double enzyme method using nuclease and proteinase K. The treated sample was then diluted further to prepare the qPCR reaction system (as shown in Table 13), and the detection was performed according to the qPCR reaction procedure (as shown in Table 14). The primers used for qPCR included an upstream primer QF and a downstream primer QR. The nucleotide sequence of the upstream primer QF was 5'-CCGTTGTCAGGCAACGTG-3', and the nucleotide sequence of the downstream primer QR was 5'-AGCTGACAGGTGGTGGCAAT-3'. The AAV purification yield was calculated based on the viral genome titers before and after sample purification. The recovery rate was calculated as: (viral genome titer in the purified sample / viral genome titer in the loading solution) × 100%. Simultaneously, the purity of the eluted samples was determined using SDS-PAGE silver staining.

[0172] The control group consisted of imported filler AVIpure AAV2 affinity resin.

[0173] Table 13 qPCR reaction system

[0174]

[0175] Table 14 qPCR reaction procedure

[0176]

[0177] The qPCR results of the affinity packing materials prepared in this application and conjugated with eight nanobodies to purify AAV2, AAV5, AAV8, and AAV9 adeno-associated viruses are shown in Table 15. AC-Load represents the sample containing adeno-associated viruses before purification. This sample has been clarified, filtered, and the buffer has been changed to the PBS equilibration solution in the purification step. AC-FT represents the flow-through buffer in the purification step. AC-E represents the virus elution buffer in the purification step. Most of the purified virus will be eluted in citric acid (pH 2.5) elution buffer. Figure 1 The image shows the SDS-PAGE silver staining results of the affinity packing material prepared in this application, which is conjugated with eight nanobodies respectively, to purify AAV2 virus. The lane markers represent molecular markers.

[0178] Table 15. qPCR detection results of AAV2 adeno-associated virus purified using affinity packing material conjugated with nanobodies.

[0179]

[0180] As shown in Table 15, Figure 1 As shown, the eight nanobodies prepared in this application, after being coupled with affinity packing material, can be used to purify AAV2 virus. During the purification process, AAV2 virus can be captured and eluted very gently; and the purity of the recovered AAV2 virus is high.

[0181] In summary, the nanobodies A2-1 to A2-8 prepared in this application can specifically bind to AAV2 adeno-associated virus and exhibit good affinity purification of AAV2 virus. Furthermore, the preparation process of the nanobodies provided in this application is short; the incubation binding time between the phage library containing the nanobodies and the antigen (AAV2 adeno-associated virus) is only 10 minutes. In actual production, this can significantly reduce production costs and facilitate mass production.

[0182] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A nanobody that specifically binds to AAV2, wherein the heavy chain variable region of the nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3, characterized in that, The amino acid sequence of the complementarity-determining region of the nanobody is selected from any one of (1), (2), (5)-(8): (1): The amino acid sequences are as shown in SEQ ID NO.1 (CDR1), SEQ ID NO.2 (CDR2), and SEQ ID NO.3 (CDR3); (2): The amino acid sequences are as shown in SEQ ID NO.8 (CDR1), SEQ ID NO.9 (CDR2), and SEQ ID NO.10 (CDR3); (5): The amino acid sequences are as shown in SEQ ID NO.26 CDR1, SEQ ID NO.27 CDR2 and SEQ ID NO.28 CDR3; (6): The amino acid sequences are as shown in SEQ ID NO.31 (CDR1), SEQ ID NO.32 (CDR2), and SEQ ID NO.33 (CDR3); (7): The amino acid sequences are as shown in SEQ ID NO.35 CDR1, SEQ ID NO.36 CDR2 and SEQ ID NO.37 CDR3; (8): The amino acid sequences are CDR1 shown in SEQ ID NO.40, CDR2 shown in SEQ ID NO.41 and CDR3 shown in SEQ ID NO.

42.

2. The nanobody according to claim 1, characterized in that, The heavy chain variable region of the nanobody also includes framework regions FR1, FR2, FR3, and FR4; The amino acid sequence of the framework region of the nanobody is selected from an amino acid sequence that has at least 90% sequence homology with one of the amino acid sequences (a), (b), (e), (f), (g), or (h); The sequence information of the amino acid sequences (a), (b), (e)-(h) is as follows: (a): Amino acid sequences as shown in SEQ ID NO.4 FR1, SEQ ID NO.5 FR2, SEQ ID NO.6 FR3 and SEQ ID NO.7 FR4; (b): Amino acid sequences as shown in SEQ ID NO.11 FR1, SEQ ID NO.12 FR2, SEQ ID NO.13 FR3 and SEQ ID NO.7 FR4; (e): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.30 FR3 and SEQ ID NO.7 FR4; (f): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.34 FR3 and SEQ ID NO.7 FR4; (g): Amino acid sequences as shown in SEQ ID NO.38 FR1, SEQ ID NO.24 FR2, SEQ ID NO.39 FR3 and SEQ ID NO.7 FR4; (h): Amino acid sequences as shown in SEQ ID NO.43 FR1, SEQ ID NO.44 FR2, SEQ ID NO.45 FR3 and SEQ ID NO.7 FR4.

3. The nanobody according to claim 1, characterized in that, The heavy chain variable region of the nanobody also includes framework regions FR1, FR2, FR3, and FR4; The amino acid sequence of the framework region of the nanobody is selected from one or more functionally conserved variants of the amino acid sequence shown in one of the amino acid sequences (a), (b), (e), (f), (g) or (h) by substitution, deletion or insertion of one or more amino acids. The sequence information of the amino acid sequences (a), (b), (e)-(h) is as follows: (a): Amino acid sequences as shown in SEQ ID NO.4 FR1, SEQ ID NO.5 FR2, SEQ ID NO.6 FR3 and SEQ ID NO.7 FR4; (b): Amino acid sequences as shown in SEQ ID NO.11 FR1, SEQ ID NO.12 FR2, SEQ ID NO.13 FR3 and SEQ ID NO.7 FR4; (e): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.30 FR3 and SEQ ID NO.7 FR4; (f): Amino acid sequences as shown in SEQ ID NO.23 FR1, SEQ ID NO.29 FR2, SEQ ID NO.34 FR3 and SEQ ID NO.7 FR4; (g): Amino acid sequences as shown in SEQ ID NO.38 FR1, SEQ ID NO.24 FR2, SEQ ID NO.39 FR3 and SEQ ID NO.7 FR4; (h): Amino acid sequences as shown in SEQ ID NO.43 FR1, SEQ ID NO.44 FR2, SEQ ID NO.45 FR3 and SEQ ID NO.7 FR4.

4. The nanobody according to claim 2, characterized in that, The amino acid sequence of the framework region of the nanobody is selected from any one of the amino acid sequences (a), (b), (e)-(h).

5. The nanobody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody is selected from an amino acid sequence having at least 90% sequence homology with one of SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52 or SEQ ID NO.

53.

6. The nanobody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody is selected from one of the amino acid sequences shown in SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52 or SEQ ID NO.53, and is a functionally conserved variant of one or more amino acid substitutions, deletions or insertions.

7. The nanobody according to claim 5, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody is selected from any one of SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52, and SEQ ID NO.

53.

8. A fusion protein, characterized in that, The nanobody as described in any one of claims 1 to 7 is linked to a tag to form a fusion protein; The label is selected from detection labels or purification labels.

9. A solid-phase carrier for the isolation and / or purification of AAV2 adeno-associated virus, characterized in that, The solid-phase support surface is coupled with the nanobody according to any one of claims 1 to 7.

10. A nucleic acid, characterized in that, It includes a nucleic acid sequence encoding the nanobody according to any one of claims 1 to 7.

11. An expression vector comprising the nucleic acid of claim 10.

12. A host cell, characterized in that, It includes the nucleic acid of claim 10 or the expression vector of claim 11.

13. An AAV2 adeno-associated virus detection kit or purification kit, characterized in that, It includes the nanobody as described in any one of claims 1 to 7, the fusion protein as described in claim 8, or the solid-phase support as described in claim 9.

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