Nanometer antibody specifically combined with AAV2 and application thereof

Abstract

The invention belongs to the technical field of nano antibodies, and particularly relates to a nano antibody specifically bound with AAV2 and application of the nano antibody. The nano antibody provided by the invention can be specifically combined with the AAV2 adeno-associated virus without being combined with other AAV serotypes such as AAV5, AAV8 and AAV9, so that high-sensitivity and high-specificity detection of the AAV2 adeno-associated virus is realized. In practical application, the nano antibody coupled solid-phase carrier provided by the invention is adopted as an affinity filler, the AAV2 adeno-associated virus can be effectively captured and mildly eluted, the virus purification and recovery effects are effectively improved, and the nano antibody coupled solid-phase carrier is suitable for high-purity recovery of the AAV2 adeno-associated virus.

Description

Technical Field

[0001] The present application belongs to the field of nanoantibody technology, and more specifically, relates to a nanoantibody that specifically binds to AAV2 and its application. Background Art

[0002] Adeno-associated virus (AAV), a member of the Parvoviridae family, is a non-enveloped, single-stranded DNA virus that is incapable of autonomous replication and infects both dividing and non-dividing cells. AAV can infect a wide variety of tissue and cell types, exhibits low or no pathogenicity and immunogenicity during host cell proliferation, and can stably express the gene it carries, making it suitable for use as a vector for in vivo gene transfer. Numerous 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 possess distinct capsid protein structures, sequences, and tissue specificities, and recognize and bind distinct cell surface receptors. Consequently, the tissue and cell types transduced, as well as the infection efficiency, vary among AAV serotypes.

[0003] AAV2, one of the most widely used serotypes in gene therapy, can infect a wide range of cell types, including dividing and non-dividing cells. It effectively transduces a wide range of cell types both in vitro and in vivo, making it an ideal vector for gene therapy. The AAV2 capsid protein has a high affinity for specific cell surface receptors (such as heparan sulfate proteoglycans and fibroblast growth factor receptor 1), making it a natural target for the retina, skeletal muscle, liver, and central nervous system. Furthermore, AAV2 vectors can maintain high levels of gene expression in vivo for up to several years, making them widely used in the field of genetic engineering drugs and of great significance for the treatment of genetic diseases. Currently, the FDA-approved drug Luxturna (voretigene neparvovec) uses an AAV2 vector to deliver the RPE65 gene and has successfully treated Leber congenital amaurosis type 2, resulting in significant improvement in visual function in patients. The development of an antibody with high affinity, good stability, and specific binding to AAV2, which can be prepared as an AAV2 affinity filler, is crucial for the enrichment, purification, detection, and quality control of AAV2 gene therapy vectors. However, the broad-spectrum antibodies currently available can simultaneously recognize multiple AAV sera, but have low affinity for specific AAV2 serotypes and poor purification effects. In addition, the antibody preparation process is complex, the preparation cycle is long, and it is not suitable for mass production, which limits the clinical application of AAV2. Summary of the Invention

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

[0005] To achieve the above object, in the first aspect, the present application provides a nanobody that specifically binds to AAV2. The variable region of the heavy chain of the nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequences of the complementarity-determining regions of the above nanobody are selected from any one of the following groups (1)-(11): (1): CDR1 with the amino acid sequence shown in SEQ ID NO.1, CDR2 with the amino acid sequence shown in SEQ ID NO.2, and CDR3 with the amino acid sequence shown in SEQ ID NO.3; (2): CDR1 with the amino acid sequence shown in SEQ ID NO.8, CDR2 with the amino acid sequence shown in SEQ ID NO.9, and CDR3 with the amino acid sequence shown in SEQ ID NO.10; (3): CDR1 with the amino acid sequence shown in SEQ ID NO.14, CDR2 with the amino acid sequence shown in SEQ ID NO.15, and CDR3 with the amino acid sequence shown in SEQ ID NO.16; (4): CDR1 with the amino acid sequence shown in SEQ ID NO.20, CDR2 with the amino acid sequence shown in SEQ ID NO.21, and CDR3 with the amino acid sequence shown in SEQ ID NO.22; (5): CDR1 with the amino acid sequence shown in SEQ ID NO.26, CDR2 with the amino acid sequence shown in SEQ ID NO.27, and CDR3 with the amino acid sequence shown in SEQ ID NO.28; (6): CDR1 with the amino acid sequence shown in SEQ ID NO.31, CDR2 with the amino acid sequence shown in SEQ ID NO.32, and CDR3 with the amino acid sequence shown in SEQ ID NO.33; (7): CDR1 with the amino acid sequence shown in SEQ ID NO.35, CDR2 with the amino acid sequence shown in SEQ ID NO.36, and CDR3 with the amino acid sequence shown in SEQ ID NO.37; (8): CDR1 with the amino acid sequence shown in SEQ ID NO.40, CDR2 with the amino acid sequence shown in SEQ ID NO.41, and CDR3 with the amino acid sequence shown in SEQ ID NO.42; (9): An amino acid sequence having at least 90% sequence homology with (1), (2), (3), (4), (5), (6), (7), or (8); (10): A functional conservative variant of the amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8) by substitution, deletion or insertion of one or more amino acids; (11): A chemically modified amino acid sequence of (1), (2), (3), (4), (5), (6), (7) or (8).

[0006] Preferably, the heavy chain variable region of the above-mentioned Nanobody further includes framework regions FR1, FR2, FR3, and FR4.

[0007] Preferably, the amino acid sequence of the framework region of the above-mentioned Nanobody is selected from any one of the groups (a) to (k): (a): The amino acid sequences are FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 5, FR3 shown in SEQ ID NO. 6, and FR4 shown in SEQ ID NO. 7; (b): amino acid sequences of FR1 shown in SEQ ID NO.11, FR2 shown in SEQ ID NO.12, FR3 shown in SEQ ID NO.13, and FR4 shown in SEQ ID NO.7; (c): amino acid sequences of FR1 shown in SEQ ID NO.17, FR2 shown in SEQ ID NO.18, FR3 shown in SEQ ID NO.19, and FR4 shown in SEQ ID NO.7; (d): amino acid sequences of FR1 shown in SEQ ID NO. 23, FR2 shown in SEQ ID NO. 24, FR3 shown in SEQ ID NO. 25, and FR4 shown in SEQ ID NO. 7; (e): amino acid sequences of FR1 shown in SEQ ID NO.23, FR2 shown in SEQ ID NO.29, FR3 shown in SEQ ID NO.30, and FR4 shown in SEQ ID NO.7; (f): amino acid sequences of FR1 shown in SEQ ID NO. 23, FR2 shown in SEQ ID NO. 29, FR3 shown in SEQ ID NO. 34, and FR4 shown in SEQ ID NO. 7; (g): amino acid sequences of FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.24, FR3 shown in SEQ ID NO.39, and FR4 shown in SEQ ID NO.7; (h): FR1 with the amino acid sequence shown in SEQ ID NO. 43, FR2 with the amino acid sequence shown in SEQ ID NO. 44, FR3 with the amino acid sequence shown in SEQ ID NO. 45, and FR4 with the amino acid sequence shown in SEQ ID NO. 7; (i): An amino acid sequence having at least 90% sequence homology with (a), (b), (c), (d), (e), (f), (g) or (h); (j): A functionally conservative variant of the amino acid sequence shown in (a), (b), (c), (d), (e), (f), (g) or (h) which has undergone one or more of substitution, deletion, insertion of one or several amino acids; (k): A chemically modified sequence of the amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8).

[0008] Further preferably, the amino acid sequence of the heavy chain variable region of the above-mentioned nanobody is selected from any one of (I)-(IV): (I): Any one of the amino acid sequences such as SEQ ID NO. 46 to SEQ ID NO. 53; (II): An amino acid sequence having at least 90% sequence homology with (I); (III): A functionally conservative variant of the amino acid sequence shown in (I) which has undergone one or more of substitution, deletion, insertion of one or several amino acids; (IV): A chemically modified sequence of the amino acid sequence shown in (I).

[0009] In the second aspect, the present application provides a multivalent construct, which comprises at least one of the above-mentioned nanobodies, or a combination thereof.

[0010] In the third aspect, the present application provides a fusion protein, which comprises one or more antigen-binding functional parts; wherein the antigen-binding functional part comprises the above-mentioned nanobody.

[0011] Preferably, the above-mentioned fusion protein further comprises a detection tag or a purification tag.

[0012] In the fourth aspect, the present application provides a solid-phase carrier for the isolation and / or purification of AAV2 adeno-associated virus, and the above-mentioned nanobody is coupled to the surface of the solid-phase carrier.

[0013] In the fifth aspect, the present application provides a nucleic acid, which comprises a nucleic acid sequence encoding the above-mentioned nanobody.

[0014] In the sixth aspect, the present application provides an expression vector comprising the above-mentioned nucleic acid.

[0015] In a seventh aspect, the present application provides a host cell comprising the above nucleic acid or the above expression vector.

[0016] In an eighth aspect, the present application provides the use of the above nanobody, the above multivalent construct, the above fusion protein or the above solid-phase carrier in the immunodetection, enrichment or purification of AAV2 adeno-associated virus.

[0017] In a ninth aspect, the present application provides an AAV2 adeno-associated virus detection kit comprising the above nanobody, the above multivalent construct, the above fusion protein or the above solid-phase carrier.

[0018] In a tenth aspect, the present application provides an AAV2 adeno-associated virus purification kit comprising the above nanobody, the above multivalent construct, the above fusion protein or the above solid-phase carrier.

[0019] Generally speaking, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: (1) The nanobody provided by the present application can specifically bind to AAV2 adeno-associated virus without binding to other AAV serotypes such as AAV5, AAV8, and AAV9, achieving highly sensitive and highly specific detection of AAV2 adeno-associated virus. In practical applications, using the nanobody provided by the present application conjugated to a solid-phase carrier as an affinity filler can effectively capture AAV2 adeno-associated virus and elute it gently, effectively improving the virus purification and recovery effect, and being suitable for the high-purity recovery of AAV2 adeno-associated virus.

[0020] (2) The preparation process of the nanobody provided by the present application is short. The incubation and binding time between the phage library containing the nanobody and the antigen (AAV2 adeno-associated virus) is only 10 min, which is significantly shorter than the conventional screening process (the incubation and binding time between the phage library containing the nanobody and the antigen is 30 min). In actual production, it can greatly shorten the production cycle of the nanobody, greatly reduce the production cost, and facilitate batch production. Description of the Drawings

[0021] Figure 1 It is the SDS-PAGE silver staining result of the affinity filler conjugated with 8 nanobodies prepared in the present application for purifying AAV2 virus. Detailed Embodiments

[0022] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0023] In the description of the embodiments of this application, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of this application should not be construed as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0024] In the description of the embodiments of the present application, unless otherwise specified, “plurality” means two or more.

[0025] The term "antibody" is used in this application in its broadest sense to include immunoglobulins or other types of molecules containing one or more antigen-binding domains that specifically bind to an antigen, and are proteins or polypeptides that exhibit binding specificity for a specific antigen. Specific examples of antibodies include intact antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, nanobodies, and multispecific antibodies. Classical antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on conservative differences in their amino acid sequences, heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable region is responsible for recognizing and binding to the antigen, while the constant region (e.g., the Fc fragment) is responsible for initiating downstream effects such as antibody-dependent cellular cytotoxicity (DCC). Within the variable regions of the heavy and light chains, there are three localized regions with a high degree of variation in amino acid composition and sequence. These regions are key locations for antibody-antigen binding and are therefore also called complementarity-determining regions (CDRs). The amino acid sequences of the CDRs can be determined using art-recognized numbering schemes, such as Kabat, Chothia, IMGT, AbM, or Contact. The three complementarity-determining regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, respectively, while the three complementarity-determining regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, respectively. Each heavy chain variable region (VH) and light chain variable region (VL) is composed of three CDRs and four FR regions, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0026] The term "nanobody" is also called "single domain antibody (sdAb)" or "V H "H antibody" refers to an antibody molecule with antigen-binding ability, including the heavy chain variable region but without the light chain.

[0027] The term "specific binding" means that the Nanobody reacts with a specific target antigen more frequently, more rapidly, longer lastingly and / or with greater affinity than with other target antigens.

[0028] The term "sequence identity" is also referred to as "sequence homology", which refers to the degree of identity between amino acid sequences or nucleotide sequences, generally expressed as a percentage.

[0029] In this application, "functionally conservative variant" or "variant" refers to an amino acid sequence in which one or more amino acid substitutions, deletions, or insertions are introduced on the basis of the parental amino acid sequence without changing the required properties (such as antigen affinity and / or antigen specificity). It can be understood that on the basis of the specific nanobody sequences provided in this application, corresponding variants of the nanobodies provided in this application can be obtained by replacing, deleting, adding a small number of amino acids and verifying or screening the binding ability or biological activity of the obtained products to the corresponding antigens, and these variants should also be included within the scope of this application.

[0030] The term "multivalent construct" refers to a construct having at least two antigen-binding sites / binding units. Multivalent constructs can be, but are not limited to, bivalent constructs, trivalent constructs, or tetravalent constructs.

[0031] The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" are used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers can contain natural and / or non-natural nucleotides. "Nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0032] The term "fusion protein" refers to a protein molecule artificially generated (e.g., by genetic engineering techniques) composed of at least two different peptide segments. These peptide segments do not exist in nature or do not exist in the same protein molecule. Common fusion proteins including antibody fragments include, but are not limited to, bispecific antibodies, enzyme-labeled antibodies for immunoassays, etc.

[0033] The term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (such as the coding sequence of a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (e.g., nucleic acid, plasmid, or virus, etc.). A vector can include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate the expression of the polynucleotide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used for colorimetric analysis, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0034] 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 above nanobody are selected from any one of the groups (1)-(11): (1): CDR1 with the amino acid sequence shown in SEQ ID NO.1, CDR2 with the amino acid sequence shown in SEQ ID NO.2, and CDR3 with the amino acid sequence shown in SEQ ID NO.3; (2): CDR1 with the amino acid sequence shown in SEQ ID NO.8, CDR2 with the amino acid sequence shown in SEQ ID NO.9, and CDR3 with the amino acid sequence shown in SEQ ID NO.10; (3): CDR1 with the amino acid sequence shown in SEQ ID NO.14, CDR2 with the amino acid sequence shown in SEQ ID NO.15, and CDR3 with the amino acid sequence shown in SEQ ID NO.16; (4): CDR1 with the amino acid sequence shown in SEQ ID NO.20, CDR2 with the amino acid sequence shown in SEQ ID NO.21, and CDR3 with the amino acid sequence shown in SEQ ID NO.22; (5): CDR1 with the amino acid sequence shown in SEQ ID NO.26, CDR2 with the amino acid sequence shown in SEQ ID NO.27, and CDR3 with the amino acid sequence shown in SEQ ID NO.28; (6): CDR1 with the amino acid sequence shown in SEQ ID NO.31, CDR2 with the amino acid sequence shown in SEQ ID NO.32, and CDR3 with the amino acid sequence shown in SEQ ID NO.33; (7): CDR1 with the amino acid sequence shown in SEQ ID NO.35, CDR2 with the amino acid sequence shown in SEQ ID NO.36, and CDR3 with the amino acid sequence shown in SEQ ID NO.37; (8): CDR1 with the amino acid sequence shown in SEQ ID NO.40, CDR2 with the amino acid sequence shown in SEQ ID NO.41, and CDR3 with the amino acid sequence shown in SEQ ID NO.42; (9): An amino acid sequence having at least 90% sequence homology with (1), (2), (3), (4), (5), (6), (7), or (8); (10): A functionally conservative variant of the amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7), or (8) that has undergone one or more of amino acid substitution, deletion, or insertion; (11): A chemically modified sequence of the amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7), or (8).

[0035] The nanobody provided by the present application can specifically bind to AAV2 adeno-associated virus and does not bind to other AAV serotypes such as AAV5, AAV8, and AAV9. It can be used for purifying AAV2 virus. During the purification process, AAV2 virus can be captured and eluted very gently, and the recovered AAV2 virus has high purity.

[0036] In some embodiments, the heavy chain variable region of the above-mentioned nanobody further includes framework regions FR1, FR2, FR3, and FR4.

[0037] In some embodiments, the amino acid sequence of the framework region of the above-mentioned nanobody is selected from any one of groups (a)-(k): (a): FR1 with the amino acid sequence shown in SEQ ID NO.4, FR2 with the amino acid sequence shown in SEQ ID NO.5, FR3 with the amino acid sequence shown in SEQ ID NO.6, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (b): FR1 with the amino acid sequence shown in SEQ ID NO.11, FR2 with the amino acid sequence shown in SEQ ID NO.12, FR3 with the amino acid sequence shown in SEQ ID NO.13, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (c): FR1 with the amino acid sequence shown in SEQ ID NO.17, FR2 with the amino acid sequence shown in SEQ ID NO.18, FR3 with the amino acid sequence shown in SEQ ID NO.19, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (d): FR1 with the amino acid sequence shown in SEQ ID NO.23, FR2 with the amino acid sequence shown in SEQ ID NO.24, FR3 with the amino acid sequence shown in SEQ ID NO.25, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (e): FR1 with the amino acid sequence shown in SEQ ID NO.23, FR2 with the amino acid sequence shown in SEQ ID NO.29, FR3 with the amino acid sequence shown in SEQ ID NO.30, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (f): FR1 with the amino acid sequence shown in SEQ ID NO.23, FR2 with the amino acid sequence shown in SEQ ID NO.29, FR3 with the amino acid sequence shown in SEQ ID NO.34, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (g): FR1 with the amino acid sequence shown in SEQ ID NO.38, FR2 with the amino acid sequence shown in SEQ ID NO.24, FR3 with the amino acid sequence shown in SEQ ID NO.39, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (h): FR1 with the amino acid sequence shown in SEQ ID NO.43, FR2 with the amino acid sequence shown in SEQ ID NO.44, FR3 with the amino acid sequence shown in SEQ ID NO.45, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (i): An amino acid sequence having at least 90% sequence homology with (a), (b), (c), (d), (e), (f), (g), or (h); (j): A functionally conservative variant of the amino acid sequence shown in (a), (b), (c), (d), (e), (f), (g), or (h) that has undergone one or more of substitution, deletion, or insertion of one or more amino acids. (k): A sequence in which the amino acid sequence shown in (a), (b), (c), (d), (e), (f), (g), or (h) has been chemically modified.

[0038] In some embodiments, the amino acid sequence of the variable region of the heavy chain of the above-mentioned nanobody is selected from any one of (I)-(IV): (I): The amino acid sequence is any one of SEQ ID NO.46 to SEQ ID NO.53; (II): An amino acid sequence having at least 90% sequence identity with (I); (III): A functionally conservative variant of the amino acid sequence shown in (I) that has undergone one or more of substitution, deletion, or insertion of one or more amino acids; (IV): A sequence in which the amino acid sequence shown in (I) has been chemically modified.

[0039] It should be noted that if an amino acid sequence has at least 90% sequence identity (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with any one of SEQ ID NO.46 to SEQ ID NO.53, and this amino acid sequence also has excellent antigen (AAV2 adeno-associated virus) affinity and antigen specificity, it is also within the scope of protection of this application.

[0040] In some embodiments, the above-mentioned chemical modifications include one or more of cyclization, N-methylation, acetylation, phosphorylation, succinylation, myristoylation, palmitoylation, glycosylation, isoprenylation, ubiquitination, biotinylation, polyethylene glycol modification, and fluorescent labeling.

[0041] Due to their small size and compact folding, nanobodies exhibit high chemical stability, solubility, and rapid tissue penetration. In addition, nanobodies can be easily converted into multivalent formats, such as bivalent, trivalent, tetravalent, or other multispecific constructs. Based on this, this application provides a multivalent construct that includes at least one of the above-mentioned nanobodies, or a combination thereof.

[0042] In some embodiments, the above-mentioned multivalent construct is a bivalent construct or a trivalent construct.

[0043] This application provides a fusion protein that includes one or more antigen-binding functional parts, wherein the antigen-binding functional part includes the above-mentioned nanobody.

[0044] In some embodiments, the above-mentioned fusion protein further comprises a detection tag or a purification tag. Among them, the detection tag is conducive to detecting the nanobody, or is conducive to detecting the immune complex formed by the nanobody and the corresponding antigen (AAV or its VP protein), and can be used to indicate the presence or content of AAV or its VP protein in the sample. The present application has no special limitation on the detection tag, including but not limited to enzymes commonly used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.; and fluorescent proteins, such as GFP. The purification tags include but are not limited to His6 tag, Flag tag, MBP tag, GST tag, SUMO tag, etc., which are conducive to separating the above-mentioned nanobody from the sample.

[0045] The present application also provides a solid-phase carrier for the isolation and / or purification of AAV2 adeno-associated virus, and the surface of the solid-phase carrier is conjugated with the above-mentioned nanobody. In some embodiments, the above-mentioned nanobody binds to functional groups such as hydroxyl groups, carboxyl groups, and amino groups on the surface of the solid-phase carrier through a coupling agent and thus is immobilized on the surface of the solid-phase carrier. The above-mentioned solid-phase carrier includes but is not limited to polymer microspheres, agarose gels, dextrans, celluloses, polycarbonates, nitrocelluloses, silica, or magnetic microspheres, etc.

[0046] In practical applications, when the above-mentioned solid-phase carrier is used for the isolation and / or purification of AAV2 adeno-associated virus, an affinity packing material can be prepared from the above-mentioned solid-phase carrier conjugated with the nanobody on the surface, and then the affinity packing material is contacted with the sample solution to adsorb the 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 realizing the isolation or purification of AAV2 adeno-associated virus.

[0047] The present application also provides a nucleic acid, which comprises a nucleic acid sequence encoding the above-mentioned nanobody.

[0048] As is well known to those skilled in the art, the nucleic acid sequence encoding the nanobody is operably linked to a vector. Based on this, the present application provides an expression vector comprising the above-mentioned nucleic acid molecule. It can be understood that the above-mentioned vector refers to any genetic element, including but not limited to plasmids, phages, transposons, cosmids, chromosomes, viruses (such as retroviruses, lentiviruses), virus particles, etc., which can replicate when combined with appropriate control elements and can transfer gene sequences to cells.

[0049] In some embodiments, the above-mentioned expression vector may further comprise other genes, such as a marker gene that allows the selection of the expression vector in a suitable host cell and under suitable conditions. In addition, the above-mentioned expression vector may further comprise expression control elements that allow the correct expression of the coding region in a suitable host. The above-mentioned expression control elements include but are not limited to promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, etc.

[0050] The present application also provides a host cell, which includes the above nucleic acid molecule or the above expression vector and can be used for preparing the above nanobody. Among them, the host cell can be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells include mammalian cells (such as primate or non-primate animal 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, as well as their derivative cells, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells.

[0051] The nanobody, multivalent construct, fusion protein, or solid-phase carrier provided by the present application can be used for the isolation and / or purification of AAV2 adeno-associated virus.

[0052] The present application also provides the use of the above nanobody, the above multivalent construct, the above fusion protein, or the solid-phase carrier in the preparation of a kit for detecting or purifying AAV2 adeno-associated virus. In the art, methods for detecting antigens using antibodies include, but are not limited to, antigen-antibody precipitation reaction, immunodiffusion experiment, immunoturbidimetry, immunoelectrophoresis (including immunoblotting), immunofluorescence technique, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), etc. Among them, the ELISA method can also be divided into types such as direct ELISA, indirect ELISA, and sandwich ELISA. These detection methods can all use the nanobody provided by the present application to detect AAV2 adeno-associated virus.

[0053] On the other hand, the present application also provides a kit for detecting AAV2 adeno-associated virus, which includes the above nanobody, the above multivalent construct, and the above fusion protein. It can be understood that when using the fusion protein for immunoassay, various enzymes (such as HRP) or fluorescent proteins (such as GFP) can be used to facilitate the detection of AAV2 adeno-associated virus.

[0054] The present application also provides a kit for purifying AAV2 adeno-associated virus, which includes the above nanobody, the above multivalent construct, or the above fusion protein, and also includes the above solid-phase carrier.

[0055] It should be understood that materials with the same or similar types, models, qualities, properties, or functions as the reagents and instruments used in the following examples can all be used to implement the present application. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified.

[0056] The main reagents and material sources used in the following examples are as follows: The lymphocyte separation solution was purchased from Solarbio Science & Technology Co., Ltd., the TRIzol™ Reagent kit was purchased from Thermo Fisher Scientific, the Taq enzyme was purchased from TAKARA Bio Inc., the rabbit anti-goat secondary antibody was purchased from GenScript Biotech Corporation, the NHS-activated 4FF affinity packing material and the SC-Ni NTA FF affinity column were both purchased from Shangchun Biotechnology (Wuhan) Co., Ltd., the iTaq Universal SYBR Green Supermix was purchased from Bio-Rad Laboratories, and the AVIpure AAV2 affinity resin was purchased from REPLIGEN Corporation.

[0057] The following are the examples: Example 1 Screening of Nanobodies Against AAV2 Adeno-Associated Virus 1.1 Preparation of Alpaca Immune Library An adult healthy alpaca was immunized with purified AAV2 adeno-associated virus, and the immunization dose was 1×10 11 virus particles per animal. A total of 3 immunizations were carried out at an interval of 2 weeks. After the immunization, 50 mL of alpaca peripheral blood was collected, and B lymphocytes were isolated using a lymphocyte separation solution (product number P8900).

[0058] 1.2 Nested PCR Amplification The total RNA of B lymphocytes was extracted using the TRIzol™ Reagent kit (product number 15596018) and reverse transcribed into cDNA. Using this cDNA as a template, the VHH gene was amplified by nested PCR. The DNA polymerase used for nested PCR was Taq enzyme (product number R001A), and the amplification program was set according to the Taq instruction manual. The primers used for nested PCR are shown in Table 1.

[0059] Table 1 Primers Used for Nested PCR

[0060] 1.3 Construction of Nanobody Phage Display Library The amplified VHH gene fragment and the phagemid display vector pComb 3XSS were digested with Sfil restriction endonuclease respectively, and then the digested VHH gene fragment and the linearized vector pComb 3XSS were ligated using T4 DNA ligase. The ligation product was recovered, and the ligated product was electrotransformed into Escherichia coli (E. coli) TG1 competent cells to construct a nanobody phage display library. The library capacity was determined to be 1.83×10 9 by the plate gradient dilution method, and the cloning efficiency of the nanobody gene inserted into the vector was 98%.

[0061] 1.4 Screening for Nanobodies against Adeno-associated Virus 2 (AAV2) According to the requirements of immunoaffinity purification, it is desirable to obtain nanobodies with "fast on and fast off" binding characteristics. Different from the conventional screening strategy (the incubation time for the nanobody phage display library and antigen binding is 30 minutes), in this application, the incubation time for the library and antigen binding is significantly shortened, and at the same time, a mild elution method is adopted to screen for nanobodies against AAV2 adeno-associated virus. The specific screening steps are as follows: First round of screening: Coat a microtiter plate with AAV2 adeno-associated virus at 100 μL / well, and the virus coating dose is 1×10 11 vp / mL. Incubate overnight at 4°C, and the next day, block with 1% bovine serum albumin (BSA), and wash the microtiter plate with PBST; add 100 μL of the above-constructed nanobody phage display library, incubate at room temperature for 10 minutes, and then wash the microtiter plate with PBST and PBS respectively; add 100 μL of citrate buffer (50 mM, pH 3.0) to the microtiter plate, gently shake for 2 minutes, and collect the eluate; infect E. coli TG1 in the logarithmic growth phase with the eluate, take a small amount of the infected E. coli TG1 and determine the titer of the eluate by the plate gradient dilution method, and amplify the remaining to obtain an AAV2-specific phage enrichment library.

[0062] Second round of screening: Use the same operation as in the "first round of screening", except that the microtiter plate is blocked with 1% ovalbumin (OVA), the library incubation time is 5 minutes, and 100 μL of the enrichment library obtained from the first round of screening is used to bind to the microtiter plate coated with AAV2 adeno-associated virus. After elution and library amplification operations, an AAV2-specific phage enrichment library is obtained.

[0063] Third round of screening: Use the same operation as in the "first round of screening", except that the library incubation time is 5 minutes, and 100 μL of the enrichment library obtained from the second round of screening is used to bind to the microtiter plate coated with AAV2 adeno-associated virus.

[0064] Fourth round of screening: Use the same operation as in the "first round of screening", except that the microtiter plate is blocked with 1% ovalbumin (OVA), the library incubation time is 5 minutes, and 100 μL of the enrichment library obtained from the third round of screening is used to bind to the microtiter plate coated with AAV2 adeno-associated virus.

[0065] Infect E. coli TG1 with the phage eluate obtained from the fourth round of screening, randomly pick monoclonal colonies from the culture dish for titer determination, inoculate them into a 2 mL deep well plate, and culture at 30°C until OD 600Approximately 0.5, induced the expression of VHH with 4 mM IPTG at the final concentration, and centrifuged to collect the bacterial culture supernatant containing the nanobody. The specific reaction of the nanobody to adeno-associated virus 2 (AAV2) was detected by enzyme-linked immunosorbent assay (ELISA), and the specific steps were as follows: Coated with 1×10 11 vp / mL of AAV2 adeno-associated virus, blocked with 5% skim milk, and then added with the appropriately diluted bacterial culture supernatant containing the nanobody, incubated at 37 °C for 1 h, washed 3 times with PBST to wash away the unbound antibodies, then added with a 1:5000 dilution of HRP-conjugated rabbit anti-goat secondary antibody, after incubation at 37 °C for 1 h, washed 3 times with PBST, and then added with TMB (3,3′,5,5′-tetramethylbenzidine) substrate solution for color development for 15 min, and the color development was terminated with 2 M H2SO4. The absorbance value of each well was read at a wavelength of 450 nm. A negative control (bacterial culture supernatant containing only the blocking agent skim milk and no nanobody) and a blank well (buffer) were set. If the OD 450 value of the well to be tested was more than 3 times that of the negative control, it was determined to be positive. The detection results of the positive clones are shown in Table 2.

[0066] Table 2 Detection results of positive clones screened in this application

[0067] As can be seen from Table 2, 8 positive clones screened in this application have affinity for AAV2 adeno-associated virus.

[0068] Example 2 Preparation and large-scale production of anti-AAV2 adeno-associated virus nanobody 2.1 Preparation of anti-AAV2 adeno-associated virus nanobody The clones corresponding to the positive wells in Example 1 were preserved, and the vectors were extracted for sequencing. The antibody sequencing work was completed by Suzhou Genewiz Co., Ltd., and the VHH coding gene sequence was obtained. After translating the DNA sequence into an amino acid sequence and comparing, the clones with the same CDR1, CDR2, and CDR3 sequences were regarded as the same antibody strain, and the clones with different CDR sequences were regarded as different antibody strains.

[0069] The anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8 were obtained by sequencing. The complementarity-determining regions CDR (CDR1~CDR3) and framework regions FR (FR1~FR4) in their heavy chain variable regions are shown in Tables 3 to 10. The amino acid sequences of the heavy chain variable regions of the anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8 are shown in Table 11, and the amino acid sequences are divided based on the IMGT numbering rules.

[0070] Table 3 Sequence information of complementarity-determining regions CDR1~CDR3 and framework regions FR1~FR4 of nanobody A2-1

[0071] Table 4 Sequence information of complementary determining regions CDR1 - CDR3 and framework regions FR1 - FR4 of nanobody A2 - 2

[0072] Table 5 Sequence information of complementary determining regions CDR1 - CDR3 and framework regions FR1 - FR4 of nanobody A2 - 3

[0073] Table 6 Sequence information of complementary determining regions CDR1 - CDR3 and framework regions FR1 - FR4 of nanobody A2 - 4

[0074] Table 7 Sequence information of complementary determining regions CDR1 - CDR3 and framework regions FR1 - FR4 of nanobody A2 - 5

[0075] Table 8 Sequence information of complementary determining regions CDR1 - CDR3 and framework regions FR1 - FR4 of nanobody A2 - 6

[0076] Table 9 Sequence information of complementary determining regions CDR1 - CDR3 and framework regions FR1 - FR4 of nanobody A2 - 7

[0077] Table 10 Sequence information of complementary determining regions CDR1 - CDR3 and framework regions FR1 - FR4 of nanobody A2 - 8

[0078] Table 11 Amino acid sequence information of the heavy - chain variable regions of nanobodies A2 - 1 to A2 - 8

[0079] 2.2 Large - scale production of anti - AAV2 adeno - associated virus nanobodies The VHH-encoding gene fusion sequence with a 6×His tag at the N-terminus was synthesized by gene synthesis. Among them, the VHH-encoding gene can be any one of nanobodies A2-1 to A2-8. Then, the fusion gene was inserted between the NcoI and NheI restriction sites of the PET25b vector. The obtained expression vector PET25b was transformed into Escherichia coli Rosetta(DE3) competent cells to obtain a VHH-expressing recombinant strain. The recombinant strain was inoculated into LB medium containing 50 μg / mL ampicillin (Amp) at an inoculation amount of 1%, and cultured with shaking at 37 °C until OD 600 reached 0.8. IPTG with a final concentration of 0.5 mM was added, and induction was carried out at 22 °C and 200 rpm for 16 h. The supernatant was discarded by centrifugation, and the cells were resuspended in 20 mL of PBS (20 mM, pH 7.4) buffer. The supernatant was discarded by centrifugation again. The washed cell mass was resuspended in 20 mL of PBS and sonicated for 3 - 5 min to break the cell suspension until the solution became clear. Then, centrifugation was carried out at 10,000 rpm for 20 min to separate the supernatant. The supernatant of the broken cells was purified by a nickel column (SC-Ni NTA FF affinity column, product number SCB0E005B). The impurities were washed with 10 column volumes (CV) of washing buffer (PBS + 10 mM imidazole), and the target protein was 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 purified nanobodies. The protein concentration was determined by the BCA method, and the purity was detected by SDS-PAGE.

[0080] The SDS-PAGE detection results showed that the purity of anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8 was greater than 95%.

[0081] Example 3 Specificity of anti-AAV2 adeno-associated virus nanobodies A2-1 to A2-8 Coat 1×10 11 vp / mL of AAV2, AAV5, AAV8, and AAV9 adeno-associated viruses, block with 5% skim milk, and add different nanobody solutions (nanobodies A2-1 to A2-8) respectively. After binding at 37 °C for 1 h, wash 3 times with PBST to wash away unbound antibodies. Then, add a 1:5000 diluted HRP-conjugated rabbit anti-goat secondary antibody. After binding at 37 °C for 1 h, wash 3 times with PBST, and then add TMB (3,3',5,5′-tetramethylbenzidine) substrate solution for color development for 15 min. Stop the color development with 2 M H2SO4. Read the absorbance value of each well at a wavelength of 450 nm. The specific detection results are shown in Table 12.

[0082] Table 12 Specificity of nanobodies A2-1 to A2-8

[0083] 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 and do not bind to other serotype viruses such as AAV5, AAV8, and AAV9, indicating that the nanobodies A2-1 to A2-8 provided in this application can specifically bind to adeno-associated virus AAV2.

[0084] Example 4 Preparation of nanobody-conjugated affinity packing material and purification of adeno-associated virus AAV2 using the same 4.1 Pre-activation of affinity packing material Take 2 g of NHS-activated 4FF affinity packing material (product number SCB0E009), and filter and wash it 3 times with pre-cooled 1 mM hydrochloric acid (0°C - 4°C), with each washing for 10 min.

[0085] 4.2 Preparation of nanobody solution The purified nanobody (nanobodies A2-1 to A2-8) solution was exchanged into the coupling buffer (sodium bicarbonate buffer, pH 8.5) using a 3KDa ultrafiltration membrane, and the protein concentration was determined by the BCA protein quantification method.

[0086] 4.3 Conjugation of nanobody to affinity packing material Add 2 g of NHS-activated 4FF affinity packing material to 6 mL of nanobody solution. The mass ratio of the affinity packing material to the nanobody is 1 g:10 mg. React at 28°C and 250 rpm for 3.5 h. After the reaction, block it overnight with 1 M ethanolamine (pH 8.0) to obtain the affinity packing material conjugated with nanobody.

[0087] 4.4 Preparation of AAV virus loading solution The harvested solution of AAV virus (adeno-associated viruses AAV2, AAV5, AAV8, AAV9) obtained after fermentation was lysed using Tween 20, and nuclease was added for incubation. The incubated fermentation broth was filtered through a 0.22 μm filter membrane to obtain the AAV supernatant. Then, the AAV supernatant was concentrated and exchanged using an ultrafiltration membrane to make the AAV sample after exchange consistent with the affinity packing material equilibration solution. The sample after exchange was passed through a 0.22 μm filter membrane, and the filtrate was collected as the AAV virus loading solution for the affinity packing material.

[0088] 4.5 Purification of adeno-associated virus AAV using the affinity packing material conjugated with nanobody Load 1 mL of the affinity packing material conjugated with nanobody prepared in step 4.3 into a chromatography column, and use the AAV2 adeno-associated virus loading solution prepared in step 4.4 for loading respectively to detect the purification effect. Before purification, equilibrate the chromatography column with PBS equilibration solution. After loading, elute the chromatography column with citric acid (pH 2.5), phosphoric acid (pH 1.9), and 4 M guanidine hydrochloride respectively. Use the qPCR method to detect the viral genome titer in the eluted sample. The specific operation is as follows: After diluting the sample 10 - 100 times with purified water, take 200 μL of the diluted sample for double enzyme digestion with nuclease and proteinase K. After treatment, dilute the sample appropriately by several times, configure the qPCR reaction system (as shown in Table 13), and perform detection according to the qPCR reaction program (as shown in Table 14). The primers used in qPCR include the upstream primer Q-F and the downstream primer Q-R. The nucleotide sequence of the upstream primer Q-F is: 5’-CCGTTGTCAGGCAACGTG-3’; the nucleotide sequence of the downstream primer Q-R is: 5’-AGCTGACAGGTGGTGGCAAT-3’. Calculate the AAV purification yield according to the viral genome titers of the sample before and after purification. The recovery rate calculation method is: (viral genome titer in the purified sample / viral genome titer in the loading solution) × 100%. At the same time, use SDS-PAGE silver staining to detect the purity of the eluted sample.

[0089] Among them, the control group is the imported packing material AVIpure AAV2 affinity resin.

[0090] Table 13 qPCR reaction system

[0091] Table 14 qPCR reaction program

[0092] The qPCR detection results of the affinity packing materials conjugated with 8 nanobodies respectively prepared in this application for purifying AAV2, AAV5, AAV8, and AAV9 adeno-associated viruses are shown in Table 15. Among them, AC-Load represents the sample before purification of the adeno-associated virus. This sample has been clarified and filtered and has been exchanged into the PBS equilibration solution in the purification step; AC-FT represents the flow-through in the purification step; AC-E represents the virus eluate in the purification step. Most of the purified virus will be eluted in the citric acid (pH 2.5) eluate. Figure 1 The SDS-PAGE silver staining detection results of the AAV2 virus purified by the affinity packing materials conjugated with 8 nanobodies respectively prepared in this application are shown. Among them, the lane Marker represents the molecular marker.

[0093] Table 15 qPCR detection results of the affinity packing material conjugated with nanobody for purifying AAV2 adeno-associated virus

[0094] As shown in Table 15, Figure 1 it can be seen that after the 8 nanobodies prepared by the present application are respectively conjugated with affinity fillers, they can be used to purify AAV2 virus. During the purification process, the AAV2 virus can be captured and eluted very gently; and the purity of the recovered AAV2 virus is high.

[0095] In summary, the nanobodies A2-1 to A2-8 prepared by the present application can specifically bind to AAV2 adeno-associated virus, and have good effect on affinity purification of AAV2 virus. In addition, the preparation process of the nanobodies provided by the present application is short, and the incubation and binding time of the phage library containing nanobodies and the antigen (AAV2 adeno-associated virus) is only 10 minutes. During actual production, it can greatly reduce the production cost and is conducive to batch production.

[0096] It is easy for those skilled in the art to understand that the above are only the preferred embodiments of the present application, and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A nanobody that specifically binds to AAV2, the variable region of the heavy chain of the nanobody comprising complementarity determining regions CDR1, CDR2, and CDR3, characterized in that, The amino acid sequences of the complementarity-determining regions of the nanobody are selected from any one of groups (1)-(11): (1): CDR1 with the amino acid sequence shown in SEQ ID NO.1, CDR2 with the amino acid sequence shown in SEQ ID NO.2, and CDR3 with the amino acid sequence shown in SEQ ID NO.3; (2): CDR1 with the amino acid sequence shown in SEQ ID NO.8, CDR2 with the amino acid sequence shown in SEQ ID NO.9, and CDR3 with the amino acid sequence shown in SEQ ID NO.10; (3): CDR1 with the amino acid sequence shown in SEQ ID NO.14, CDR2 with the amino acid sequence shown in SEQ ID NO.15, and CDR3 with the amino acid sequence shown in SEQ ID NO.16; (4): CDR1 with the amino acid sequence shown in SEQ ID NO.20, CDR2 with the amino acid sequence shown in SEQ ID NO.21, and CDR3 with the amino acid sequence shown in SEQ ID NO.22; (5): CDR1 with the amino acid sequence shown in SEQ ID NO.26, CDR2 with the amino acid sequence shown in SEQ ID NO.27, and CDR3 with the amino acid sequence shown in SEQ ID NO.28; (6): CDR1 with the amino acid sequence shown in SEQ ID NO.31, CDR2 with the amino acid sequence shown in SEQ ID NO.32, and CDR3 with the amino acid sequence shown in SEQ ID NO.33; (7): CDR1 with the amino acid sequence shown in SEQ ID NO.35, CDR2 with the amino acid sequence shown in SEQ ID NO.36, and CDR3 with the amino acid sequence shown in SEQ ID NO.37; (8): CDR1 with the amino acid sequence shown in SEQ ID NO.40, CDR2 with the amino acid sequence shown in SEQ ID NO.41, and CDR3 with the amino acid sequence shown in SEQ ID NO.42; (9): An amino acid sequence having at least 90% sequence homology with (1), (2), (3), (4), (5), (6), (7) or (8); (10): A functionally conservative variant of the amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8) that has undergone one or more of substitution, deletion, insertion of one or several amino acids; (11): A chemically modified sequence of the amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8).

2. The nanobody according to claim 1, characterized in that, The variable heavy chain of the nanobody further includes framework regions FR1, FR2, FR3, FR4; The amino acid sequences of the framework regions of the nanobody are selected from any one of groups (a)-(k): (a): FR1 with the amino acid sequence shown in SEQ ID NO.4, FR2 with the amino acid sequence shown in SEQ ID NO.5, FR3 with the amino acid sequence shown in SEQ ID NO.6, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (b): FR1 with the amino acid sequence shown in SEQ ID NO.11, FR2 with the amino acid sequence shown in SEQ ID NO.12, FR3 with the amino acid sequence shown in SEQ ID NO.13, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (c): FR1 with the amino acid sequence shown in SEQ ID NO.17, FR2 with the amino acid sequence shown in SEQ ID NO.18, FR3 with the amino acid sequence shown in SEQ ID NO.19, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (d): FR1 with the amino acid sequence shown in SEQ ID NO.23, FR2 with the amino acid sequence shown in SEQ ID NO.24, FR3 with the amino acid sequence shown in SEQ ID NO.25, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (e): FR1 with the amino acid sequence shown in SEQ ID NO.23, FR2 with the amino acid sequence shown in SEQ ID NO.29, FR3 with the amino acid sequence shown in SEQ ID NO.30, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (f): FR1 with the amino acid sequence shown in SEQ ID NO.23, FR2 with the amino acid sequence shown in SEQ ID NO.29, FR3 with the amino acid sequence shown in SEQ ID NO.34, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (g): FR1 with the amino acid sequence shown in SEQ ID NO.38, FR2 with the amino acid sequence shown in SEQ ID NO.24, FR3 with the amino acid sequence shown in SEQ ID NO.39, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (h): FR1 with the amino acid sequence shown in SEQ ID NO.43, FR2 with the amino acid sequence shown in SEQ ID NO.44, FR3 with the amino acid sequence shown in SEQ ID NO.45, and FR4 with the amino acid sequence shown in SEQ ID NO.7; (i): An amino acid sequence having at least 90% sequence homology with (a), (b), (c), (d), (e), (f), (g) or (h); (j): A functionally conservative variant of the amino acid sequence shown in (a), (b), (c), (d), (e), (f), (g) or (h) that has undergone one or more of substitution, deletion, insertion of one or several amino acids; (k): A sequence obtained by chemically modifying the amino acid sequence shown in (1), (2), (3), (4), (5), (6), (7) or (8); Preferably, the amino acid sequence of the heavy chain variable region of the nanobody is selected from any one of (I)-(IV): (I): The amino acid sequence is any one of SEQ ID NO.46 - SEQ ID NO.53; (II): An amino acid sequence having at least 90% sequence homology with (I); (III): A functionally conservative variant of the amino acid sequence shown in (I) that has undergone one or more of substitution, deletion, insertion of one or several amino acids; (IV): A sequence obtained by chemically modifying the amino acid sequence shown in (I).

3. A multivalent construct, characterized in that, It includes at least one of the nanobodies described in claim 1 or 2, or a combination thereof.

4. A fusion protein comprising one or more antigen-binding functional portions, characterized in that, The antigen-binding functional part comprises the nanobody described in claim 1 or 2; Preferably, the fusion protein further comprises a detection tag or a purification tag.

5. A solid-phase carrier for AAV2 adeno-associated virus isolation and / or purification, characterized in that, The surface of the solid-phase carrier is conjugated with the nanobody described in claim 1 or 2.

6. A nucleic acid, characterized in that, It comprises a nucleic acid sequence encoding the nanobody described in claim 1 or 2.

7. An expression vector comprising the nucleic acid described in claim 6.

8. A host cell, characterized in that, It comprises the nucleic acid described in claim 6 or the expression vector described in claim 7.

9. Use of the nanobody described in claim 1 or 2, the multivalent construct described in claim 3, the fusion protein described in claim 4 or the solid-phase carrier described in claim 5 in the immunodetection, enrichment or purification of adeno-associated virus 2 (AAV2).

10. An AAV2 adeno-associated virus detection kit or purification kit, characterized in that, It comprises the nanobody described in claim 1 or 2, the multivalent construct described in claim 3, the fusion protein described in claim 4 or the solid-phase carrier described in claim 5.

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