Nanometer antibody targeting beta-amyloid protein and application thereof

Abstract

The invention relates to the field of biomedicine and molecular biology, in particular to a nanometer antibody targeting beta-amyloid protein and application of the nanometer antibody. The invention provides a nano antibody for targeting beta-amyloid protein. The nano antibody comprises three complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 comprises a sequence as shown in SEQ ID NO. 1; the amino acid sequence of the CDR2 comprises a sequence as shown in SEQ ID NO. 2; the amino acid sequence of the CDR3 comprises a sequence as shown in SEQ ID NO.3; the nano antibody can target and combine various forms of Abeta proteins, can be used for ELISA detection of Abeta proteins, and has the advantages of low synthesis cost, stable properties and the like.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and molecular biology, and particularly to a nanobody targeting amyloid-beta and its uses. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease mainly characterized by memory loss and cognitive impairment. With the aging of the global population, the number of AD patients is increasing continuously. At present, the etiology and development mechanism of AD are not fully understood, but it is known to be related to multiple genetic and environmental factors. Although there are some treatment methods that can temporarily relieve symptoms, so far, there is no treatment that can cure or significantly delay the progression of AD.

[0003] Monoclonal antibody therapy is currently a hot topic in AD treatment research, especially antibodies against key molecules in the AD pathological process. These molecules include amyloid-beta (Aβ) and tau protein, which abnormally aggregate in the brains of AD patients to form plaques and tangles. Monoclonal antibodies against these molecules can specifically bind and remove them, thus promising to slow down the progression of AD. However, current monoclonal antibody therapies face multiple challenges including high cost, limited ability to penetrate the blood-brain barrier (BBB), and potential immunogenicity.

[0004] Compared with traditional monoclonal antibodies, nanobodies (also known as single-domain antibodies or VHHs) offer a new treatment possibility. Due to their small size, nanobodies can more easily penetrate the BBB, which is a key advantage in AD treatment because many large molecule drugs cannot effectively enter the brain. In addition, the production cost of nanobodies is relatively low and can be expressed through relatively simple biological systems such as bacteria or yeast. Their high stability and ease of engineering also provide flexibility for customized design against specific disease markers. These characteristics make nanobodies strong candidates for the treatment of diseases such as AD. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a nanobody targeting amyloid-beta and its uses, wherein the nanobody targeting amyloid-beta specifically binds to amyloid-beta and is used for the detection of amyloid-beta and its application in the diagnosis and treatment of AD.

[0006] For this purpose, the present invention provides the following technical solutions:

[0007] A nanobody targeting amyloid-beta, comprising three complementarity-determining regions CDR1, CDR2, and CDR3;

[0008] The amino acid sequence of CDR1 comprises the sequence shown in SEQ ID NO.1;

[0009] The amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.2;

[0010] The amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO.3.

[0011] Optionally, the amino acid sequence of CDR1 is SEQ ID NO.1 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith;

[0012] and / or, the amino acid sequence of CDR2 is SEQ ID NO.2 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith;

[0013] and / or, the amino acid sequence of CDR3 is SEQ ID NO.3 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith.

[0014] Optionally, it further comprises four framework regions FR1, FR2, FR3 and FR4;

[0015] The amino acid sequence of FR1 comprises the sequence shown in SEQ ID NO.4;

[0016] The amino acid sequence of FR2 comprises the sequence shown in SEQ ID NO.5;

[0017] The amino acid sequence of FR3 comprises the sequence shown in SEQ ID NO.6;

[0018] The amino acid sequence of FR4 comprises the sequence shown in SEQ ID NO.7.

[0019] Optionally, the amino acid sequence of FR1 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith;

[0020] and / or, the amino acid sequence of FR2 is the sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith;

[0021] And / or, the amino acid sequence of FR3 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith;

[0022] And / or, the amino acid sequence of FR4 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith.

[0023] Optionally, the nanobody targeting β-amyloid is one of the following A1) or A2):

[0024] A1), comprising the sequence shown in SEQ ID NO.8;

[0025] A2), a nanobody obtained by connecting a protein tag to the N-terminus and / or C-terminus of the sequence in A1).

[0026] A polynucleotide, a nucleotide sequence encoding the nanobody targeting β-amyloid.

[0027] An expression vector or recombinant vector, comprising the polynucleotide.

[0028] A host cell, the host cell comprising:

[0029] Expressing the nanobody targeting β-amyloid;

[0030] And / or, comprising the polynucleotide;

[0031] And / or, comprising the expression vector or recombinant vector.

[0032] A polypeptide molecule, containing the amino acid sequence of the nanobody targeting β-amyloid.

[0033] The nanobody targeting β-amyloid, the polynucleotide, the expression vector or recombinant vector, the host cell or the polypeptide molecule has the following uses:

[0034] (1), in the use of detecting β-amyloid in non-disease diagnosis;

[0035] (2), in the use of preparing a product for detecting β-amyloid;

[0036] (3), in the use of preparing a product for diagnosing β-amyloid-mediated diseases;

[0037] (4), in the use of preparing a drug for preventing, delaying or treating β-amyloid-mediated diseases

[0038] (5) Use in the preparation of a product for evaluating or screening the efficacy of a drug in treating Alzheimer's disease.

[0039] Optionally, the β-amyloid protein includes Aβ1-42, Aβ1-40, soluble aggregates addls derived from Aβ, and / or fibrils derived from Aβ.

[0040] Optionally, the product includes a reagent, a kit, a test strip, a detection device, or a pharmaceutical composition;

[0041] Optionally, the reagent includes an enzyme-linked immunosorbent assay (ELISA) detection reagent, a Western blot reagent, or an immunohistochemical detection reagent.

[0042] An ELISA kit for detecting β-amyloid protein, comprising the nanobody targeting β-amyloid protein;

[0043] Optionally, it further includes a reagent for binding or paired detection with the nanobody targeting β-amyloid protein;

[0044] Optionally, the reagent includes a reagent for ELISA detection;

[0045] Optionally, the reagent for paired detection includes a 4G8 antibody or a 3D6 antibody.

[0046] A pharmaceutical composition for treating Alzheimer's disease, comprising the nanobody targeting β-amyloid protein and a pharmaceutically acceptable excipient.

[0047] Optionally, the excipient includes any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent, or a buffer.

[0048] The technical solution of the present invention has the following advantages:

[0049] 1. A nanobody targeting β-amyloid protein provided by the present invention contains three complementarity-determining regions CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 contains the sequence shown in SEQ ID NO.1; the amino acid sequence of CDR2 contains the sequence shown in SEQ ID NO.2; the amino acid sequence of CDR3 contains the sequence shown in SEQ ID NO.3; the above-mentioned nanobody can target and bind various forms of Aβ protein, and this nanobody can be used for ELISA detection of Aβ protein, having the advantages of low synthesis cost and stable properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0051] Figure 1 For Example 1 of the present invention, SDS-PAGE electrophoresis was used to detect the expression of nanobody ZxNb4 induced by 1 mM IPTG and the purification by Ni-NTA affinity chromatography; M is the protein molecular weight standard (unit: Kda); Lane 1 is the whole cell protein without IPTG induction; Lane 2 is the whole cell protein induced by IPTG; Lane 3 is the supernatant after cell lysis; Lane 4 is the precipitate after cell lysis; Lane 5 is the total lysate; Lane 6 is the flow-through; Lane 7 is the eluate with 10 mM imidazole; Lane 8 is the eluate with 30 mM imidazole; Lane 9 is the eluate with 300 mM imidazole.

[0052] Figure 2 For Example 1 of the present invention, western blot was used to detect the expression of nanobody ZxNb4 induced by 1 mM IPTG and the purification by Ni-NTA affinity chromatography; Lane 1 is the whole cell protein without IPTG induction; Lane 2 is the whole cell protein induced by IPTG; Lane 3 is the supernatant after cell lysis; Lane 4 is the precipitate after cell lysis; Lane 5 is the total lysate; Lane 6 is the flow-through; Lane 7 is the eluate with 10 mM imidazole; Lane 8 is the eluate with 30 mM imidazole; Lane 9 is the eluate with 300 mM imidazole;

[0053] Figure 3 For Example 2 of the present invention, indirect ELISA was used to detect the binding ability of nanobody ZxNb4 to Aβ1-42, Aβ1-40, and soluble aggregates (addls) derived from Aβ.

[0054] Figure 4 For Example 3 of the present invention, sandwich ELISA was used to detect the ability of nanobody ZxNb4 as a capture antibody to pair with 4G8 and 3D6 antibodies to detect Aβ1-40 monomers and insoluble fibrils formed by Aβ1-42.

[0055] Figure 5 The recombinant plasmid inserted with the ZxNb4 gene in Example 1 of the present invention. Specific Embodiments

[0056] The following embodiments are provided to better understand the present invention further. It is not limited to the described best mode, and does not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0057] For those embodiments where specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0058] As used herein, the term "Aβ" can replace β-amyloid, amyloid-β, amyloid beta, A-beta, A-β. β-amyloid is a peptide of 36 - 43 amino acids, including all wild-type and mutant forms of all types, especially human Aβ. The most common subtypes of human Aβ are Aβ1 - 40 and Aβ1 - 42. In human cerebrospinal fluid and blood, the content levels of Aβ1 - 40 are 10 times and 1.5 times higher than those of Aβ1 - 42 respectively. Aβ1 - 42 has stronger toxicity and is more prone to aggregation, thus forming the core of Aβ precipitation and triggering neurotoxic effects. Under pathological conditions, the production of Aβ in the brains of AD patients is 4 - 10 times that under normal conditions. The aggregation of a large amount of Aβ forms neuritic plaques (senile plaques), and the formation of senile plaques leads to the death of a large number of cells in the hippocampus, which indicates that the extended two amino acids not only increase the hydrophobicity of Aβ, making it more prone to aggregation, but also improve the stability of the aggregates, and can selectively deposit in amyloid plaques at an early stage.

[0059] As used herein, the soluble aggregates (addls) formed by Aβ monomers are such that the soluble addl pool present in the human brain extends to cerebrospinal fluid. The increase in addl levels is correlated with the presence of AD and is a common biomarker of AD.

[0060] As used herein, the insoluble aggregates fibrils formed by Aβ monomers are such that in AD, the self-aggregation of amyloid precursor protein hydrolysis product Aβ42 peptide chains and other hydrolysis product mutants will lead to the aggregation of misfolded fibers (fibrils), and the fibers have strong cytotoxicity, which will cause the death of neurons. Fibrils are one of the pathological morphologies for diagnosing AD.

[0061] As used herein, the terms "nanobody", "nanobody targeting β-amyloid", "ZxNb4", "nanobody ZxNb4" are used interchangeably and all refer to a nanobody that specifically recognizes and binds to β-amyloid. A nanobody is the variable region of a heavy chain antibody and typically contains three complementarity-determining regions CDR1, CDR2, and CDR3, and four framework regions FR1, FR2, FR3, and FR4.

[0062] As used herein, "specifically recognize and bind to β-amyloid" or "target β-amyloid" is not limited to an antigenic determinant, epitope, part, domain, subunit, or conformation of β-amyloid. An "epitope" refers to the antibody-binding site in an antigen that is specifically recognized by an antibody.

[0063] As used herein, "affinity" is the relative degree of antibody binding.

[0064] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences.

[0065] As used herein, a "vector" refers to a construct that is capable of delivering, preferably expressing in a host cell, one or more genes or sequences of interest. Vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors complexed with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes.

[0066] As used herein, "pharmaceutically acceptable" means that a carrier, diluent, or excipient, etc., is compatible with the other components of the formulation and is substantially harmless to the recipient.

[0067] In a first aspect, the present invention provides a nanobody targeting β-amyloid, comprising three complementarity-determining regions CDR1, CDR2, and CDR3;

[0068] The amino acid sequence of CDR1 comprises the sequence shown in SEQ ID NO.1;

[0069] The amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.2;

[0070] The amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO.3.

[0071] In the present invention, in the case where the nanobody targets β-amyloid, the nanobody can specifically recognize and bind to the antigenic determinant, epitope, part, domain, subunit or conformation of β-amyloid, but is not limited to targeting the antigenic determinant, epitope, part, domain, subunit or conformation of β-amyloid. The β-amyloid specifically recognized and bound by the nanobody includes all wild-type and mutant forms of all types. In addition, the β-amyloid can be in monomeric form, polymeric form or protein complex form.

[0072] In some preferred but non-limiting specific embodiments, the amino acid sequence of CDR1 is SEQ ID NO.1 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith; and / or, the amino acid sequence of CDR2 is SEQ ID NO.2 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith; and / or, the amino acid sequence of CDR3 is SEQ ID NO.3 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith.

[0073] In some non-limiting specific embodiments, the nanobody further includes four framework regions FR1, FR2, FR3 and FR4;

[0074] The amino acid sequence of FR1 contains the sequence shown in SEQ ID NO.4;

[0075] The amino acid sequence of FR2 contains the sequence shown in SEQ ID NO.5;

[0076] The amino acid sequence of FR3 contains the sequence shown in SEQ ID NO.6;

[0077] The amino acid sequence of FR4 contains the sequence shown in SEQ ID NO.7.

[0078] In some preferred but non-limiting specific embodiments, the amino acid sequence of FR1 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith; and / or, the amino acid sequence of FR2 is the sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith; and / or, the amino acid sequence of FR3 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith; and / or, the amino acid sequence of FR4 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith

[0079] In some more preferred embodiments, the nanobody targeting β-amyloid is one of the following A1) or A2):

[0080] A1), comprising the sequence shown in SEQ ID NO.8;

[0081] A2), a nanobody obtained by linking a protein tag to the N-terminus and / or C-terminus of the sequence in A1).

[0082] In some preferred but non-limiting specific embodiments, in A1), the nanobody is the sequence shown in SEQ ID NO.8 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% sequence identity therewith.

[0083] In some preferred but non-limiting specific embodiments, in A2), a functional polypeptide or protein, such as a protein tag composed of six histidines, can be fused to the N-terminus and / or C-terminus of the nanobody of the present invention.

[0084] The nanobody of the present invention can substitute, delete, and / or add at least one amino acid based on the specific amino acid sequences listed above, and the resulting nanobody has the activity of binding to β-amyloid protein. The number of the substituted, deleted, or added amino acids can be any value, such as 1, 5, 10, 15, and above, etc., so that the sequence identity between the changed amino acid sequence and its corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above. In the art, when conservative substitutions are made with amino acids having similar or close properties, the function of the protein is usually not changed. For example, amino acids with similar properties are substituted in the CDR region and / or FR region. The amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues are or are not encoded by the genetic code. Therefore, the nanobody with conservative substitutions using amino acids having similar or close properties is within the protection scope of the present invention.

[0085] In a second aspect, the present invention provides a polynucleotide, which is a nucleotide sequence encoding the nanobody targeting β-amyloid protein.

[0086] As used herein, the terms "polynucleotide" and "nucleic acid molecule" can be used interchangeably, including DNA molecules or RNA molecules. The DNA molecule can be single-stranded or double-stranded.

[0087] Due to the degeneracy of the genetic code, a large number of polynucleotides can be obtained for encoding the nanobody of the present invention. Therefore, in the case where a specific amino acid sequence has been identified, those skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. According to the preference of the host cell used in the actual preparation process, after codon optimization, more preferred polynucleotides can be selected.

[0088] The polynucleotide can be obtained by conventional methods, such as PCR amplification method or artificial synthesis method, etc. Currently, the polynucleotide sequence can be completely obtained by chemical synthesis.

[0089] In a third aspect, the present invention provides a vector, which contains the polynucleotide. The vector can be transformed into a host cell to express the polynucleotide or the nanobody.

[0090] In a fourth aspect, the present invention provides a host cell, which includes: expressing the nanobody targeting β-amyloid protein; and / or, containing the polynucleotide; and / or, containing the expression vector or recombinant vector.

[0091] The host cells of the present invention can be prokaryotic cells, lower eukaryotic cells or higher eukaryotic cells. Prokaryotic cells such as bacterial cells, lower eukaryotic cells such as yeast cells, and higher eukaryotic cells such as mammalian cells. Representative examples include Escherichia coli, yeast cells, etc.

[0092] The transformation of the vector into the host cell can be carried out by conventional methods well-known to those skilled in the art. For example, the CaCl2 method, electroporation method, calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. The obtained transformants can be cultured by conventional methods well-known to those skilled in the art, and the culture medium can be a conventional culture medium. The nanobodies produced by the transformants can be separated and purified by physical, chemical and other methods, and can be carried out by conventional methods well-known to those skilled in the art such as salting out, centrifugation, cell disruption, chromatography, etc.

[0093] In the fifth aspect, the present invention provides a polypeptide molecule containing the amino acid sequence of the nanobody targeting β-amyloid protein.

[0094] The polypeptide molecule of the present invention can be a polypeptide molecule after fusion of the nanobody with other functional polypeptides, proteins, etc., or can be in the form of a composition containing at least one nanobody of the present invention and other polypeptides, proteins, antibodies, etc. For example, one of the nanobodies is a monovalent nanobody, and can be a multivalent nanobody composed of multiple different nanobodies.

[0095] In the sixth aspect, the present invention provides the following uses of the nanobody targeting β-amyloid protein, the polynucleotide, the expression vector or recombinant vector, the host cell or the polypeptide molecule:

[0096] (1) Use in detecting β-amyloid protein for non-disease diagnosis;

[0097] (2) Use in preparing products for detecting β-amyloid protein;

[0098] (3) Use in preparing products for diagnosing diseases mediated by β-amyloid protein;

[0099] (4) Use in preparing drugs for preventing, delaying or treating diseases mediated by β-amyloid protein

[0100] (5) Use in preparing products for evaluating or screening the efficacy of drugs in treating Alzheimer's disease.

[0101] In some preferred but non-limiting specific embodiments, the β-amyloid protein includes Aβ1-42, Aβ1-40, soluble aggregates addls derived from Aβ, and / or fibrils derived from Aβ.

[0102] In some preferred but non-limiting specific embodiments, the β-amyloid protein-mediated diseases include Alzheimer's disease.

[0103] In the above uses, the products involved include reagents, kits, test strips, detection devices, or pharmaceutical compositions.

[0104] In some preferred but non-limiting specific embodiments, the reagent includes an enzyme-linked immunosorbent assay (ELISA) detection reagent, a Western blot reagent, or an immunohistochemical detection reagent. Among them, the ELISA can be an indirect ELISA or a sandwich ELISA. For example, in the indirect ELISA, the nanobody of the present invention can be used as the primary antibody, or in the sandwich ELISA, the nanobody of the present invention can be used as the capture antibody and coated on the microplate.

[0105] In a seventh aspect, the present invention provides a detection kit for β-amyloid protein, including the nanobody targeting β-amyloid protein described above.

[0106] In some preferred but non-limiting specific embodiments, the detection kit for β-amyloid protein can be used to detect, diagnose, or monitor β-amyloid protein-mediated diseases.

[0107] In some preferred but non-limiting specific embodiments, the detection kit for β-amyloid protein further includes a reagent for binding or paired detection with the nanobody targeting β-amyloid protein.

[0108] In some preferred but non-limiting specific embodiments, the reagent includes a reagent for enzyme-linked immunosorbent assay detection; for example, the reagent can be a reagent related to indirect ELISA (including but not limited to coated antigen, washing solution, blocking solution, secondary antibody, chromogenic solution, etc.), or a reagent related to sandwich ELISA (including but not limited to antigen, washing solution, blocking solution, primary antibody, secondary antibody, chromogenic solution, etc. For example, the antibodies used for paired detection with the nanobody include but not limited to 4G8 antibody or 3D6 antibody).

[0109] In an eighth aspect, the present invention provides a pharmaceutical composition for treating Alzheimer's disease, including the nanobody targeting β-amyloid protein described above, and a pharmaceutically acceptable excipient.

[0110] The excipients include any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

[0111] Aβ1-42 and Aβ1-40 belong to β-amyloid protein. Addls are soluble aggregates derived from Aβ, and fibrils are insoluble aggregates. Both Aβ1-42 and Aβ1-40 monomers are synthesized by Qiangyao, and addls and fibrils are obtained by incubating Aβ1-42 monomers according to conventional methods.

[0112] The TBST buffer was purchased from Beyotime.

[0113] The ELISA blocking buffer is a commercially available product.

[0114] The secondary antibody anti His-HRP was purchased from GenScript.

[0115] The TMB chromogenic solution was purchased from Beyotime.

[0116] The stop solution was purchased from Beyotime.

[0117] The dilution buffers for 4G8 antibody and 3D6 antibody are both commercially available products. The 4G8 antibody was purchased from biolegend, and the 3D6 antibody was purchased from the United States. 4G8 and 3D6 were diluted with 1% BSA TBST.

[0118] The secondary antibody anti mouse-HRP was purchased from a commercially available product of jackson.

[0119] Example 1 Expression and Purification of Nanobody

[0120] The nanobody targeting β-amyloid protein in the present invention is: QVQLQESGGGLVQAGGSLRLSCAASGNIFRGVPMGWYRQAPGKERELVAGISTGSTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVLGRFRYYFNYWGQGTQVTVSS (as shown in SEQ ID NO.8);

[0121] Among them, it contains three complementary determining regions CDR1, CDR2 and CDR3. The CDR1 sequence is NIFRGVP, the CDR2 sequence is LVAGISTGSTTN, and the CDR3 sequence is AVLGRFRYYFNY, as shown in SEQ ID NO.1-3 respectively;

[0122] It also contains four framework regions FR1, FR2, FR3 and FR4. The sequence of FR1 segment is QVQLQESGGGLVQAGGSLRLSCAASG, the sequence of FR2 segment is MGWYRQAPGKERE, the sequence of FR3 segment is YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC, and the sequence of FR4 segment is WGQGTQVTVSS, as shown in SEQ ID NO.4 - 7 respectively.

[0123] The above - mentioned nanobody was named ZxNb4. The gene sequence encoding nanobody ZxNb4 is as shown in SEQ ID NO.9. The gene sequence of ZxNb4 (the gene sequence was synthesized by GenScript) was inserted into the expression vector pet30a(+) (the plasmid was synthesized by GenScript, and the plasmid map is as Figure 5 shown. When constructing, the gene sequence of the tag protein HHHHHH was ligated to one end of the gene sequence of ZxNb4) and transformed into Escherichia coli BL21(DE3) cells. The heat - shock method was used for transformation: 1 ng of plasmid was added to 100 μl of BL21(DE3) competent cells, gently mixed, incubated on ice for 30 min, heat - shocked at 42 °C for 60 s, then placed on ice for 3 min, 1 ml of antibiotic - free LB medium was added, and cultured in a shaker at 220 rpm and 37 °C for 1 h. Centrifuged at 12000 rpm for 2 min, 900 μl of the supernatant was discarded. After mixing the precipitate, 20 μl was taken and evenly spread on an LB plate (amp 100 μg / ml), and cultured overnight at 37 °C in an inverted position for 15 h to obtain successfully transformed monoclonal colonies. The successfully transformed monoclonal colonies were picked into 5 ml of LB (amp 100 μg / ml) medium and cultured overnight, and then the obtained culture solution was transferred to 1 L of LB (amp 100 μg / ml) medium at a ratio of 1:100 (v / v) and cultured at 37 °C until the OD 600 was about 0.6, 1 mM IPTG (isopropyl - β - D - thiogalactoside) was added for induction, induced at 25 °C for 15 h, and the bacteria were collected by centrifugation at 8000 g for 10 min. The expression of nanobody in bacteria before and after adding IPTG was detected by 12% SDS - PAGE electrophoresis and western blot. The results are as Figure 1-2 shown. In Figure 1 , M is the protein molecular weight standard; lane 1 is the whole - cell protein without IPTG induction; lane 2 is the whole - cell protein after IPTG induction; Figure 2 in Figure 1 , lane 1 is the whole - cell protein without IPTG induction; lane 2 is the whole - cell protein after IPTG induction; in Figure 1 , there is obvious protein expression of the nanobody of the present invention at 15 kDa. By comparing lane 2 with lane 1, it was found that the expression level of the nanobody increased after adding IPTG.

[0124] The collected bacteria after centrifugation were resuspended with 50 ml of lysis buffer (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 10 mM imidazole, 10 v / v% glycerol). The bacteria were sonicated at 200 w and 4 °C to obtain the total lysate, which was centrifuged at 12,000 rpm for 15 min. The supernatant and precipitate were collected to obtain the supernatant and precipitate after cell disruption, respectively. The expression of nanobody in the total lysate, the supernatant after cell disruption, and the precipitate after cell disruption was detected by 12% SDS-PAGE electrophoresis and Western blot. The results are as Figure 1-2 shown. Lane 3 is the supernatant after cell disruption, lane 4 is the precipitate after cell disruption, and lane 5 is the total lysate. By comparison, it can be seen that the nanobody was successfully induced, and most of the nanobody was in the supernatant of the lysate without forming inclusion bodies.

[0125] Take 5 ml of Ni-NTA packing material and load it into the chromatography column. After washing with lysis buffer (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 10 mM imidazole, 10 v / v% glycerol), the Ni-NTA packing material was resuspended with lysis buffer and added to the total lysate in (1) and incubated at 4 °C for 1 h with rotation.

[0126] The incubated lysate was reloaded into the chromatography column, and the flow-through was collected. It was washed with washing buffer 1 (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 10 mM imidazole, 10 v / v% glycerol) and washing buffer 2 (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 30 mM imidazole, 10 v / v% glycerol) for 5 to 6 column volumes. The eluate of washing buffer 1 (eluted with 10 mM imidazole) and the eluate of washing buffer 2 (eluted with 30 mM imidazole) were collected. It was eluted with 15 ml of elution buffer (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 300 mM imidazole, 10 v / v% glycerol) to elute the target protein and collect the eluate (eluted with 300 mM imidazole). The obtained flow-through and the collected eluates were detected by 12% SDS-PAGE electrophoresis and Western blot. The results are as Figure 1-2 shown. By comparison, it can be seen that the nanobody with His tag was successfully purified. The detected concentration was 2 mg / ml, and the total yield was 20 mg / L of bacteria.

[0127] Example 2 Indirect ELISA to Detect the Affinity of ZxNb4 with Aβ1-42, Aβ1-40, and Addls

[0128] This embodiment provides a method for indirectly detecting the affinity of ZxNb4 with Aβ1-42, Aβ1-40, and addls by ELISA, including the following steps:

[0129] (1) Antigen coating: Prepare 200 nM dilutions of Aβ1-42, Aβ1-40, and addls respectively, add 25 μL per well to a 96-well plate, and incubate overnight on a shaker at 4°C;

[0130] (2) Washing: Discard the antigen diluent, add 150 μL of TBST buffer to each well of the 96-well plate, gently tap the 96-well plate on a paper towel to remove the liquid, and repeat washing the 96-well plate three times;

[0131] (3) Blocking: Add 100 μL of ELISA blocking buffer per well to the 96-well plate, and incubate on a shaker at 300 rpm for 1 h at room temperature;

[0132] (4) Washing: Discard the blocking solution, add 150 μL of TBST buffer to each well of the 96-well plate, gently tap the 96-well plate on a paper towel to remove the liquid, and repeat washing the 96-well plate three times;

[0133] (5) Incubation with primary antibody: Prepare a gradient dilution of ZxNb4 with an initial concentration of 100000 ng / mL and a final concentration of 10 0 ng / mL, with a 3-fold serial dilution. Add 25 μL of the dilution per well to the 96-well plate, set 2 replicates for each concentration, and incubate on a shaker at room temperature for 1 h;

[0134] (6) Washing: Discard the primary antibody diluent, add 150 μL of TBST buffer to each well of the 96-well plate, gently tap the 96-well plate on a paper towel to remove the liquid, and repeat washing the 96-well plate four times;

[0135] (7) Incubation with secondary antibody: Dilute the secondary antibody anti His-HRP at 1:1000, add 25 μL per well to the 96-well plate, and incubate on a shaker at 300 rpm for 1 h at room temperature;

[0136] (8) Washing: Discard the secondary antibody diluent, add 150 μL of TBST buffer to each well of the 96-well plate, gently tap the 96-well plate on a paper towel to remove the liquid, and repeat washing the 96-well plate four times;

[0137] (9) Color development: Add 50 μl of TMB chromogenic solution to each well and place at room temperature for 15 min;

[0138] (10) Termination: Add 50 μl of termination solution to each well;

[0139] (11) Detection: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 450 nm within 3 - 5 min after color development is terminated.

[0140] The detection results are as Figure 3 shown. It can be concluded that ZxNb4 can bind to Aβ1-40 monomer, Aβ1-42 monomer and soluble aggregates addls formed by Aβ1-42 with high affinity, and there is no significant difference in the binding ability to these three forms of Aβ in indirect ELISA.

[0141] Example 3 Sandwich ELISA for Detecting the Affinity of ZxNb4 with Aβ1-40 and Fibrils

[0142] (1) Coating with capture antibody: Prepare a 10 μg / ml ZxNb4 dilution, add 25 μL per well to a 96-well plate, and incubate overnight on a shaker at 4°C.

[0143] (2) Washing: Discard the antibody dilution, add 150 μL of TBST to each well of the 96-well plate, tap the 96-well plate gently on a paper towel to remove the liquid, and repeat the washing of the 96-well plate three times.

[0144] (3) Blocking: Add 100 μL of ELISA blocking buffer per well to the 96-well plate, and incubate on a shaker at 300 rpm at room temperature for 1 h.

[0145] (4) Washing: Discard the blocking solution, add 150 μL of TBST to each well of the 96-well plate, tap the 96-well plate gently on a paper towel to remove the liquid, and repeat the washing of the 96-well plate three times.

[0146] (5) Antigen incubation: Prepare gradient dilutions of Aβ1-40 and fibrils with an initial concentration of 200 nM and a final concentration of 0 nM, add 25 μL per well to the 96-well plate, and incubate on a shaker at room temperature for 1 h.

[0147] (6) Incubation with primary antibody dilution (detection antibody): Prepare 4g8 antibody and 3D6 antibody dilutions at 1 μg / ml respectively, add 25 μL of the antibody dilution per well to the 96-well plate, set 2 replicates for each concentration, and incubate on a shaker at room temperature for 1 h.

[0148] (7) Washing: Discard the primary antibody dilution, add 150 μL of TBST to each well of the 96-well plate, tap the 96-well plate gently on a paper towel to remove the liquid, and repeat the washing of the 96-well plate four times.

[0149] (8) Incubation with secondary antibody: Dilute the secondary antibody anti mouse-HRP at 1:40000, add 25 μL per well to the 96-well plate, and incubate on a shaker at 300 rpm at room temperature for 1 h.

[0150] (9) Washing: Discard the secondary antibody dilution, add 150 μL of TBST to each well of the 96-well plate, tap the 96-well plate gently on a paper towel to remove the liquid, and repeat the washing of the 96-well plate four times.

[0151] (10) Color development: Add 50 μl of TMB color development solution to each well and place at room temperature for 15 min; Termination: Add 50 μl of termination solution to each well;

[0152] (11) Detection: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 450 nm within 3 - 5 min after color development termination.

[0153] The detection results are as Figure 4 shown. It can be concluded that ZxNb4 can be combined with two Aβ antibodies for sandwich ELISA detection to identify insoluble fibrils formed by Aβ1 - 42, but not the monomers of Aβ1 - 40.

[0154] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A nanobody targeting β-amyloid protein, characterized in that, It contains three complementarity-determining regions CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 contains the sequence shown in SEQ ID NO.1; The amino acid sequence of CDR2 contains the sequence shown in SEQ ID NO.2; The amino acid sequence of CDR3 contains the sequence shown in SEQ ID NO.

3.

2. The nanobody targeting β-amyloid protein according to claim 1, characterized in that, The amino acid sequence of the said CDR1 is SEQ ID NO.1 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith; and / or, the amino acid sequence of the said CDR2 is SEQ ID NO.2 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith; and / or, the amino acid sequence of the said CDR3 is SEQ ID NO.3 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith.

3. The nanobody targeting β-amyloid protein according to claim 1 or 2, characterized in that, It further includes four framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 contains the sequence shown in SEQ ID NO.4; The amino acid sequence of FR2 contains the sequence shown in SEQ ID NO.5; The amino acid sequence of FR3 contains the sequence shown in SEQ ID NO.6; The amino acid sequence of FR4 contains the sequence shown in SEQ ID NO.

7.

4. The nanobody targeting β-amyloid protein according to claim 3, characterized in that, The amino acid sequence of the said FR1 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith; and / or, the amino acid sequence of FR2 is the sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith; and / or, the amino acid sequence of FR3 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith; and / or, the amino acid sequence of FR4 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 80%, preferably at least 90%, more preferably 95%, even more preferably at least 99% or more sequence identity therewith.

5. The nanobody targeting β-amyloid protein according to any one of claims 1-4, characterized in that, The nanobody targeting β-amyloid protein is the following A1) or A2): A1), containing the sequence shown in SEQ ID NO.8; A2), the nanobody obtained by connecting a protein tag to the N-terminus and / or C-terminus of the sequence in A1).

6. A biological material, characterized in that, It includes: 1), a polynucleotide, the nucleotide sequence encoding the nanobody targeting β-amyloid protein according to any one of claims 1-5; 2), a vector, containing the polynucleotide according to claim 6; 3) a host cell, the host cell includes: An anti-β-amyloid nanobody according to any one of claims 1-5; and / or, comprising the polynucleotide described above; and / or, comprising the vector described above; 4), A polypeptide molecule containing the amino acid sequence of the anti-β-amyloid nanobody according to any one of claims 1-5.

7. The anti-β-amyloid nanobody according to any one of claims 1-5 and the biomaterial according to claim 6 have the following uses: (1), Use in detecting β-amyloid for non-disease diagnosis; (2), Use in preparing a product for detecting β-amyloid; (3), Use in preparing a product for diagnosing β-amyloid-mediated diseases; (4), Use in preparing a drug for preventing, delaying or treating β-amyloid-mediated diseases; (5), Use in preparing a product for evaluating or screening the efficacy of drugs for treating Alzheimer's disease.

8. The use according to claim 7, wherein The β-amyloid includes Aβ1-42, Aβ1-40, Aβ-derived soluble aggregates addls and / or Aβ-derived fibrils; and / or, the β-amyloid-mediated disease includes Alzheimer's disease.

9. Use according to claim 7 or 8, characterized in that, The product includes a reagent, a kit, a test strip, a detection device or a pharmaceutical composition; Optionally, the reagent includes an enzyme-linked immunosorbent assay detection reagent, a western blot reagent or an immunohistochemical detection reagent.

10. A detection kit for beta-amyloid protein, characterized in that, Includes the anti-β-amyloid nanobody according to any one of claims 1-5.

11. The detection kit for β-amyloid protein according to claim 10, characterized in that, Also includes a reagent for binding or paired detection with the anti-β-amyloid nanobody; Optionally, the reagent includes a reagent for enzyme-linked immunosorbent assay detection; Optionally, the reagent for paired detection includes a 4G8 antibody or a 3D6 antibody.

12. A pharmaceutical composition for treating Alzheimer's disease, characterized in that, Includes the anti-β-amyloid nanobody according to any one of claims 1-5, and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition for treating Alzheimer's disease according to claim 12, characterized in that, The excipient includes any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

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