Anti-botulinum toxin type b neutralizing antibodies and related biological materials and uses

Abstract

This invention discloses an anti-botulinum toxin type B neutralizing antibody and its related biomaterials and applications. This invention belongs to the field of biotechnology, specifically relating to an anti-botulinum toxin type B neutralizing antibody and its related biomaterials and applications. The nanobody or antigen-binding fragment containing the nanobody of this invention, targeting human adenovirus, has three complementary determinants CDR1, CDR2, and CDR3; wherein the amino acid sequence of CDR1 is SEQ ID No. 2, the amino acid sequence of CDR2 is SEQ ID No. 3, and the amino acid sequence of CDR3 is SEQ ID No. 4. The nanobody B9 is fused with the Fc fragment (hFc) of human immunoglobulin to obtain a fusion protein, and the resulting B9-hFc can effectively block 20×LD. 50 Botulinum toxin type B poisoning, its IC50 50 It is 1.25 nM.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy biotechnology pharmaceuticals, specifically relating to neutralizing antibodies against botulinum toxin type B and related biomaterials and applications. Background Technology

[0002] Botulinum neurotoxin (BoNT), an exotoxin produced by anaerobic Clostridium bacteria, is currently the most potent known protein. Based on antigenicity, botulinum toxin can be classified into seven types, A-G, with types A, B, E, and F being the main types causing human poisoning. Botulinum toxin blocks the release of neurotransmitters through the respiratory tract, digestive tract, wounds, or eyes, leading to foodborne and iatrogenic botulism. The main symptoms include muscle paralysis, difficulty breathing, and flaccid paralysis, posing a significant biosafety risk.

[0003] Botulinum toxin poisoning has a low incidence rate in the general population, but it is highly toxic and has a high mortality rate. Commonly used preventative vaccines abroad are the pentavalent and heptavalent PBT vaccines, while no such vaccines are currently available in China. Antitoxin is an important and commonly used treatment for poisoning; bivalent and heptavalent antitoxin serums are available abroad, and the Lanzhou Institute of Biomedical Research in China has produced AF-type monovalent antitoxin serum. However, this therapy has many drawbacks, including the potential for severe serum sickness and allergic reactions, and the limited availability of serum restricts its application.

[0004] Antibodies, as an important treatment for botulism, have a dual role in short-term prevention and treatment of poisoning. Currently, most antibody drugs targeting botulinum toxin are still in the molecular discovery stage, with no approved drugs available for use. Therefore, there is an urgent need to develop antibody drugs to enrich the treatment options for botulism. Nanobodies (Nb), which have been developed in recent years, play an important role in disease diagnosis and treatment. They are heavy-chain antibodies naturally found in camelids. Compared to ordinary antibodies, they do not contain light chains and have a long CDR3 region, possessing advantages such as small molecular weight, strong penetrability, high specificity, high expression levels, and exceptionally high modifiability. With these advantages, nanobodies are being rapidly applied to the treatment of various diseases, from viral infections to cancer, and have a promising future in therapeutic drug development.

[0005] Therefore, there is an urgent need in this field to apply nanobody technology to develop highly efficient and safe anti-botulinum toxin neutralizing nanobodies to replace commercially available serum products and meet human needs. Summary of the Invention

[0006] The technical problem to be solved by this invention is to develop a specific nanobody that can efficiently neutralize botulinum toxin, providing candidate antibodies for the diagnosis and prevention of botulism and enriching the means of preventing and treating botulism.

[0007] To address the aforementioned problems, the present invention provides a nanobody targeting botulinum toxin type B or an antigen-binding fragment containing the nanobody.

[0008] The present invention provides a nanobody targeting botulinum toxin type B or an antigen-binding fragment containing the nanobody, wherein the nanobody has three complementary determinant clusters CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is shown in SEQ ID No. 2, the amino acid sequence of CDR2 is shown in SEQ ID No. 3 and the amino acid sequence of CDR3 is shown in SEQ ID No. 4.

[0009] The CDRs mentioned above are sequences defined based on the results of the IMGT system analysis.

[0010] The nanobodies described herein typically comprise a VHH consisting of four framework regions (FRs) and three complementarity-determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antigen-binding fragment contains at least a portion of the nanobodies, which is sufficient to confer the ability of the fragment to specifically bind botulinum toxin type B.

[0011] The four frame regions can be FR1, FR2, FR3 and FR4.

[0012] The amino acid sequence of FR1 is SEQ ID No. 5;

[0013] The amino acid sequence of FR2 is SEQ ID No. 6;

[0014] The amino acid sequence of FR3 is SEQ ID No. 7;

[0015] The amino acid sequence of FR4 is SEQ ID No. 8.

[0016] In the nanobodies or antigen-binding fragments described above, the nanobodies may be any of the following:

[0017] A1) Nanobody with an amino acid sequence as shown in SEQ ID No. 1;

[0018] A2) Nanobodies obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1.

[0019] The protein tag refers to a polypeptide or protein that is fused with the target protein for expression, detection, tracing, and / or purification. The protein tag may be a His tag, Flag tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, or the Fc fragment of immunoglobulin G, etc.

[0020] In this invention, the protein tag is the Fc segment (hFc) of human immunoglobulin G.

[0021] In the above-mentioned nanobody, the nanobody is composed of the complementary determinant cluster region and the framework region.

[0022] In this invention, the term "antibody" refers to a heterotetraglycoprotein of approximately 150,000 Daltons with identical structural features, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.

[0023] The light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0024] In this invention, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning, referring to...

[0025] By cloning the variable region of the antibody heavy chain, a single-domain antibody (VHH) consisting of only one variable region of the heavy chain is constructed. It is the smallest antigen-binding fragment with full function.

[0026] The antigen-binding fragments mentioned above may be complete antibodies, fusion antibodies, antibody-drug conjugates, Fab fragments, Fv fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies (ScFv), or minimum recognition units (MRUs) containing the nanobody.

[0027] The term "Fab fragment" refers to a heterodimer composed of a heavy chain (Fd) and a complete light chain linked by disulfide bonds, containing only one antigen-binding site. The aforementioned heavy chain (Fd) refers to approximately half of the H chain portion of the Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0028] The term "Fv fragment" refers to a vector containing VH and VL genes that can be constructed separately, co-transfected into cells to express them separately, and then assembled into a functional Fv antibody; alternatively, a stop codon can be set between VH and VL in the vector to express two small protein fragments, which can then be bound together by non-covalent bonds to form an Fv antibody (Fv fragment).

[0029] The term "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab' fragments to form the F(ab')2 molecule.

[0030] The term "F(ab')2 segment" contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by the disulfide bond between the two heavy chains.

[0031] In this invention, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments in the variable regions of the light and heavy chains, called complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partially β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0032] In some embodiments, the nanobodies of the present invention may be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of those backbone regions, as long as they substantially maintain antigen binding and specificity.

[0033] In this invention, the nanobody is named nanobody B9.

[0034] This invention also provides biomaterials related to the nanobodies described above, wherein the biomaterials may be any of the following:

[0035] B1) Nucleic acid molecules that encode the nanobodies or antigen-binding fragments described above;

[0036] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0037] B3) A recombinant vector containing the nucleic acid molecules described in B1);

[0038] B4) A recombinant vector containing the expression cassette described in B2);

[0039] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0040] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0041] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0042] B8) Recombinant microorganisms containing the recombinant vector described in B4).

[0043] In the above-mentioned biological materials, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0044] In the aforementioned biological materials, the expression cassette (B2) refers to DNA capable of expressing the nanobody in host cells. This DNA may include not only a promoter to initiate transcription of the nanobody-encoding gene, but also a terminator to terminate transcription of the nanobody-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0045] Recombinant vectors containing the expression cassette can be constructed using existing expression vectors.

[0046] In the aforementioned biological materials, the carrier may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0047] In the above-mentioned biomaterials, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule described in B1) into the nanobody-hFc fusion protein expression vector pTSE-hFc.

[0048] In the above-mentioned biological materials, the microorganisms may be bacteria (such as Escherichia coli), yeast, algae, or fungi.

[0049] In the above-mentioned biomaterials, the nucleic acid molecule described in B1) may be a nucleic acid molecule encoding the nanobody described above. In the nucleic acid molecule, the encoding gene of CDR1 is nucleotides 70-93 of SEQ ID No. 9, the encoding gene of CDR2 is nucleotides 145-165 of SEQ ID No. 9, and the encoding gene of CDR3 is nucleotides 280-333 of SEQ ID No. 9.

[0050] In the above-mentioned biological materials, the nucleic acid molecule described in B1) can be any of the following:

[0051] C1) DNA molecules with nucleotide sequences as shown in SEQ ID No. 9;

[0052] C2) hybridizes with the DNA molecule defined by C1) under stringent conditions and encodes the DNA molecule of the nanobody;

[0053] A DNA molecule encoding the nanobody has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to any of the defined DNA sequences in C3) and C1)-C2).

[0054] The stringent conditions can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1 ...7% SDS, 0.5M Na3PO4, and 1mM EDTA; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS. Hybridize in a mixed solution of SDS, 0.5M Na3PO4 and 1mM EDTA, and wash at 65°C with 0.1×SSC and 0.1% SDS; alternatively, hybridize in a solution of 6×SSC and 0.5% SDS at 65°C, and then wash once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.

[0055] Those skilled in the art can readily mutate the nucleotide sequence of the nanobody B9 encoding gene described in B1) of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or more identity with the nucleotide sequence of B9 described in B1) of the present invention, as long as they encode the nanobody and possess nanobody B9 activity, are derived from and equivalent to the nucleotide sequence of the present invention.

[0056] The present invention also provides a method for preparing the above-mentioned nanobody, which may include the following steps: introducing a nucleic acid molecule encoding the nanobody described above into a recipient cell to obtain a transgenic cell expressing the nanobody, culturing the transgenic cell to obtain the nanobody.

[0057] Furthermore, the nucleic acid molecule encoding the nanobody is the nucleic acid molecule described above.

[0058] In the above method, the nucleotide sequence of the nucleic acid molecule encoding the nanobody described above can be any of the following:

[0059] C1) DNA molecules with nucleotide sequences as shown in SEQ ID No. 9;

[0060] C2) hybridizes with the DNA molecule defined by C1) under stringent conditions and encodes the DNA molecule of the nanobody;

[0061] A DNA molecule encoding the nanobody has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to any of the defined DNA sequences in C3) and C1)-C2).

[0062] Furthermore, the recipient cell may be a microbial cell, such as bacteria (e.g., Escherichia coli), yeast, algae, or fungi.

[0063] In a specific embodiment of the present invention, the receptor cell may specifically be FreeStyle. TM HEK293-F cells.

[0064] The present invention also provides a nanobody fusion protein, wherein the nanobody fusion protein is formed by fusing the aforementioned nanobody or antigen-binding fragment with another molecule, wherein the other molecule may include the Fc domain of an immunoglobulin, a fluorescent protein, or a VHH with different specificities.

[0065] In a specific embodiment of the present invention, the other molecule is the Fc domain of a human immunoglobulin.

[0066] In a specific embodiment of the present invention, the amino acid sequence of the Fc domain of the human immunoglobulin described above is positions 125-351 of SEQ ID No. 11.

[0067] In specific embodiments of the present invention, the nanobody fusion protein described above may be any of the following:

[0068] The M1 amino acid sequence is shown in SEQ ID No. 11 of the fusion protein;

[0069] M3 is a protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 11.

[0070] In the above-mentioned nanobody fusion protein, the nucleic acid molecule encoding the fusion protein can be any of the following:

[0071] D1) A DNA molecule with a nucleotide sequence as shown in SEQ ID No. 10;

[0072] D2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined by D1) and encodes the fusion protein;

[0073] A DNA molecule encoding the fusion protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to any of the defined DNA sequences in D3 and D1-D2).

[0074] The present invention also provides an ELISA detection kit targeting botulinum toxin type B, the kit comprising the nanobody, the biomaterial, or the fusion protein described above.

[0075] This invention also provides any of the following applications:

[0076] E1) The application of the nanobodies or antigen-binding fragments mentioned above in the preparation of products for detecting botulinum toxin type B;

[0077] E2) The application of the aforementioned biomaterials in the preparation of products for detecting botulinum toxin type B;

[0078] E3) The application of the preparation method described above in the preparation of products for detecting botulinum toxin type B;

[0079] E4) The application of the aforementioned kit in the preparation of products for detecting botulinum toxin type B;

[0080] E5) The application of the nanobodies mentioned above in the preparation of products bound to botulinum toxin type B;

[0081] E6) The application of any of the biomaterials mentioned above in the preparation of products in combination with botulinum toxin type B;

[0082] E7) Application of the preparation method described above in the preparation of products combined with botulinum toxin type B;

[0083] E8) Application of the aforementioned kit in the preparation of products conjugated with botulinum toxin type B;

[0084] E9) The application of the nanobodies or antigen-binding fragments mentioned above in the preparation of botulinum toxin type B detection reagents;

[0085] E10) The application of the nanobodies or antigen-binding fragments mentioned above in the preparation of botulinum toxin type B diagnostic reagents;

[0086] E11) The application of the nanobodies or antigen-binding fragments described above in the preparation of drugs for the prevention and / or treatment of botulinum toxin type B.

[0087] The above products may be medicines.

[0088] This invention includes not only complete antibodies, but also fragments of the aforementioned nanobodies or fusion proteins formed by antibodies and other sequences, possessing immunomodulatory activity. Therefore, this invention also includes polypeptides such as fragments, derivatives, and analogs of the aforementioned nanobodies that maintain the same biological function or activity as the antibodies of this invention. The polypeptides may be as follows: D1) a single-chain antibody containing the aforementioned nanobodies; D2) a Fab containing the aforementioned nanobodies; D3) a complete antibody containing the aforementioned nanobodies; D4) a fusion antibody containing the aforementioned nanobodies; D5) an antibody-drug conjugate containing the aforementioned nanobodies.

[0089] D4) The fusion antibody of the nanobody may be: M1) a nanobody fusion protein with an amino acid sequence as shown in SEQ ID No. 11.

[0090] As those skilled in the art will recognize, the conjugates and fusion antibody expression products comprise: conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the nanobodies or fragments thereof.

[0091] The present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0092] This invention prepares a phage nanobody display library by immunizing camels with antigens and then screens the resulting nanobody molecules capable of neutralizing botulinum toxin type B. Nanobodies have the advantages of rapid preparation, simple structure, and ease of modification into multispecific antibodies, and are expected to be developed into highly efficient and safe anti-botulinum toxin type B neutralizing nanobodies to replace commercially available serum products and meet human needs. Attached Figure Description

[0093] Figure 1 This image shows the binding of some phage clones to the target antigen or control antigen after the third round of screening using Phage-ELISA. Odd-numbered columns represent the target antigen, and even-numbered columns represent the control antigen.

[0094] Figure 2 The purified anti-botulinum toxin type B nanobody hFc fusion protein B9-hFc was detected by SDS-PAGE electrophoresis. In the image, a represents the SDS-PAGE result of B9-hFc reductively; b represents the SDS-PAGE result of B9-hFc non-reductively; and M represents the marker band.

[0095] Figure 3 To detect the binding activity of the anti-botulinum toxin type B nanobody hFc fusion protein B9-hFc.

[0096] Figure 4 This study aimed to specifically detect the B9-hFc fusion protein of the anti-botulinum toxin type B nanobody. AHc, BHc, EHc, and FHc are the Hc antigens of the four serotypes of botulinum toxin (A, B, E, and F), respectively; 7F is the fiber antigen of adenovirus.

[0097] Figure 5 To evaluate the neutralizing activity of the anti-botulinum toxin type B nanobody hFc fusion protein B9-hFc. BoNT / B represents botulinum toxin type B, Ab represents the anti-botulinum toxin type B nanobody B9 fusion protein with human immunoglobulin hFc B9-hFc, and BAT represents a standard solution of equine botulinum toxin type B. Detailed Implementation

[0098] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0099] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0100] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0101] The botulinum toxin type B (BoNT / B) and equine botulinum antitoxin standard solution (BAT) used in the following examples were purchased from the National Institutes for Food and Drug Control.

[0102] The pTSE-hFc in the following examples was obtained by linking the gene of the Fc domain of human immunoglobulin G to the pCMV vector. pTSE-hFc has been described in: Xie Qing, Li Zhiying, Zhang Wei, et al. Screening and identification of antibodies against protective antigen V of plague bacteria [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03):266-271. The public can obtain this biological material from the applicant. This biological material is only for repeating the experiments of this invention and shall not be used for other purposes.

[0103] The NEN-SCFV described in the following examples was obtained by modifying the genes of the phage surface protein pIII and the arabinose operon by linking them to a pET vector. NEN-SCFV has been described in: Chen L, Lu J, Yue J, Wang R, Du P, Yu Y, Guo J, Wang X, Jiang Y, Cheng K, Yang Z and Zheng T (2023) A humanized antihumanadenovirus 55 monoclonal antibody with good neutralization ability. Front. Immunol. 14:1132822. doi:10.3389 / fimmu.2023.1132822.Epub2023Mar16.PMID:37006289; PMCID:10060833. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and shall not be used for any other purpose.

[0104] The Hc antigens BoNT / A-Hc, BoNT / B-Hc, BoNT / E-Hc, and BoNT / F-Hc of the four serotypes of botulinum toxin (A, B, E, and F) in the following examples were prepared in our laboratory. The preparation method is described in: Shi DY, Liu FJ, Li ZY, Mao YY, Lu JS, Wang R, Pang XB, Yu YZ, Yang ZX. Development and evaluation of atetravalent botulinum vaccine. Hum Vaccin Immunother. 2022 Nov30;18(5):2048621.doi:10.1080 / 21645515.2022.2048621.Epub 2022Apr 18. PMID:35435814; PMCID:PMC9196761. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and may not be used for any other purpose.

[0105] The adenovirus fiber antigen in the following examples was prepared in our laboratory, and the preparation method is described in: Chen L, Lu J, Yue J, Wang R, Du P, Yu Y, Guo J, Wang X, Jiang Y, Cheng K, Yang Z, Zheng T. A humanized anti-human adenovirus 55 monoclonal antibody with good neutralization ability. Front Immunol. 2023 Mar 16; 14:1132822. doi:10.3389 / fimmu.2023.1132822.PMID:37006289; PMCID:PMC10060833. The public can obtain this biological material from the applicant. This biological material is only for repeating the experiments of the present invention and shall not be used for other purposes.

[0106] The irrelevant control antibody (T23) in the following examples was prepared in our laboratory. The preparation method was as follows: camels were immunized with tetanus TL-HN protein; camel peripheral blood lymphocytes were isolated; the VHH gene fragment was amplified by nested PCR; a specific nanobody phage library was constructed; and nanobody molecules that specifically bind to the TL-HN protein were screened. One of these antibodies was named T23. This biological material is available to the public from the applicant. This biological material is only for repeating the experiments of this invention and may not be used for other purposes.

[0107] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0108] Example 1: Construction of an anti-botulinum toxin type B nanobody library

[0109] 1. Camel Immunity

[0110] BoNT / B-Hc antigen was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881), vortexed and emulsified, and then injected subcutaneously at multiple sites into healthy adult Bactrian camels. Booster immunizations were then performed every two weeks thereafter. Except for the first immunization which used Freund's complete adjuvant, all other immunizations used Freund's incomplete adjuvant (Sigma, F5506).

[0111] 2. Isolation of peripheral blood lymphocytes from camels

[0112] 120-150 mL of peripheral blood was collected from camels that had undergone five immunizations and placed into anticoagulant tubes to separate peripheral blood lymphocytes (PBMCs). The whole blood sample was gently diluted and mixed with lymphocyte separation medium (STEMCELL, 07851) to form a well-defined mixture, thus separating the peripheral blood lymphocytes from the camel blood. After centrifugation of the whole blood and lymphocyte separation medium mixture, the liquid in the centrifuge tube separated into four layers from top to bottom: a plasma layer, a PBMC layer, a lymphocyte separation medium layer, and a red blood cell layer.

[0113] 3. Nested PCR amplification of the VHH gene fragment

[0114] Total RNA was extracted from PBMCs using the OMEGA EZNA Total RNA kit I (OMEGA, R6834), and then cDNA was obtained by reverse transcription using the Invitrogen Superscript III First-strand synthesis system for RT-PCR kit (Invitrogen, 18080-051). First-round PCR: Using the synthesized cDNA as a template, the designed IgG-specific upstream primer CALL001 and downstream primer CALLOO2 were used to amplify the antibody CH2 region sequence. Using the recovered first-round PCR product as a template, the designed primers VHH-F and VHH-R were used for a second-round PCR amplification to amplify the VHH fragment.

[0115] Table 1. Primer sequences used in the two rounds of PCR

[0116] CALL001 GTCCTGGCTGCTCTTCTACAAGG CALL002 GGTACGTGCTGTTGAACTGTTCC VHH-F cggCCATGGcGGTCCTGGCTGCTCTTCTACA VHH-R tcccGCGGCCGCTGAGGAGAYGGTGACCWGGGT

[0117] 4. Electro-connection products

[0118] The vector NEN-SCFV and the amplified VHH gene were digested using restriction endonucleases NcoⅠ and NotⅠ. The ligation products were constructed using T4 ligase.

[0119] The ligation product was transformed into E. coli TG1 competent cells using electroporation to construct an anti-botulinum toxin B-specific phage antibody library. The library was serially diluted to determine its capacity and transformation efficiency. The library was identified as having a capacity of 4 × 10⁻⁶ cells / year. 8 PFU. To test the accuracy of the library, 48 clones were randomly selected for colony PCR. The sequence alignment results showed that the VHH fragment insertion rate reached 100% and the sequences were all different.

[0120] Example 2: Screening of a phage library of anti-botulinum toxin type B specific nanobodies

[0121] Using BoNT / B-Hc as the antigen, a solid-phase screening was performed using a constructed nanobody phage library to obtain anti-botulinum toxin type B specific nanobodies.

[0122] The constructed anti-botulinum toxin B specific nanobody phage library was transferred to 2YT-GA medium (1L 2YT medium contains 16g Typtone, 10g Yeast Extract, 5g NaCl, 100μg / mL ampicillin, and 20% glucose) and cultured to the logarithmic growth phase. M13KO7 helper phage was added at a ratio of MOI≈10. After infection at room temperature for 30 min, the culture was carried out at 37℃ and 150rpm for 30 min. The deep well plate was centrifuged at 4000rpm at room temperature for 15 min, and the supernatant was discarded. The culture was then presented overnight using 2YT-KAA medium (1L 2YT medium contains 16g Typtone, 10g Yeast Extract, 5g NaCl, 100μg / mL kanamycin, 20% glucose, and a final concentration of 1mM arabinose). The supernatant of the culture was collected on the second day, and the phage was concentrated with 20% PEG / NaCl solution (1L of solution contains 200g PEG6000 and 146.25g NaCl) to obtain high-titer antibody library presentation products for subsequent screening. Specific nanobodies were screened using a nanobody phage library for solid-phase screening. BoNT / B-Hc protein was coated with 0.05M NaHCO3 coating buffer (pH=9.6) onto immunotubes and incubated overnight at 4°C. The next day, the immunotubes were washed twice with PBS for 3 min each time, blocked with blocking buffer (2% bovine serum albumin) at room temperature for 2 h, and then the nanobody phage library solution was added and incubated at room temperature for 2 h. The tubes were then shaken at 200 rpm for 20 min. The tubes were then washed five times with PBST (1 L PBS solution containing 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, and 0.1% Tween-20). After washing, 1 mL of elution buffer (0.1M Glycine-HCl, pH=9.6) was added. 2.2) Elute at 400 rpm for 20 min under shaking conditions; remove the eluent from the target antigen immunotherapy tube and add 20-60 μL of neutralization solution (1M Tris-HCl, pH 8.0) for neutralization; infect Escherichia coli TG1 in the logarithmic growth phase, let stand at room temperature for 30 min, and then culture at 37℃ and 150 rpm for 30 min to produce and purify phages for the next round of screening. Repeat the same screening process 3 times, and the enrichment results are shown in Table 2.

[0123] Table 2. Enrichment analysis of the anti-botulinum toxin phage nanobody library screening.

[0124] 1 <![CDATA[5.0×10 11 ]]> <![CDATA[8.4×10 5 ]]> <![CDATA[1.7×10 -6 ]]> 2 <![CDATA[1.2×10 11 ]]> <![CDATA[2.0×10 8 ]]> <![CDATA[1.7×10 -3 ]]> 3 <![CDATA[1.2×10 11 ]]> <![CDATA[3.2×10 9 ]]> <![CDATA[2.7×10 -2 ]]>

[0125] After the above three rounds of screening, single clones with clear spacing and regular shape were selected from the phage-grown plates and inoculated into 96-well deep-well plates containing 250 μL of 2YT-GA medium per well. Two clones were left uninoculated or inoculated with other antibodies as negative control wells. After the bacterial culture was cultured at 37°C to the logarithmic growth phase, M13KO7 helper phage was added at a ratio of MOI≈10, that is, 100 μL was added to each well of the deep-well plate containing a single phage clone. After static infection at room temperature for 30 min, the plate was cultured at 37°C and 150 rpm for 30 min. The deep-well plate was centrifuged at 2000 rpm at room temperature for 10 min, and the supernatant was discarded. Expression was induced using arabinose with a final concentration of 1 mM and cultured overnight at 28°C and 220 rpm to obtain phage particles displaying nanobodies.

[0126] Example 3: Identification of botulinum toxin type B specific nanobodies using phage-ELISA.

[0127] Using BoNT / B-Hc protein as the antigen, ELISA plates were coated. The antigen concentration was diluted to 2 ng / μL with 0.05 M NaHCO3 coating buffer, with a volume of approximately 200 ng / well. Adjacent columns of the antigen were coated with 2% BSA antigen as a negative control. Coating was carried out overnight at 4°C. The next day, the ELISA plates were removed, washed 6 times with PBST on a plate washer, and then blocked with blocking buffer (30 g skim milk powder added to 1 L PBS) at 200 μL / well at 37°C for 2 h. The overnight induced monoclonal bacterial culture was centrifuged at 4°C and 3000 rpm for 10 min. 125 μL of the supernatant from each bacterial culture was added to a 96-well deep-well plate containing 125 μL of blocking buffer and pre-bound for 30 min. 100 μL of the blocked induced expression supernatant was added to the corresponding ELISA plates coated with the target antigen and control antigen, and incubated at 37°C for 1.5 h.

[0128] After washing the plate 6 times with PBST using a plate washer, the HRP-labeled anti-M13 mouse monoclonal antibody (Sino Bioligical, 1973-MM05T-H) was diluted 4000 times with blocking buffer and added to the ELISA plate at 100 μL / well. The plate was incubated at 37°C for 45 min. The plate was then washed 6 times with PBST using a plate washer. A chromogenic buffer was prepared (10 mL of the buffer contained 1 mL of 10× OPD, 9 mL of a mixture of 0.2 M Na₂HPO₄ and 0.1 M citric acid, and 10 μL of 30% hydrogen peroxide). This buffer was added to the ELISA plate at 100 μL / well and incubated in the dark for 15-20 minutes. A 2 M H₂SO₄ stop solution was added to stop the reaction at 50 μL / well. The absorbance was read using a microplate reader at dual wavelengths of 492 / 630 nm. Monoclonal antibodies with an absorbance ratio greater than 5 between the antigen group and the negative control group were considered positive clones. Some Phage-ELISA results are shown below. Figure 1 Odd-numbered columns represent the target antigen, and even-numbered columns represent the control antigen. Positive clones were sent to a biotechnology service company for sequencing to obtain the DNA sequence of the inserted fragment, ultimately yielding the sequence of a bacteriophage clone B9 that specifically binds to BoNT / B-Hc.

[0129] The B9 amino acid sequence is shown in SEQ ID No. 1: it includes a framework region (FR: FR1, FR2, FR3, FR4) and a complementarity-determining region (CDR: CDR1, CDR2, CDR3). The four parts of the framework region are denoted as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively; the three parts of the complementarity-determining region are denoted as SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively.

[0130] Among them, SEQ ID No. 1:

[0131]

[0132] SEQ ID No. 2: VSGYTNDN

[0133] SEQ ID No. 3: TYDIGDP

[0134] SEQ ID No.4:AVGRADWDASLRAASYPY

[0135] SEQ ID No.5: QPQVQLLESGGGSVQAGGSLRLSCT

[0136] SEQ ID No.6:MAWFRQAPGKEREGVAA

[0137] SEQ ID No.7: MYADSVQGRFTSKDTAKNILYLQMNSLKPDDTALYYC

[0138] SEQ ID No. 8: WGQGTLVTVSS

[0139] The gene encoding the above-mentioned anti-botulinum toxin type B nanobody B9 has the nucleotide sequence shown in SEQ ID No. 9.

[0140] SEQ ID No. 9:

[0141]

[0142]

[0143] Example 4: Preparation of anti-botulinum toxin type B nanobodies

[0144] 1. Construction of eukaryotic expression plasmid pTSE-B9-hFc for anti-botulinum toxin type B nanobody hFc fusion protein

[0145] Based on the gene sequence of the anti-botulinum toxin type B nanobody (SEQ ID No. 9), its carboxyl terminus was linked to the hFc segment of human immunoglobulin to form the anti-botulinum toxin type B nanobody hFc fusion protein. The nucleotide sequence encoding this fusion protein is shown in SEQ ID No. 10, and the amino acid sequence of the fusion protein is shown in SEQ ID No. 11.

[0146] The obtained B9 gene sequence was cloned into the pTSE-hFc expression vector using basic PCR amplification, enzyme digestion, and ligation techniques. A single clone was selected for sequencing verification. After successfully inserting the amplified VHH gene fragment into the corresponding vector, a eukaryotic expression plasmid was constructed, and the resulting recombinant plasmid was named pTSE-B9-hFc.

[0147] The structure of the recombinant vector pTSE-B9-hFc is described as follows: A DNA fragment with the sequence SEQ ID No. 10 was inserted between the recognition sites of restriction endonucleases Sal I and Nhe I in the pTSE-hFc vector, while keeping the other sequences of the pTSE-hFc vector unchanged.

[0148] 2. Expression and purification of anti-botulinum toxin nanobody hFc fusion protein

[0149] The constructed pTSE-B9-hFc expression plasmid was transfected into FreeStyle using the FectoPRO DNA Transfection Reagent (Polyplus, 116-001). TMIn HEK293-F cells (Invitrogen, R79007), cell viability was monitored daily starting 72 hours later. When cell viability decreased from 95-100% to 80-85%, the cell supernatant was collected for purification to obtain the nanobody hFc fusion protein B9-hFc.

[0150] SDS-PAGE electrophoresis analysis of the purified antibody yielded the following results: Figure 2 As shown: the molecular weight of the antibody is as expected, and the band size of the nanobody hFc fusion protein under reduction conditions is approximately 40 kDa. Figure 2 In section a), the band size under non-reducing conditions is approximately 80 kDa. Figure 2 (b)

[0151] Example 5: Evaluation of the characteristics of the nanobody hFc fusion protein

[0152] The binding activity of the nanobody hFc fusion protein B9-hFc to BoNT / B-Hc protein was detected by ELISA. The experimental method is as follows:

[0153] BoNT / B-Hc protein was diluted to 2 μg / mL with carbonate coating buffer (pH 9.6), and 100 μL was added to each well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. The coating solution was discarded, and the plate was washed with PBST (1 L of PBS solution contains 8.0 g NaCl, 0.2 g KCl, 1.42 g Na₂HPO₄, 0.27 g KH₂PO₄, and 0.1% KCl). Wash 6 times with Tween-20 solution, blot dry any remaining liquid in the plate, add 200 μL / well of blocking buffer (3% skim milk powder), and incubate at 37°C for 2 h. Serially dilute the antibody for binding activity assay 2-fold with blocking buffer to an initial dilution of 12.5 μg / mL. Discard the blocking buffer, wash 6 times with PBST, blot dry any remaining liquid, add 100 μL / well of the B9-hFc antibody to be tested, and incubate at 37°C for 1.5 h. Then, dilute the plate with blocking buffer at a 1:400 ratio. HRP-labeled goat anti-human IgG was diluted 0-fold, the primary antibody was discarded, and the cells were washed 6 times with PBST. 100 μL of the diluted solution was added to each well and incubated at 37°C for 45 min. The secondary antibody was discarded, and the cells were washed 6 times with PBST. 100 μL of peroxidase substrate chromogenic solution was added to each well and incubated in the dark for 15-20 min. The colorimetric effect was observed, and 50 μL of 2M sulfuric acid was added to each well to terminate the reaction after complete color development. The optical density was measured using a microplate reader at 492 nm / 630 nm. The concentration required for 50% antibody binding of the antigen protein (EC50) was calculated. 50 ), to assess their binding ability.

[0154] The results are as follows Figure 3As shown, the analysis revealed the half-maximal effective concentration (EC50) of B9-hFc binding to BoNT / B-Hc proteins. 50 The value is 0.557 nM.

[0155] The specificity of nanobodies was identified using an ELISA assay, the method of which is as follows:

[0156] BoNT / A-Hc, BoNT / B-Hc, BoNT / E-Hc, BoNT / F-Hc, and adenovirus Fiber antigen (7F) were diluted to 1 μg / mL using carbonate coating buffer. 100 μL / well was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plate was washed with PBST (0.1% Tween-20) six times. After coating the 96-well plate, the remaining liquid in the wells was blotted dry. Blocking buffer (3% skim milk powder) was added to each well at 200 μL / well, and the plate was blocked at 37°C for 2 hours. Primary antibody solution was prepared, diluted to 1 μg / mL. The blocking buffer was discarded, and the plate was washed with PBST. After 6 washes, the plate was dried, and 100 μL of B9-hFc was added to each well. The plate was incubated at 37°C for 1.5 h. HRP-labeled goat anti-human IgG was diluted 1:4000 with blocking buffer, the primary antibody was discarded, and the plate was washed 6 times with PBST. 100 μL of the solution was added to each well and incubated at 37°C for 45 min. The secondary antibody was discarded, and the plate was washed 6 times with PBST. 100 μL of peroxidase substrate chromogenic solution was added to each well and incubated in the dark for 15-20 min. The color development was observed, and 50 μL of 2M sulfuric acid was added to each well to stop the reaction after complete color development. The optical density was measured using a microplate reader at 492 nm / 630 nm dual wavelengths, and the data were analyzed using GraphPad Prism 8 software.

[0157] The results are as follows Figure 4 As shown, B9-hFc specifically binds to BoNT / B-Hc antigens, but does not bind to other antigens, or its binding activity is very weak.

[0158] Example 6: Evaluation of the neutralizing activity of anti-botulinum toxin nanobodies

[0159] The neutralizing activity of the antibody was determined by injecting a mixture of the antibody and a lethal dose of botulinum toxin type B into KM (Kunming) mice in vitro. Four KM mice (18-20g each) were purchased from Beijing Spaford Biotechnology Co., Ltd. The evaluation method is as follows:

[0160] 1. Sample preparation:

[0161] Diluent: KH2PO4 0.7g, Na2HPO4·12H2O 2.4g, NaCl 6.8g, gelatin 2g, add water to 1L, autoclave;

[0162] Botulinum toxin solution: Dilute type B botulinum toxin (purchased from the China National Institutes for Food and Drug Control) to 100 LD with diluent. 50 / mL;

[0163] B9-hFc solution: is a solution obtained by dissolving the fusion protein B9-hFc prepared in Example 4 in a diluent.

[0164] Equine botulinum antitoxin standard solution (BAT): This solution is obtained by dissolving equine anti-botulinum toxin serum (purchased from the China National Institutes for Food and Drug Control) in a diluent.

[0165] 2. The specific experimental grouping scheme is as follows:

[0166] (1) BoNT / B solution 20×LD 50 Group: Each KM mouse was intraperitoneally injected with 200 μL, which is 100 × LD50. 50 A solution of botulinum toxin per mL, ensuring that each injection contains a toxin dose of 20 × LD50. 50 / Only.

[0167] (2) BoNT / B 20×LD 50 +B9-hFc-2μg group: The above botulinum toxin solution was mixed with the B9-hFc solution, and the volume of each group was increased to 2.5mL with diluent. After mixing thoroughly, the mixture was incubated at 37℃ for 30min to obtain BoNT / B+B9-hFc solution. This solution was injected intraperitoneally into KM mice, 500μL per mouse, so that the dose of BoNT / B was 20×LD. 50 / ani, so that the dose of B9-hFc in the injection solution is 2μg / ani.

[0168] (3) BoNT / B 20×LD 50 +B9-hFc-1μg group: The difference between this group and the BoNT / B+B9-hFc-2μg group is that the dose of B9-hFc is 1μg / animal, and the rest of the operation is the same as BoNT / B 20×LD. 50 +B9-hFc-2μg group.

[0169] (4) BoNT / B 20×LD 50 +B9-hFc-0.5μg group: The difference between this group and the BoNT / B+B9-hFc-2μg group is that the dose of B9-hFc is 0.5μg / animal, and the other procedures are the same as those for BoNT / B 20×LD. 50 +B9-hFc-2μg group.

[0170] (5) BoNT / B 20×LD 50+B9-hFc-0.25μg group: The difference between this group and the BoNT / B+B9-hFc-2μg group is that the dose of B9-hFc is 0.25μg / animal; the other procedures are the same as for BoNT / B 20×LD. 50 +B9-hFc-2μg group.

[0171] (6) BoNT / B 20×LD 50 +B9-hFc-0.125μg group: The difference between this group and the BoNT / B+B9-hFc-2μg group is that the dose of B9-hFc is 0.125μg / animal; the other procedures are the same as for BoNT / B 20×LD. 50 +B9-hFc-2μg group.

[0172] (7) BoNT / B 20×LD 50 +B9-hFc-0.0625μg group: The difference between this group and the BoNT / B+B9-hFc-2μg group is that the dose of B9-hFc is 0.0625μg / animal; the other procedures are the same as for BoNT / B 20×LD. 50 +B9-hFc-2μg group.

[0173] (8) BoNT / B 20×LD 50 +BAT-0.2IU group: Botulinum toxin solution was mixed with equine botulinum antitoxin standard solution, and the volume of each group was increased to 2.5mL with diluent. After mixing thoroughly, the mixture was incubated at 37℃ for 30min to obtain BoNT / B+BAT solution. This solution was injected intraperitoneally into KM mice, 500μL per mouse, to achieve a botulinum toxin dose of 20×LD50. 50 / animal, the dosage of equine botulinum antitoxin BAT is 0.2 IU / animal.

[0174] Four mice were injected into each experimental group, and their health status and survival were monitored for 7 days.

[0175] The results are as follows Figure 5 As shown, 0.25 μg of B9-hFc can completely neutralize 20 × LD. 50 Lethal dose of BoNT / B.

[0176] Example 7: Evaluation of the preventive and therapeutic effects of anti-botulinum toxin nanobodies

[0177] 1. To evaluate whether the anti-botulinum toxin nanobody has a protective effect against BoNT challenge, and whether this protective effect is dose-dependent in a mouse model, KM mice (n=4 per group, 18-20g) were purchased from Beijing Spaford Biotechnology Co., Ltd. The evaluation method is as follows:

[0178] Diluent: KH2PO4 0.7g, Na2HPO4·12H2O 2.4g, NaCl 6.8g, gelatin 2g, add water to 1L, autoclave;

[0179] Botulinum toxin solution: Dilute type B botulinum toxin (purchased from the China National Institutes for Food and Drug Control) with diluent to a concentration of 40 or 200 × 10⁻⁶ LD⁻¹. 50 / mL;

[0180] B9-hFc solution: is a solution obtained by dissolving B9-hFc prepared in Example 4 in a diluent;

[0181] Equine botulinum antitoxin standard (BAT) solution: This solution is obtained by dissolving equine anti-botulinum toxin serum (purchased from the China National Institutes for Food and Drug Control) in a diluent.

[0182] Irrelevant antibody (T23) solution: This is a solution obtained by dissolving irrelevant antibody (T23, a fusion protein of nanobody against tetanus TL-HN protein and hFc prepared in our laboratory) in a diluent.

[0183] The prevention evaluation experiment was repeated three times, with the following groupings for each experiment:

[0184] A, BoNT / B 20×LD 50 / each + antibody -0.025mg / kg

[0185] ①BoNT / B 20×LD 50 / mouse +B9-hFc 0.025mg / kg group: Mice were treated with 100μL of B9-hFc solution (i.e., an injection dose of 0.025mg / kg) via the tail vein. 24, 48, and 72 hours later, KM mice were intraperitoneally injected with 500μL of 40×LD50. 50 / mL botulinum toxin solution, injection dose is 20×LD 50 / mouse. Four mice were injected in each experimental group, and their health status and survival were monitored for 7 days.

[0186] ②BoNT / B 20×LD 50 / animal + BAT-0.025mg / kg group: Replace B9-hFc solution with 100μL of BAT solution (i.e., injection dose of 0.025mg / kg / animal), and perform the remaining procedures as with BoNT / B 20×LD 50 / group +B9-hFc-0.025mg / kg

[0187] ③BoNT / B 20×LD 50 / animal +T23-0.025mg / kg+ group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.025mg / kg), and perform the remaining procedures as with BoNT / B 20×LD 50 / group +B9-hFc-0.025mg / kg

[0188] ④PBS group: Replace the antibody solution with an equal volume of PBS, and perform the remaining operations as with BoNT / B 20×LD. 50 / group +B9-hFc-0.025mg / kg

[0189] B, BoNT / B 100×LD 50 / each + antibody -0.025mg / kg

[0190] ①BoNT / B 100×LD 50 / mouse +B9-hFc 0.025mg / kg group: Mice were treated with 100μL of B9-hFc solution (i.e., an injection dose of 0.025mg / kg) via the tail vein. 24, 48, and 72 hours later, KM mice were intraperitoneally injected with 500μL of 200×LD50. 50 / mL botulinum toxin solution, injection dose is 100×LD 50 / mouse. Four mice were injected in each experimental group, and their health status and survival were monitored for 7 days.

[0191] ②BoNT / B 100×LD 50 / v +T23-0.025mg / kg+ group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.025mg / kg), and perform the remaining procedures as with BoNT / B 100×LD 50 / group +B9-hFc-0.025mg / kg

[0192] C. BoNT / B 20×LD 50 / each + antibody -0.125mg / kg

[0193] ①BoNT / B 20×LD 50 / mouse +B9-hFc 0.125mg / kg group: Mice were treated with 100μL of B9-hFc solution (i.e., an injection dose of 0.125mg / kg) via the tail vein. 24, 48, and 72 hours later, KM mice were intraperitoneally injected with 500μL of 40×LD50. 50 / mL botulinum toxin solution, injection dose is 20×LD 50 / mouse. Four mice were injected in each experimental group, and their health status and survival were monitored for 7 days.

[0194] ②BoNT / B 20×LD 50 / v +T23-0.125mg / kg group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.125mg / kg), and perform the remaining procedures as with BoNT / B 20×LD 50 / group +B9-hFc-0.125mg / kg

[0195] D, BoNT / B 100×LD 50 / each + antibody -0.125mg / kg

[0196] ①BoNT / B 100×LD 50 / mouse +B9-hFc 0.125mg / kg group: Mice were treated with 100μL of B9-hFc solution (i.e., injection dose of 0.125mg / kg) via the tail vein. 24, 48, and 72 hours later, KM mice were intraperitoneally injected with 500μL of 200×LD50. 50 / mL botulinum toxin solution, injection dose is 100×LD 50 / mouse. Four mice were injected in each experimental group, and their health status and survival were monitored for 7 days.

[0197] ②BoNT / B 100×LD 50 / animal + BAT-0.125mg / kg group: Replace B9-hFc solution with 100μL of BAT solution (i.e., injection dose of 0.125mg / kg / animal), and perform the remaining procedures as with BoNT / B 100×LD 50 / group +B9-hFc-0.125mg / kg

[0198] ③BoNT / B 100×LD 50 / v +T23-0.125mg / kg+ group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.125mg / kg), and perform the remaining procedures as with BoNT / B 100×LD 50 / group +B9-hFc-0.125mg / kg

[0199] The results are shown in Table 1. The low-dose (0.025 mg / kg) B9-hFc antibody group effectively counteracted 20×LD within 3 days after antibody injection. 50 BoNT / B attack, and against 100×LD 50 BoNT / B challenge can achieve complete protection. In the high-dose (0.125 mg / kg) B9-hFc antibody group, protection against 100×LD50 was achieved within 3 days after antibody injection. 50 BoNT / B attacks are effective and can provide complete protection.

[0200] Table 3. Evaluation of the preventive effect of anti-botulinum toxin nanobodies

[0201]

[0202]

[0203] Note: a. KM mice in each experimental group treated with B9-hFc, BAT, T23, or PBS were intraperitoneally injected with 20×LD at different time points. 50 Or 100×LD 50 Dosage of BoNT / B.

[0204] b KM mice in each experimental group were given 0.025 or 0.125 mg / kg B9-hFc, 0.125 or 0.125 mg / kg BAT, 0.025 or 0.125 mg / kg T23, or PBS for prevention experiments.

[0205] c Mice were challenged with different doses of BoNT / B at 24, 48, and 72 hours after being injected with the specified dose of antibody.

[0206] d Each experimental group consisted of four KM mice. The final survival rate of the mice after seven days was recorded.

[0207] 2. To evaluate whether the anti-botulinum toxin nanobody has a therapeutic effect in mice exposed to BoNT / B, KM mice (n=4 per group, 18-20g) were used, purchased from Beijing Spaford Biotechnology Co., Ltd. The evaluation method is as follows:

[0208] The preparation methods for the diluent, B9-hFc solution, equine botulinum antitoxin standard (BAT) solution, and irrelevant antibody (T23) solution are as described in step (1) of Example 7.

[0209] Botulinum toxin solution: Dilute type B botulinum toxin (purchased from the China National Institutes for Food and Drug Control) to 10 or 40 LD with diluent. 50 / mL.

[0210] The experimental groups are as follows:

[0211] A, BoNT / B 5×LD 50 / each + antibody -0.025mg / kg

[0212] 1) BoNT / B 5×LD 50 / mouse +B9-hFc-0.025mg / kg group: KM mice were intraperitoneally injected with 500μL of 10×LD50 50 / mL botulinum toxin solution, injection dose is 5×LD 50 / mouse. Mice were divided into four groups (n=4 per group) at 0.5 hours, 1 hour, 2 hours, and 3 hours after injection. Each group of mice was injected with 100 μL of B9-hFc solution (0.025 mg / kg) 0.5, 1, 2, or 3 hours after the initial injection of botulinum toxin solution. The health status and survival of the mice were monitored for 7 days.

[0213] 2) BoNT / B 5×LD 50 / animal + BAT-0.025mg / kg group: 100μL of equine anti-botulinum toxin serum solution (i.e., injection dose of 0.025mg / kg) was used instead of B9-hFc solution, and the remaining procedures were the same as for BoNT / B 5×LD. 50 / group +B9-hFc-0.025mg / kg

[0214] 3) BoNT / B 5×LD 50 / v +T23-0.025mg / kg group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.025mg / kg), and perform the remaining procedures as with BoNT / B 5×LD 50 / group +B9-hFc-0.025mg / kg

[0215] 4) PBS group: Replace the B9-hFc solution with an equal volume of PBS, and perform the remaining operations as with BoNT / B 5×LD. 50 / group +B9-hFc-0.025mg / kg

[0216] B, BoNT / B 20×LD 50 / each + antibody -0.025mg / kg

[0217] 1) BoNT / B 20×LD 50 / mouse +B9-hFc-0.025mg / kg group: KM mice were intraperitoneally injected with 500μL 40×LD 50 / mL botulinum toxin solution, injection dose is 20×LD 50 / mouse. Mice were divided into four groups (n=4 per group) at 0.5 hours, 1 hour, 2 hours, and 3 hours after injection. Each group of mice was injected with 100 μL of B9-hFc solution (0.025 mg / kg) 0.5, 1, 2, or 3 hours after the initial injection of botulinum toxin solution. The health status and survival of the mice were monitored for 7 days.

[0218] 2) BoNT / B 20×LD 50 / v +T23-0.025mg / kg group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.025mg / kg), and perform the remaining procedures as with BoNT / B 5×LD 50 / group +B9-hFc-0.025mg / kg

[0219] C, BoNT / B 5×LD 50 / each + antibody -0.125mg / kg

[0220] 1) BoNT / B 5×LD 50 / mouse +B9-hFc-0.125mg / kg group: KM mice were intraperitoneally injected with 500μL of 10×LD50 50 / mL botulinum toxin solution, injection dose is 5×LD 50 / mouse. Mice were divided into four groups (n=4 per group) after injection: 0.5-hour, 1-hour, 2-hour, and 3-hour groups. Each group of mice was injected with 100 μL of B9-hFc solution (0.125 mg / kg) 0.5, 1, 2, or 3 hours after the initial injection of botulinum toxin solution. The health status and survival of the mice were monitored for 7 days.

[0221] 2) BoNT / B 5×LD 50 / v +T23-0.125mg / kg group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.125mg / kg), and perform the remaining procedures as with BoNT / B 5×LD 50 / group +B9-hFc-0.125mg / kg

[0222] D, BoNT / B 20×LD 50 / each + antibody -0.125mg / kg

[0223] 1) BoNT / B 20×LD 50 / mouse +B9-hFc-0.125mg / kg group: KM mice were intraperitoneally injected with 500μL 40×LD 50 / mL botulinum toxin solution, injection dose is 20×LD 50 / mouse. Mice were divided into four groups (n=4 per group) after injection: 0.5-hour, 1-hour, 2-hour, and 3-hour groups. Each group of mice was injected with 100 μL of B9-hFc solution (0.125 mg / kg) 0.5, 1, 2, or 3 hours after the initial injection of botulinum toxin solution. The health status and survival of the mice were monitored for 7 days.

[0224] 2) BoNT / B 20×LD 50 / animal + BAT-0.125mg / kg group: 100μL of equine anti-botulinum toxin serum solution (i.e., injection dose of 0.125mg / kg / animal) was used instead of B9-hFc solution, and the remaining procedures were the same as BoNT / B 20×LD 50 / group +B9-hFc-0.125mg / kg

[0225] 3) BoNT / B 20×LD 50 / v +T23-0.125mg / kg group: Replace B9-hFc solution with 100μL of T23 solution (i.e., injection dose of 0.125mg / kg), and perform the remaining procedures as with BoNT / B 20×LD 50 / group +B9-hFc-0.125mg / kg

[0226] The results are shown in Table 4. High-dose BoNT / B challenge (20×LD) 50 The B9-hFc treatment group showed effectiveness within 3 hours of exposure, with optimal protection within 1 hour, achieving complete protection. For low-dose BoNT / B challenge (5×LD), 50 Treatment with B9-hFc 0.025 mg / kg within 3 hours of exposure can achieve complete protection.

[0227] Table 4. Evaluation of the therapeutic effect of anti-botulinum toxin nanobodies

[0228]

[0229] Note: a KM mice in each experimental group treated with B9-hFc, BAT, T23, or PBS were first intraperitoneally injected with 5×LD50. 50 Or 20×LD 50 Dosage of BoNT / B; b KM mice in each experimental group were treated with 0.025 or 0.125 mg / kg B9-hFc, 0.125 or 0.125 mg / kg BAT, 0.025 or 0.125 mg / kg T23, or PBS at different time points after challenge. c Mice were injected with different doses of antibody or PBS at 0.5, 1, 2 and 3 hours after being challenged with a specified dose of toxin. d Each experimental group consisted of four KM mice. The final survival rate of the mice after seven days was recorded.

[0230] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A nanobody targeting botulinum toxin type B, characterized in that, The nanobody has three complementarity-determining regions, CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is shown in SEQ ID No. 2, the amino acid sequence of CDR2 is shown in SEQ ID No. 3, and the amino acid sequence of CDR3 is shown in SEQ ID No.

4.

2. The nanobody according to claim 1, characterized in that, The nanobody is a nanobody with an amino acid sequence as shown in SEQ ID No.

1.

3. A nanobody targeting botulinum toxin type B, characterized in that, The nanobody is a nanobody obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1; The protein tags are His tags, Flag tags, MBP tags, HA tags, myc tags, GST tags, and / or SUMO tags.

4. A biomaterial relating to the nanobody of claim 1 or 2, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the nanobody of claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecules described in B1); B4) A recombinant vector containing the expression cassette described in B2); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1); B6) Recombinant microorganisms containing the expression cassette described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) Recombinant microorganisms containing the recombinant vector described in B4).

5. The biomaterial according to claim 4, characterized in that, B1) The nucleic acid molecule is a nucleic acid molecule encoding the nanobody of claim 1 or 2, wherein the encoding gene of CDR1 is nucleotide 70-93 of SEQ ID No. 9, the encoding gene of CDR2 is nucleotide 145-165 of SEQ ID No. 9, and the encoding gene of CDR3 is nucleotide 280-333 of SEQ ID No.

9.

6. The biomaterial according to claim 4 or 5, characterized in that, B1) The nucleic acid molecule is any of the following: C1) DNA molecules with nucleotide sequences as shown in SEQ ID No. 9; The DNA sequence defined by C2) and C1) has more than 80% homology and encodes the DNA molecule of the nanobody.

7. A method for preparing the nanobody according to claim 1 or 2, comprising the following steps: Nucleic acid molecules encoding the nanobody of claim 1 or 2 are introduced into recipient cells to obtain transgenic cells expressing the nanobody, and the transgenic cells are cultured to obtain the nanobody.

8. The preparation method according to claim 7, characterized in that, The nucleic acid molecule is any one of the following: C1) DNA molecules with nucleotide sequences as shown in SEQ ID No. 9; The DNA sequence defined by C2) and C1) has more than 80% homology and encodes the DNA molecule of the nanobody.

9. A nanobody fusion protein, characterized in that, The nanobody fusion protein is formed by fusing the nanobody described in claim 1 or 2 with another molecule, wherein the other molecule is the Fc domain of an immunoglobulin.

10. The nanobody fusion protein according to claim 9, characterized in that, The fusion protein is a fusion protein with an amino acid sequence as shown in SEQ ID No.

11.

11. An ELISA detection kit targeting botulinum toxin type B, characterized in that, The kit comprises any of the nanobodies described in claims 1-3, any of the biomaterials described in claims 4-6, or the fusion protein described in claim 9 or 10.

12. The use of the nanobody described in any one of claims 1-3 in the preparation of botulinum toxin type B detection reagent.

13. The use of the nanobody according to any one of claims 1-3 in the preparation of botulinum toxin type B diagnostic reagent.

14. The use of the nanobody according to any one of claims 1-3 in the preparation of a medicament for the prevention and / or treatment of botulinum toxin type B poisoning.

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