Anti-botulinum toxin type A neutralizing antibody A16 and its related biomaterials and applications

Abstract

This invention discloses an anti-botulinum toxin type A neutralizing antibody A16 and its related biomaterials and applications. This invention belongs to the field of biotechnology, specifically relating to anti-botulinum toxin type A neutralizing antibody A16 and its related biomaterials and applications. The nanobody targeting botulinum toxin type A or the antigen-binding fragment containing the nanobody of this invention has three complementary determinants CDR1, CDR2, and CDR3; wherein 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. The fusion protein A16-hFc obtained by fusing the nanobody A16 with the Fc fragment (hFc) of human immunoglobulin can effectively block botulinum toxin type A infection, resisting 20LD... 50 ED50 of botulinum toxin 50 The value is 1.1 μg.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to anti-botulinum toxin type A neutralizing antibody A16 and its related biomaterials and applications. Background Technology

[0002] Botulinum neurotoxin (BoNT) is a potent exotoxin known to humans, produced by Clostridium botulinum under anaerobic conditions. The seven main serotypes of botulinum toxin (A, B, C, D, E, F, and G) have a molecular weight of 150 kDa, each consisting of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC) linked by disulfide bonds. The HC, as a crucial binding site, enables irreversible binding of the toxin to nerve cells at the neuromuscular junction and plays a significant role in mediating transmembrane translocation. Serotypes A, B, E, and F can cause botulism in humans, manifesting as flaccid muscle paralysis, dizziness, and dyspnea. Poisoning caused by any serotype will produce similar symptoms and may lead to death due to respiratory failure or cardiac arrest.

[0003] To date, no small molecule drugs have been approved for the treatment of botulism. Antitoxin therapy is currently the only specific and available method for treating botulism, with polyclonal IgG equine antitoxin being the most effective treatment. Human anti-botulinum immunoglobulin obtained from vaccinated donors has been developed for treating infant botulism, but these therapies have side effects such as serum sickness and hypersensitivity reactions, and their production is not easily scaled up. Compared with traditional antiserum therapy, monoclonal antibodies are a safer alternative for treating poisoning, with advantages such as better patient tolerability, safety, and repeatability, showing promising development prospects. Botulinum poisoning has a rapid onset, and post-exposure vaccination is ineffective, making neutralizing antibodies urgently needed for preventative treatment. Therefore, research on botulism has become increasingly focused worldwide.

[0004] Camels produce unconventional heavy-chain-only antibodies (HCAbs) that bind to antigens based on their variable heavy-chain domains (nanobodies or VHH antibodies). Nanobodies offer unique advantages, such as smaller molecular weight, better tissue penetration, and simpler expression in bacteria. Furthermore, nanobodies can be easily engineered into multimeric forms to enhance their functionality or half-life. Currently, various forms of VHH are used to evaluate and treat common respiratory diseases. The inherent advantages of nanobodies make them an ideal alternative to traditional antibodies, gradually emerging as a new force in therapeutics and clinical diagnostics.

[0005] Botulinum toxin type A (VHH) is the most dangerous serotype posing a serious threat to humans due to its high potency and long duration of action, with a 50% lethal dose of only 0.1-1 ng / kg. Despite the urgent need for treatments for VHH poisoning, the feasibility of using VHH for this purpose has not been fully explored. Therefore, there is an urgent need in this field to develop highly effective and safe anti-VHH neutralizing nanobottlebodies using nanobody technology 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 A or an antigen-binding fragment containing the nanobody.

[0008] The present invention provides a nanobody targeting botulinum toxin type A 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 A.

[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, and 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. Each 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 interfaces between the variable regions of the light and heavy chains.

[0023] In this invention, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning, referring to...

[0024] Cloning the variable region of the antibody heavy chain constructs a single-domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0025] 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.

[0026] 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).

[0027] 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).

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] In this invention, the nanobody is named nanobody A16.

[0033] This invention also provides biomaterials related to the nanobodies described above, wherein the biomaterials may be any of the following:

[0034] B1) Nucleic acid molecules that encode the nanobodies or antigen-binding fragments described above;

[0035] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0036] B3) A recombinant vector containing the nucleic acid molecules described in B1);

[0037] B4) A recombinant vector containing the expression cassette described in B2);

[0038] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0039] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0040] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0041] B8) Recombinant microorganisms containing the recombinant vector described in B4).

[0042] 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.

[0043] 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.

[0044] Recombinant vectors containing the expression cassette can be constructed using existing expression vectors.

[0045] In the aforementioned biological materials, the carrier may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0046] 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.

[0047] In the above-mentioned biological materials, the microorganisms may be bacteria (such as Escherichia coli), yeast, algae, or fungi.

[0048] 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 76-99 of SEQ ID No. 9, the encoding gene of CDR2 is nucleotides 151-174 of SEQ ID No. 9, and the encoding gene of CDR3 is nucleotides 289-336 of SEQ ID No. 9.

[0049] In the above-mentioned biological materials, the nucleic acid molecule described in B1) can be any of the following:

[0050] C1) DNA molecules with nucleotide sequences as shown in SEQ ID No. 9;

[0051] C2) hybridizes with the DNA molecule defined by C1) under stringent conditions and encodes the DNA molecule of the nanobody;

[0052] A DNA molecule encoding the nanobody having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in C3) and C1) or C2).

[0053] 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.

[0054] Those skilled in the art can readily mutate the nucleotide sequence of the nanobody A16 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 A16 described in B1) of the present invention, as long as they encode the nanobody and possess nanobody A16 activity, are derived from and equivalent to the nucleotide sequence of the present invention.

[0055] 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.

[0056] Furthermore, the nucleic acid molecule encoding the nanobody is the nucleic acid molecule described above.

[0057] In the above method, the nucleotide sequence of the nucleic acid molecule encoding the nanobody described above can be any of the following:

[0058] C1) DNA molecules with nucleotide sequences as shown in SEQ ID No. 9;

[0059] C2) hybridizes with the DNA molecule defined by C1) under stringent conditions and encodes the DNA molecule of the nanobody;

[0060] 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).

[0061] The full-length nucleotide sequence or fragments of the antibody described in this invention can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For example, artificial synthesis is used to synthesize the relevant sequence, especially when the fragment length is short. Generally, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0062] Furthermore, the recipient cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Specifically, they can be: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; Drosophila S2 or Sf9 insect cells; CHO, COS7, 293 animal cells, etc.

[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-hFc fusion protein, wherein the nanobody-hFc 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 126-352 of SEQ ID No. 11.

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

[0068] The N1 amino acid sequence of the fusion protein is shown in SEQ ID No. 11;

[0069] The 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 (N2) 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-hFc 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%, 95%, 90%, 85%, or 80% homology with the DNA sequence defined in D3) or D1) or D2).

[0074] The present invention also provides an ELISA detection kit targeting botulinum toxin type A, 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 A;

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

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

[0079] E4) Application of the aforementioned kit in the preparation of products for detecting botulinum toxin type A;

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

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

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

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

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

[0085] E10) The application of the nanobodies or antigen-binding fragments mentioned above in the preparation of botulinum toxin type A 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 A.

[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: N1) a nanobody-hFc 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 it to obtain nanobody molecules capable of neutralizing botulinum toxin type A. Nanobodies have the advantages of rapid preparation, simple structure, and easy modification into multispecific antibodies. They are expected to be developed into highly efficient and safe anti-botulinum toxin type A 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 expression and purification of the anti-botulinum toxin type A nanobody-hFc fusion protein A16-hFc were detected by SDS-PAGE electrophoresis. M represents the marker band, lane 1 shows the results of non-reducing SDS-PAGE of A16-hFc, and lane 2 shows the results of reducing SDS-PAGE of A16-hFc.

[0095] Figure 3 To detect the binding activity of anti-botulinum toxin type A nanobody-hFc fusion protein A16-hFc.

[0096] Figure 4 This study aimed to specifically detect the A16-hFc fusion protein of the anti-botulinum toxin type A nanobody-hFc. AHc, BHc, EHc, and FHc are the Hc antigens of the four serotypes of botulinum toxin (A, B, E, and F), respectively; THc is the Hc antigen of tetanus toxin; and AL-HN is the L-HN antigen of botulinum toxin type A.

[0097] Figure 5 To evaluate the neutralizing activity of the anti-botulinum toxin type A nanobody-hFc fusion protein A16-hFc. BoNT / A represents botulinum toxin type A, Ab represents the Fc fusion protein A16-hFc of the anti-botulinum toxin type A nanobody A16 and human immunoglobulin, and BAT-A represents a standard solution of equine-derived botulinum toxin type A. 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, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0101] The botulinum toxin type A (BoNT / A) and equine botulinum antitoxin standard solution (BAT-A) used in the following examples were purchased from the China 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 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 is 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, Zheng TA humanized antihuman adenovirus 55 monoclonal antibody with good neutralization ability. Front. Immunol. 2023; 14:1132822. doi:10.3389 / fimmu.2023.1132822. This biological material is available to the public from the applicant and is intended solely for the replication of 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; 18(5):2048621. doi:10.1080 / 21645515.2022.2048621. The public can obtain this biological material from the applicant. This biological material is only for repeating the experiments of this invention and cannot be used for other purposes.

[0105] The Hc antigen of tetanus toxin in the following examples was prepared in our laboratory, and the preparation method is described in: Liu XY, Wei DK, Li ZY, Lu JS, Xie XM, Yu YZ, Pang XB. Immunogenicity and immunoprotection of the functional TL-HN fragment derived from tetanustoxin. Vaccine. 2023 Oct8:S0264-410X(23)01102-7. doi:10.1016 / j.vaccine.2023.09.032. The biological material is available to the public from the applicant and is only for repeating the experiments of the present invention and shall not be used for other purposes.

[0106] The L-HN antigen of botulinum toxin type A in the following examples was prepared in our laboratory, and the preparation method is described in: Liu FJ, Shi DY, Mao YY, Xiong XH, Lu JS, Pang XB, Dong XJ, Yang ZX, Yu YZ. Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A. Vaccine. 2020; 38(14):2978-2983. doi:10.1016 / j.vaccine.2020.02.060. 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.

[0107] 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.

[0108] The following examples used GraphPad Prism 8 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.

[0109] Example 1: Construction of an anti-F type botulinum toxin nanobody library

[0110] 1. Camel Immunity

[0111] BoNT / A-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).

[0112] 2. Isolation of peripheral blood lymphocytes from camels

[0113] Peripheral blood of 150 mL was collected from camels that had undergone five immunizations and placed into an anticoagulant tube for peripheral blood lymphocyte separation (PBMC). The whole blood sample was gently diluted and mixed with lymphocyte separation medium (STEMCELL, 07851) to form a well-defined mixture, thus separating the PBMC 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.

[0114] 3. Nested PCR amplification of the VHH gene fragment

[0115] 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).

[0116] Round 1 PCR: Using the synthesized cDNA as a template, the antibody CH2 region sequence was amplified by PCR using the designed IgG-specific upstream primer CALL001 and downstream primer CALLOO2; using the recovered first-round PCR product as a template, the second-round PCR amplification was performed using the designed primers VHH-F and VHH-R to amplify the VHH fragment.

[0117] Table 1. Primer sequences used in the two rounds of PCR

[0118] Primer name Primer sequence (5'-3') CALL001 GTCCTGGCTGCTCTTCTACAAGG CALL002 GGTACGTGCTGTTGAACTGTTCC VHH-F cggCCATGGcGGTCCTGGCTGCTCTTCTACA VHH-R tcccGCGGCCGCTGAGGAGAYGGTGACCWGGGT

[0119] 4. Electro-connection products

[0120] 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.

[0121] The ligation products were transformed into E. coli TG1 competent cells (Beijing Huayueyang Biotechnology Co., Ltd., WG1220) using electroporation to construct an anti-Botulinum toxin type F specific phage antibody library. The library was serially diluted to determine its capacity and transformation efficiency. The library's capacity was found to be 4 × 10⁻⁶. 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.

[0122] Example 2: Screening of a phage library of anti-botulinum toxin type A specific nanobodies

[0123] Using BoNT / A-Hc as the antigen, a solid-phase screening was performed using a constructed nanobody phage library to obtain anti-botulinum toxin type A specific nanobodies.

[0124] Prepare the following solution:

[0125] 2YT-GA medium (1L): 16g Typtone, 10g Yeast Extract, 5g NaCl, 100μg / mL Ampicillin, 20% Glucose, and the remainder is water.

[0126] 2YT-KAA medium (1L): 16g Typtone, 10g Yeast Extract, 5g NaCl, 100μg / mL Kanamycin, 20% Glucose, final concentration 1mM Arabinose, the remainder being water.

[0127] 20% PEG / NaCl solution (1L): 200g PEG6000, 146.25g NaCl, and the remainder is water.

[0128] PBST solution (1L): 8.0g NaCl, 0.2g KCl, 1.42g Na2HPO4, 0.27g KH2PO4, 0.1% Tween-20, the remainder being water.

[0129] The constructed anti-botulinum toxin A specific nanobody phage library was transferred to 2YT-GA medium and cultured to the logarithmic growth phase. M13KO7 helper phage (NEW ENGLAND BioLabs, N0315S) was added at an MOI ≈ 10. After static infection at room temperature for 30 min, the culture was incubated at 37℃ and 150 rpm for 30 min. The deep-well plate was centrifuged at 4000 rpm at room temperature for 15 min, and the supernatant was discarded. The culture was then incubated overnight on 2YT-KAA medium. The supernatant was collected the next day, and the phage was concentrated with 20% PEG / NaCl solution to obtain a high-titer antibody library presentation product for subsequent screening. Specific nanobodies were screened using a nanobody phage library for solid-phase screening. BoNT / A-Hc protein was coated with 0.05M NaHCO3 coating buffer (pH=9.6) 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 washed 10 times with PBST, followed by 5 washes with PBS. After washing, 1 mL of elution buffer (0.1M Glycine-HCl, pH 2.2) was added, and the tubes were eluted at 400 rpm for 20 min. The elution buffer from the target antigen immunotube was removed, and 20-60 μL of neutralization buffer (1M Tris-HCl, pH 2.2) was added. 8.0) Neutralize; infect Escherichia coli TG1 in the logarithmic growth phase, let stand at room temperature for 30 min, then culture at 37℃ and 150 rpm for 30 min, produce and purify phage for the next round of screening, repeat the same screening process 3 times, and the enrichment results are shown in Table 2.

[0130] Table 2. Enrichment analysis of the anti-botulinum toxin phage nanobody library screening.

[0131] Number of filters Input amount (pfu) Output (pfu) Output / Input 1 <![CDATA[5.0×10 11 ]]> <![CDATA[9.0×10 5 ]]> <![CDATA[1.8×10 -6 ]]> 2 <![CDATA[1.2×10 11 ]]> <![CDATA[5.0×10 6 ]]> <![CDATA[4.2×10 -5 ]]> 3 <![CDATA[1.2×10 11 ]]> <![CDATA[6.6×10 8 ]]> <![CDATA[5.5×10 -3 ]]>

[0132] 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.

[0133] Example 3: Identification of type A botulinum toxin-specific nanobodies using phage-ELISA.

[0134] Using BoNT / A-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 and washed 6 times with PBST on a plate washer. Blocking buffer (30 g skim milk powder added to 1 L PBS) was applied at 200 μL / well, and the plates were blocked 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.

[0135] 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, the sequence of a bacteriophage clone A16 that specifically binds to BoNT / B-Hc was obtained.

[0136] The A16 amino acid sequence is shown in SEQ ID No. 1: it includes a frame region (FR: FR1, FR2, FR3, FR4) and a complementarity-determining region (CDR: CDR1, CDR2, CDR3). The four parts of the frame 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.

[0137] Among them, SEQ ID No. 1:

[0138] QVPLVESGGGSVQAGGSLRLSCAASGDTYGSYCMGWFRQAPGKERERVAMIWPGTGHTYNADSVKGRFTV SHDKAKNTLYLQMNSLKPEDTAMYYCAATFAGECASWDGMDYWGKGTQVTVSS;

[0139] SEQ ID No. 2: GDTYGSYC;

[0140] SEQ ID No. 3: IWPGTGHT;

[0141] SEQ ID No.4:AATFAGECASWDGMDY;

[0142] SEQ ID No.5: QVPLVESGGGSVQAGGSLRLSCAAS;

[0143] SEQ ID No.6: MGWFRQAPGKERERVAM;

[0144] SEQ ID No.7: YNADSVKGRFTVSHDKAKNTLYLQMNSLKPEDTAMYYC;

[0145] SEQ ID No. 8: WGKGTQVTVSS.

[0146] The gene encoding the above-mentioned anti-botulinum toxin type A nanobody A16 has the nucleotide sequence shown in SEQ ID No. 9.

[0147] SEQ ID No. 9:

[0148] CAGGTGCCGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGACACCTACGGTAGCTACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGCGGGTCGCAATGATTTGGCCTGGTACTGGTCACACATACAATGCCGA CTCCGTGAAGGGCCGATTCACCGTCTCCCATGACAAAGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAACCTTCGCGGGGGAGTGCGCGTCGTGGGATGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA.

[0149] Example 4: Preparation of anti-botulinum toxin type A nanobody-hFc fusion protein

[0150] 1. Construction of eukaryotic expression plasmid pTSE-A16-hFc for anti-botulinum toxin type A nanobody-hFc fusion protein

[0151] Based on the gene sequence of the anti-botulinum toxin type A nanobody (SEQ ID No. 9), its carboxyl terminus was linked to the hFc segment of human immunoglobulin to form the anti-botulinum toxin type A 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.

[0152] SEQ ID No.10:

[0153]

[0154] SEQ ID No. 11:

[0155] QVPLVESGGGSVQAGGSLRLSCAASGDTYGSYCMGWFRQAPGKERERVAMIWPGTGHTYNADSVKGRFTVSHDKAKNTLYLQMNSLKPEDTAMYYCAATFAGECASWDGMDYWGKGTQVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0156] The obtained A16 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-A16-hFc.

[0157] The structure of the recombinant vector pTSE-A16-hFc is described as follows: A DNA fragment with the sequence SEQ ID No. 9 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.

[0158] 2. Expression and purification of anti-botulinum toxin nanobody-hFc fusion protein

[0159] The constructed pTSE-A16-hFc expression plasmid was transfected into FreeStyle using the FectoPRO DNA Transfection Reagent (Polyplus, 116-001). TM In 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 A16-hFc.

[0160] 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 lane 2 of the middle swimming pool, the band size under non-reducing conditions is approximately 80 kDa. Figure 2 Middle lane 1).

[0161] Example 5: Characterization of Nanobody-hFc Fusion Protein

[0162] The binding activity between the nanobody-hFc fusion protein A16-hFc and the BoNT / A-Hc protein was detected by ELISA. The experimental method is as follows:

[0163] BoNT / A-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 50 μg / mL. Discard the blocking buffer. Wash 6 times with PBST, blot dry any remaining liquid, add 100 μL / well of the A16-hFc antibody to be tested, and incubate at 37°C for 1.5 h. Then, dilute the antibody with blocking buffer at a ratio of 1:4000. HRP-labeled goat anti-human IgG was diluted proportionally, the primary antibody was discarded, and the cells were washed 6 times with PBST. 100 μL of the diluted antibody 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. The optical density was measured using a microplate reader at 492 nm / 630 nm. The concentration required for 50% binding of the antigen protein (EC50) was calculated. 50 ), to assess their binding ability.

[0164] The results are as follows Figure 3 As shown, the analysis revealed the half-maximal effective concentration (EC50) of A16-hFc binding to BoNT / A-Hc protein. 50 The value is 11.86 nM.

[0165] The specificity of the nanobody-hFc fusion protein was identified by ELISA. The experimental method is as follows:

[0166] BoNT / A-Hc, BoNT / B-Hc, BoNT / E-Hc, BoNT / F-Hc, BoNT / AL-HN, and TeNT-Hc were diluted to 2 μg / mL using carbonate coating buffer, and 100 μL / well was added to a 96-well ELISA plate. The plate was incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plate was washed 6 times with PBST (0.1% Tween-20). The remaining liquid in the wells was blotted dry, and blocking buffer (3% skim milk powder) was added to 200 μL / well. The plate was incubated at 37°C for 2 hours. A16-hFc protein primary antibody solution was prepared, diluted to 2 μg / mL, and the blocking buffer was discarded. After washing 6 times with PBST and patting dry, add 100 μL / well of A16-hFc and incubate at 37°C for 1.5 h. Dilute HRP-labeled goat anti-human IgG with blocking buffer at a ratio of 1:4000, discard the primary antibody, wash 6 times with PBST, add 100 μL / well of A16-hFc and incubate at 37°C for 45 min. Discard the secondary antibody, wash 6 times with PBST, add 100 μL / well of peroxidase substrate chromogenic solution, and incubate in the dark for 15-20 min. Observe the color development effect. After complete color development, add 50 μL / well of 2M sulfuric acid to stop the reaction. Measure the optical density at 492 nm / 630 nm using a microplate reader. Analyze the data using GraphPad Prism 8 software.

[0167] The results are as follows Figure 4 As shown, A16-hFc specifically binds to BoNT / A-Hc antigens, but does not bind to other antigens or has very weak binding activity.

[0168] Example 6: Evaluation of the neutralizing activity of the anti-botulinum toxin nanobody-hFc fusion protein

[0169] The neutralizing activity of the antibody was determined by injecting a mixture of the antibody and a lethal dose of botulinum toxin type A 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:

[0170] 1. Sample preparation:

[0171] Diluent: KH2PO4 0.7g, Na2HPO4·12H2O 2.4g, NaCl 6.8g, gelatin 2g, add water to 1L, autoclave;

[0172] Botulinum toxin solution: Dilute type A botulinum toxin (purchased from the China National Institutes for Food and Drug Control) to 100×LD using diluent. 50 / mL;

[0173] A16-hFc solution: is a solution obtained by dissolving the fusion protein A16-hFc prepared in Example 4 in a diluent.

[0174] Equine botulinum antitoxin standard solution (BAT-A): 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.

[0175] 2. The specific experimental grouping scheme is as follows:

[0176] (1) BoNT / A 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.

[0177] (2) BoNT / A 20×LD 50 +A16-hFc-10μg group: The above botulinum toxin solution was mixed with the A16-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 / A+A16-hFc solution. This solution was injected intraperitoneally into KM mice, 500μL per mouse, so that the dose of BoNT / A was 20×LD50. 50 / animal, so that the dose of A16-hFc in each injection solution is 10μg / animal.

[0178] (3) BoNT / A 20×LD 50 +A16-hFc-5μg group: The difference between this group and the BoNT / A+A16-hFc-10μg group is that the dose of A16-hFc is 5μg / animal, and the rest of the operation is the same as BoNT / A 20×LD. 50 +A16-hFc-10μg group.

[0179] (4) BoNT / A 20×LD 50 +A16-hFc-2.5μg group: The difference between this group and the BoNT / A+A16-hFc-10μg group is that the dose of A16-hFc is 2.5μg / animal; the rest of the procedures are the same as for BoNT / A 20×LD. 50 +A16-hFc-10μg group.

[0180] (5) BoNT / A 20×LD 50 +A16-hFc-1.25μg group: The difference between this group and the BoNT / A+A16-hFc-10μg group is that the dose of A16-hFc is 1.25μg / animal, and the rest of the operation is the same as BoNT / A20×LD. 50+A16-hFc-10μg group.

[0181] (6) BoNT / A 20×LD 50 +A16-hFc-0.625μg group: The difference between this group and the BoNT / A+A16-hFc-10μg group is that the dose of A16-hFc is 0.625μg / animal. The other procedures are the same as those for BoNT / A20×LD. 50 +A16-hFc-10μg group.

[0182] (7) BoNT / A 20×LD 50 +BAT-A-0.2 IU group: Botulinum toxin solution was mixed with equine botulinum antitoxin standard solution, and the volume of each group was increased to 2.5 mL with diluent. After thorough mixing, the mixture was incubated at 37°C for 30 min to obtain BoNT / A+BAT-A solution. This solution was injected intraperitoneally into KM mice, 500 μL per mouse, to achieve a botulinum toxin dose of 20 × LD50. 50 / animal, the dosage of equine botulinum antitoxin BAT-A is 0.2 IU / animal.

[0183] Four mice were injected into each experimental group, and their health status and survival were monitored for 7 days.

[0184] The results are as follows Figure 5 As shown, 2.5 μg of A16-hFc can completely neutralize 20 × LD. 50 Lethal dose of BoNT / A.

[0185] 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 camel-derived nanobody targeting botulinum toxin type A, characterized in that, 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.

2. The nanobody according to claim 1, characterized in that, The nanobody is either A1) or A2) below: A1) Nanobody with an amino acid sequence as shown in SEQ ID No. 1; 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.

3. 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) Recombinant microorganisms containing the nucleic acid molecules described in B1).

4. The biomaterial according to claim 3, characterized in that, B1) The nucleic acid molecule is a nucleic acid molecule encoding the nanobody of claim 1 or 2, wherein the coding gene of CDR1 is the nucleotide sequence of SEQ ID No. 9 from position 76 to 99, the coding gene of CDR2 is the nucleotide sequence of SEQ ID No. 9 from position 151 to 174, and the coding gene of CDR3 is the nucleotide sequence of SEQ ID No. 9 from position 289 to 336.

5. The biomaterial according to claim 3 or 4, characterized in that, B1) The nucleic acid molecule is a DNA molecule with a nucleotide sequence as shown in SEQ ID No.

9.

6. 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.

7. A nanobody-hFc fusion protein, characterized in that, The nanobody-hFc fusion protein is formed by fusing the nanobody described in claim 1 or 2 with the Fc domain of human immunoglobulin.

8. The nanobody-hFc fusion protein according to claim 7, characterized in that, The fusion protein is any one of the following: The N1 amino acid sequence of the fusion protein is shown in SEQ ID No. 11; The 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 (N2) 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.

9. An ELISA detection kit targeting botulinum toxin type A, characterized in that, The kit contains the nanobody of claim 1 or 2 or the fusion protein of claim 7 or 8.

10. Any of the following applications: E1) The use of the nanobody according to claim 1 or 2 in the preparation of a product for detecting botulinum toxin type A; E2) The use of the biomaterial described in any one of claims 3-5 in the preparation of products for detecting botulinum toxin type A; E3) Application of the preparation method according to claim 6 in the preparation of products for detecting botulinum toxin type A; E4) Use of the kit according to claim 9 in the preparation of a product for detecting botulinum toxin type A; E5) The use of the nanobody of claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of botulinum toxin type A infection.

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