Anti-botulinum toxin type f neutralizing antibody f13 and related biological materials and uses thereof

Abstract

The application discloses an anti-F botulinum toxin neutralizing antibody F13 and related biological materials and application thereof, and belongs to the field of immunotherapy, and particularly relates to the anti-F botulinum toxin neutralizing antibody F13 and related biological materials and application thereof. The nanobody or antigen binding fragment containing the nanobody of the application targeting the F botulinum toxin has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is 26-33 of SEQ ID No. 1, the amino acid sequence of CDR2 is 51-58 of SEQ ID No. 1, and the amino acid sequence of CDR3 is 97-120 of SEQ ID No. 1. The fusion protein F13-hFc obtained by fusing the nanobody F13 with the Fc segment (hFc) of human immunoglobulin can effectively block the poisoning caused by the F botulinum toxin, the resistance of the fusion protein F13-hFc to the F botulinum toxin is 20 LD 50 of ED 50 is 0.0016 nM.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunotherapy, and particularly relates to anti-F botulinum toxin neutralizing antibody F13 and related biological materials and applications. BACKGROUND

[0002] Botulinum toxin is the most toxic natural protein known, and its serotypes are mainly divided into A-G seven types, among which A, B, E and F are the main types causing human poisoning. Botulinum toxin poisoning is a potentially fatal disease, and serologically different types of neurotoxin can prevent the release of acetylcholine at the neuromuscular junction, leading to paralysis. Botulinum poisoning occurs very quickly, and vaccination after exposure is also useless, so there is an urgent need for neutralizing antibodies for prevention and treatment, and therefore the research on botulinum poisoning has multiplied in the world.

[0003] Antibody therapy is a method for treating botulinum poisoning, and the only treatment method is antitoxin. At present, equine serum antitoxin and human immunoglobulin are respectively permitted to be used for treating adult and infant botulinum poisoning, but the application of the two has side effects such as serum sickness, hypersensitivity including asystole, which limits their application in treatment and recovery after treatment. At present, high-efficiency antitoxin based on monoclonal antibodies is being developed as an effective alternative, and most of the botulinum antibody drugs are in the molecular discovery stage, and no drug has been approved for use, so it is urgent to develop antibody drugs to enrich the treatment means for botulinum poisoning.

[0004] In the 1990s, a unique "heavy chain only" antibody was found in camel serum, which binds to antigens through a variable region, and this variable region is called VHH or nanobody (Nb). Therapeutic nanobodies are in the early development stage, and the special structure endows nanobodies with small size, strong stability and strong antigen binding affinity, making them ideal substitutes for traditional antibodies, and widely used in disease diagnosis and treatment, with broad prospects.

[0005] Although botulinum poisoning caused by F-type botulinum neurotoxin is relatively rare, the gene encoding BoNT / F is highly differentiated by up to 25%, which is the most diversified type among the seven main serotypes. The lack of treatment related to F-type botulinum poisoning highlights the need for treatment of botulinum poisoning, and there is an urgent need to develop antibodies with good therapeutic effect. Therefore, there is an urgent need in the art to apply nanobody technology to develop efficient and safe anti-F botulinum toxin neutralizing nanobodies to replace the serum products supplied on the market to meet the needs of human beings. SUMMARY

[0006] The technical problem to be solved by the present application is to develop a specific nanobody capable of efficiently neutralizing botulinum toxin, to provide a candidate antibody for botulinum poisoning diagnosis and prevention, and to enrich the means for preventing and treating botulinum poisoning.

[0007] To solve the above problems, the present application provides a nanobody targeting botulinum toxin type F or an antigen-binding fragment containing the nanobody.

[0008] The nanobody targeting botulinum toxin type F or the antigen-binding fragment containing the nanobody provided by the present application, wherein the nanobody has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID No. 1 at positions 26-33, the amino acid sequence of the CDR2 is shown in SEQ ID No. 1 at positions 51-58, and the amino acid sequence of the CDR3 is shown in SEQ ID No. 1 at positions 97-120.

[0009] The above CDR is a sequence defined according to the analysis results of the IMGT system.

[0010] The nanobody described herein generally includes a VHH composed of four framework regions (FRs) and three complementarity determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and the antigen-binding fragment contains at least a portion of the nanobody, which is sufficient to endow the fragment with the ability to specifically bind to botulinum toxin type F.

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

[0012] The amino acid sequence of the FR1 is SEQ ID No. 1 at positions 1-25;

[0013] The amino acid sequence of the FR2 is SEQ ID No. 1 at positions 34-50;

[0014] The amino acid sequence of the FR3 is SEQ ID No. 1 at positions 59-96;

[0015] The amino acid sequence of the FR4 is SEQ ID No. 1 at positions 121-131.

[0016] In a specific embodiment, the SEQ ID No. 1 can be as follows:

[0017] QVQLQESGGGSVQAGGSLRLSCAASGYIYGSNYMGWFRQAPEKEREGIAAIYAGGGSTYYADSVKGRFTISLDNAKATLYLQMNNLKPEDTAMYYCAAVDDPGLVVADTEYILQALAFGSTGQGTQVTVSS.

[0018] In the above-mentioned nanobody or antigen-binding fragment, the nanobody can be any one of the following:

[0019] A1) a nanobody having an amino acid sequence as shown in SEQ ID No. 1;

[0020] A2) a nanobody obtained after connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1.

[0021] The protein tag refers to a polypeptide or protein fused with the target protein for the purpose of facilitating the expression, detection, tracing and / or purification of the target protein. The protein tag can be a His tag, a Flag tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, an Fc fragment of immunoglobulin G, etc.

[0022] In the present application, the protein tag is an Fc fragment of human immunoglobulin G (hFc).

[0023] In the above-mentioned nanobody, the nanobody is composed of the complementarity determining region and the framework region.

[0024] The term "antibody" in the present application refers to a heterotetrasaccharide protein of about 150,000 daltons having the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain through a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regular interval intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), 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. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0025] One end of the light chain has a variable region (VL), and the other end has a constant region; 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. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0026] In the present invention, "variable" means that certain portions of the variable domains in antibodies differ in sequence, which confers the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable domains of the light chain and the heavy chain. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of the heavy and light chains each comprise four FR regions, joined by three CDRs, which are generally termed CDR1, CDR2 and CDR3, in sequence from the N-terminus. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant domains are not directly involved in binding of antibodies to antigen, but exhibit various effector functions, such as participation in antibody-dependent cellular cytotoxicity.

[0027] In the present invention, the terms "single domain antibody (VHH)", "nanobody" have the same meaning, referring to

[0028] The variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with full function.

[0029] The above antigen binding fragment can be a complete antibody, a fusion antibody, an antibody drug conjugate, a Fab fragment, a Fv fragment, a Fab' fragment, a F(ab')2 fragment, a single chain antibody (ScFv), or a minimum recognition unit (MRU) containing the nanobody.

[0030] The term "Fab fragment" is a heterodimer composed of the heavy chain Fd and the complete light chain, bound by disulfide bonds, containing only one antigen binding site. The above heavy chain Fd refers to about 1 / 2 of the H chain portion in Fab (about 225 amino acid residues, including VH, CH1 and part of the hinge region).

[0031] The term "Fv fragment" refers to a vector containing VH and VL genes, respectively, co-transfected into cells to express, respectively, and then assembled into a functional Fv antibody; or a stop codon is set between VH and VL in the vector, two small molecular protein fragments are expressed, respectively, and then combined by non-covalent bond to form Fv antibody (Fv fragment).

[0032] The term "Fab' fragment" contains one light chain and a portion of one heavy chain containing the VH domain and the CH1 domain and the region between CH1 and CH2, so that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0033] The term "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CHI and CH2 domains, whereby a disulfide bond between the two heavy chains is formed. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0034] In some embodiments, the Nanobodies described herein can be truncated at the N- or C-terminus to include only a portion of FR1 and / or FR4, or lack one or both of those framework regions, so long as antigen binding and specificity are substantially maintained.

[0035] In the present application, the Nanobody is designated Nanobody F13.

[0036] The present application also provides biological materials related to the Nanobody described above, which can be any of the following:

[0037] B1) a nucleic acid molecule encoding the Nanobody or antigen binding fragment described above;

[0038] B2) an expression cassette comprising the nucleic acid molecule of B1);

[0039] B3) a recombinant vector comprising the nucleic acid molecule of B1);

[0040] B4) a recombinant vector comprising the expression cassette of B2);

[0041] B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

[0042] B6) a recombinant microorganism comprising the expression cassette of B2);

[0043] B7) a recombinant microorganism comprising the recombinant vector of B3);

[0044] B8) a recombinant microorganism comprising the recombinant vector of B4).

[0045] In the above 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, etc.

[0046] In the above biological materials, the expression cassette of B2) refers to DNA that is capable of expressing the Nanobody in a host cell, which can include not only a promoter that initiates transcription of the Nanobody-encoding gene, but also a terminator that terminates transcription of the Nanobody-encoding gene. Further, the expression cassette can also include an enhancer sequence.

[0047] In the above biological material, the vector can be a plasmid, cosmid, bacteriophage or viral vector. The recombinant vector containing the expression cassette can be constructed using an existing expression vector.

[0048] In the above biological material, the recombinant vector can be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into the nanobody fusion protein expression vector pTSE-hFc.

[0049] The vector pTSE-hFc is obtained by linking the gene of the Fc domain of human immunoglobulin to the pCMV vector.

[0050] In the present application, the recombinant vector can be the recombinant vector pTSE-F13-hFc, the structure of which is described as follows: a DNA fragment with the sequence of SEQ ID No. 3 is inserted between the recognition sites of restriction endonuclease Sal I and Nhe I of the pTSE-hFc vector, and the other sequences of the pTSE-hFc vector remain unchanged to obtain the recombinant vector.

[0051] In the above biological material, the host cell contains the nucleic acid molecule or vector of the nanobody or antigen-binding fragment as described above. The host cell includes but is not limited to prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, animal cells (such as mammalian cells, for example, mouse cells, human cells), insect cells and plant cells, etc.

[0052] In the present application, the cell can be FreeStyle TM HEK293-F cells.

[0053] In the above biological material, the nucleic acid molecule of B1) can be a nucleic acid molecule encoding the nanobody described above, wherein the coding gene of the CDR1 is the nucleotide sequence from 76 to 99 of SEQ ID No. 2, the coding gene of the CDR2 is the nucleotide sequence from 151 to 174 of SEQ ID No. 2, and the coding gene of the CDR3 is the nucleotide sequence from 289 to 360 of SEQ ID No. 2.

[0054] In the above biological material, the nucleic acid molecule of B1) can be any of the following:

[0055] C1) a DNA molecule with the nucleotide sequence shown in SEQ ID No. 2;

[0056] C2) a DNA molecule that hybridizes to the DNA molecule defined in C1) under stringent conditions and encodes the nanobody;

[0057] C3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with any of the DNA sequences defined in C1 ) - C2) and encoding said Nanobody.

[0058] In a particular embodiment, said SEQ ID No. 2 can be as follows:

[0059] CAGGTACAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTACGGTAGCAACTACATGGGCTGGTTCCGCCAGGCTCCAGAGAAGGAGCGCGAGGGAATCGCAGCTATTTATGCTGGTGGTGGTAGCACATACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCTAGACAACGCCAAGGCCACATTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGTGGACGACCCCGGACTGGTGGTAGCGGATACTGAGTATATCCTCCAGGCCCTTGCTTTTGGTTCCACGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0060] The stringent conditions can be as follows: hybridization at 50°C in a mixture of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in 2xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in lxSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in 0.5xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 65°C in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 65°C in a solution of 6xSSC, 0.5% SDS, followed by rinsing the membrane once each with 2xSSC, 0.1% SDS and lxSSC, 0.1% SDS.

[0061] The nucleotide sequence of the gene encoding the nanobody F13 according to B1) of the present application can be easily mutated by a person of ordinary skill in the art using known methods, such as methods of directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of F13 according to B1) of the present application, as long as they encode the nanobody and have the activity of nanobody F13, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0062] The present application also provides a method for preparing the nanobody described above, which can comprise 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, and culturing the transgenic cell to obtain the nanobody.

[0063] Further, the nucleic acid molecule encoding the nanobody is the nucleic acid molecule described above.

[0064] In the method described above, the nucleotide sequence of the nucleic acid molecule encoding the nanobody described above can be specifically any one of the following:

[0065] C1) a DNA molecule having the nucleotide sequence shown in SEQ ID No. 1;

[0066] C2) a DNA molecule hybridizing to the DNA molecule defined in C1) under stringent conditions and encoding the nanobody;

[0067] C3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology to the DNA sequence defined in C1) or C2) and encoding said Nanobody.

[0068] Further, the recipient cell can be a prokaryotic cell such as an E. coli cell, and a eukaryotic cell such as a yeast cell, an animal cell (such as a mammalian cell, e.g. a mouse cell, a human cell), an insect cell and a plant cell, etc.

[0069] In particular embodiments of the application, the recipient cell can be E. coli TG1 or FreeStyle TM HEK293-F cells.

[0070] The present application also provides a Nanobody fusion protein, which is a fusion of the Nanobody or antigen binding fragment as described above with another molecule, which can include the Fc domain of an immunoglobulin, a fluorescent protein or a VHH with a different specificity.

[0071] In particular embodiments of the application, the other molecule is the Fc domain of a human immunoglobulin.

[0072] In particular embodiments of the application, the amino acid sequence of the Fc domain of a human immunoglobulin as described above is positions 367-594 of SEQ ID No. 4.

[0073] In particular embodiments of the application, the Nanobody fusion protein as described above can be any one of the following:

[0074] M1) a fusion protein having an amino acid sequence as set forth in SEQ ID No. 4;

[0075] M2) a fusion protein having more than 75% identity to the protein set forth in M1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of M1);

[0076] M3) a protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the amino acid sequence set forth in SEQ ID No. 4.

[0077] In the Nanobody fusion protein as described above, the nucleic acid molecule encoding the fusion protein can be any one of the following:

[0078] D1) a DNA molecule having a nucleotide sequence as set forth in SEQ ID No. 3;

[0079] D2) a DNA molecule hybridizing to the DNA molecule defined in D1) under stringent conditions and encoding the fusion protein;

[0080] D3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequences defined in D1) or D2) and encoding said fusion protein.

[0081] In a particular embodiment, the Nanobody fusion protein is F13-hFc.

[0082] The present application also provides an ELISA detection kit for targeting botulinum toxin type F, said kit comprising the Nanobody, the biological material or the fusion protein as described hereinbefore.

[0083] The present application also provides the use of any one of:

[0084] E1) the Nanobody or antigen binding fragment as described hereinbefore for the manufacture of a product for detecting botulinum toxin type F;

[0085] E2) the biological material as described hereinbefore for the manufacture of a product for detecting botulinum toxin type F;

[0086] E3) the method of manufacture as described hereinbefore for the manufacture of a product for detecting botulinum toxin type F;

[0087] E4) the kit as described hereinbefore for the manufacture of a product for detecting botulinum toxin type F;

[0088] E5) the Nanobody as described hereinbefore for the manufacture of a product that binds to botulinum toxin type F;

[0089] E6) the biological material as described hereinbefore for the manufacture of a product that binds to botulinum toxin type F;

[0090] E7) the method of manufacture as described hereinbefore for the manufacture of a product that binds to botulinum toxin type F;

[0091] E8) the kit as described hereinbefore for the manufacture of a product that binds to botulinum toxin type F;

[0092] E9) the Nanobody or antigen binding fragment as described hereinbefore for the manufacture of a detection reagent for botulinum toxin type F;

[0093] E10) the Nanobody or antigen binding fragment as described hereinbefore for the manufacture of a diagnostic reagent for botulinum toxin type F;

[0094] E11) the Nanobody or antigen binding fragment as described hereinbefore for the manufacture of a medicament for preventing and / or treating botulinum toxin type F.

[0095] The product can be a medicament.

[0096] The present application not only includes the complete antibody, but also the fragments of the nanobody with immunological activity or the fusion proteins formed by the antibody and other sequences. Therefore, the present application also includes the polypeptides such as the fragments, derivatives and analogues of the nanobody which maintain the same biological function or activity as the antibody of the present application. The polypeptides can be as follows: D1) single-chain antibody containing the nanobody described above; D2) Fab containing the nanobody described above; D3) complete antibody containing the nanobody described above; D4) fusion antibody containing the nanobody described above; D5) antibody drug conjugate containing the nanobody described above.

[0097] D4) The fusion antibody of the nanobody can be the nanobody fusion protein with the amino acid sequence as shown in SEQ ID No. 4.

[0098] As known by those skilled in the art, the conjugate and fusion antibody expression product include the conjugate formed by the combination of the antibody or fragment thereof of the present application and the drug, toxin, cytokine, radionuclide, enzyme and other diagnostic or therapeutic molecules. The present application also includes the cell surface marker or antigen combined with the nanobody or fragment thereof.

[0099] The present application includes any protein or protein conjugate and fusion expression product (i.e. immunological conjugate and fusion expression product) with the heavy chain containing the variable region, as long as the variable region is the same as or at least 90% homologous, preferably at least 95% homologous to the heavy chain variable region of the antibody of the present application.

[0100] The present application immunizes the camel with the antigen, prepares the phage nanobody display library and screens to obtain the nanobody F13-hFc capable of neutralizing the botulinum toxin type F. The nanobody has the advantages of rapid preparation, simple structure and easy modification into the multi-specific antibody, and is expected to be developed into the efficient and safe nanobody for neutralizing the botulinum toxin type F, which is used to replace the serum preparation supplied in the market to meet the needs of human beings. BRIEF DESCRIPTION OF DRAWINGS

[0101] Figure 1 Part of the phage clones after the third round of screening are identified by Phage-ELISA for the binding with the target antigen or the control antigen. The odd columns are the target antigens; the even columns are the control antigens.

[0102] Figure 2 The nanobody-hFc fusion protein F13-hFc for anti-botulinum toxin type F is detected by SDS-PAGE electrophoresis after expression and purification. M is the Marker band, lane 1 is the non-reducing SDS-PAGE detection result of F13-hFc, and lane 2 is the reducing SDS-PAGE detection result of F13-hFc.

[0103] Figure 3 To detect the binding activity of anti-F botulinum toxin nanobody-hFc fusion protein F13-hFc.

[0104] Figure 4 To detect the specificity of anti-F botulinum toxin nanobody-hFc fusion protein F13-hFc. Wherein AHc, BHc, EHc, FHc are Hc antigens of A, B, E, F four serotypes of botulinum toxin respectively; THc is the Hc antigen of tetanus toxin; FL-HN is the L-HN antigen of F botulinum toxin.

[0105] Figure 5 To evaluate the neutralization activity of anti-F botulinum toxin nanobody-hFc fusion protein F13-hFc. Wherein BoNT / F is F botulinum toxin, Ab is anti-F botulinum toxin nanobody F13 and human immunoglobulin Fc fusion protein F13-hFc, and BAT-F is a standard solution of horse-derived F botulinum antitoxin. DETAILED DESCRIPTION

[0106] The application will be further described in detail below with specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0107] The experimental methods in the following examples are all routine methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0108] The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged, unless otherwise specified.

[0109] The F botulinum toxin (BoNT / F) and the standard solution of horse-derived botulinum antitoxin (BAT-F) in the following examples are both purchased from China Institute for Food and Drug Control.

[0110] NEN-SCFV in the following examples is engineered by linking the genes of phage surface protein pIII and the arab sugar operon on the 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 antihuman adenovirus 55 monoclonal antibody with good neutralization ability. Front. Immunol. 14: 1132822. doi: 10.3389 / fimmu.2023.1132822. The biological material is available from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0111] pTSE-hFc in the following examples is engineered by linking the gene of the Fc domain of human immunoglobulin on the pCMV vector, and pTSE-hFc has been described in: Xie Q, Li ZY, Zhang W, et al. Screening and identification of antibodies against protective antigens of Yersinia pestis [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03): 266-271. The biological material is available from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0112] The Hc antigen of tetanus toxin in the following examples is prepared by the laboratory, and the preparation method has been 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 tetanus toxin. Vaccine. 2023: S0264-410X(23)01102-7. doi: 10.1016 / j.vaccine.2023.09.032. The biological material is available from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0113] The Hc antigens of A, B, E, F serotypes of botulinum toxin in the following examples, AHc, BHc, EHc, FHc, were prepared in the laboratory, and the preparation method has been described in Shi DY, Liu FJ, Li ZY, Mao YY, Lu JS, Wang R, Pang XB, Yu YZ, Yang ZX. Development and evaluation of a tetravalent botulinum vaccine. Hum Vaccin Immunother. 2022; 18(5): 2048621. doi: 10.1080 / 21645515.2022.2048621. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the invention and cannot be used for other purposes.

[0114] The L-HN antigen of F serotype of botulinum toxin in the following examples was prepared in the laboratory, and the preparation method has been described in Li ZY, Li BL, Lu JS, Liu XY, Tan X, Wang R, Du P, Yu, S, Xu Q, Pang XB, Yu YZ, Yang ZX. Biological and Immunological Characterization of a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F. Toxins 2023; 15(3), 200. doi:10.3390 / toxins15030200. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the invention and cannot be used for other purposes.

[0115] The irrelevant control antibody (T23) in the following examples was prepared in the laboratory by immunizing camels with tetanus TL-HN protein, isolating camel peripheral blood lymphocytes, amplifying VHH gene fragments by nested PCR, constructing a specific nanobody phage library and screening to obtain a nanobody molecule that specifically binds to TL-HN protein, and one of the antibodies was named T23. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the invention and cannot be used for other purposes.

[0116] The following examples use GraphPad Prism 8 software to process data, and the experimental results are expressed as mean ± standard deviation, using One-way ANOVA test, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference, P<0.001 (*** ) indicates extremely significant difference.

[0117] Example 1, Construction of Anti-F Botulinum Toxin Nanobody Library

[0118] 1. Camel immunization

[0119] BoNT / F-Hc antigen was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881), shaken and emulsified, and after complete emulsification, it was injected into healthy adult bactrian camels by subcutaneous multiple point injection. After that, booster immunization was performed every two weeks, and Freund's incomplete adjuvant (Sigma, F5506) was used for immunization except for the first time.

[0120] 2. Isolation of peripheral blood lymphocytes from camels

[0121] Peripheral blood 150 mL from camels immunized five times was collected into an anticoagulant tube to isolate peripheral blood lymphocytes (PBMC). After diluting the whole blood sample gently, it was mixed with lymphocyte separation medium (STEMCELL, 07851) to form a mixture with a clear interface between the two, so as to separate peripheral blood lymphocytes from camel blood. After centrifugation of the mixture of whole blood and lymphocyte separation medium, the liquid in the centrifuge tube was divided into four layers from top to bottom: plasma layer, PBMC layer, lymphocyte separation medium layer and red blood cell layer.

[0122] 3. Nested PCR amplification of VHH gene fragments

[0123] OMEGA E.Z.N.A Total RNA kit I kit (OMEGA, R6834) was used to extract total RNA from PBMC, and then Invitrogen Superscript Ⅲ First-strand synthesis system for RT-PCR kit (Invitrogen, 18080-051) was used for reverse transcription to obtain cDNA.

[0124] First round of PCR: using synthesized cDNA as template, IgG specific upstream primer CALL001 and downstream primer CALLOO2 were used for PCR amplification of antibody CH2 region sequence; using the first round of PCR product as template, primer VHH-F and VHH-R were used for second round of PCR amplification to amplify VHH fragments.

[0125] Table 1, primer sequences used in two-round PCR

[0126]

[0127] 4, electrotransformation of ligation product

[0128] The vector NEN-SCFV and the VHH gene amplified in step 3 were digested with restriction endonuclease Ncol and Notl. The ligation product was constructed by T4 ligase ligation.

[0129] The ligation product was transformed into TG1 competent cells (Beijing Huayueyang Biotechnology Co., Ltd., WG1220) using electroporation technology to construct an anti-F botulinum toxin specific phage antibody library. The phage antibody library was diluted by doubling dilution to determine its capacity and transformation efficiency. E. coli TG1 competent cells (Beijing Huayueyang Biotechnology Co., Ltd., WG1220) using electroporation technology to construct an anti-F botulinum toxin specific phage antibody library. The phage antibody library was diluted by doubling dilution to determine its capacity and transformation efficiency.

[0130] The capacity of the antibody library was identified to be 4 x 10 8 pfu. To detect the accuracy of the library, 48 clones were randomly selected for colony PCR, and the sequence alignment results showed that the VHH fragment insertion rate was 100% and the sequences were different.

[0131] Example 2, screening of anti-F botulinum toxin specific nanobody phage library

[0132] The following medium and solution were prepared:

[0133] 2YT-GA medium (1 L): 16 g Typtone, 10 g Yeast Extract, 5 g NaCl, 100 μg / mL ampicillin, 20% glucose, and the rest is water.

[0134] 2YT-KAA medium (1 L): 16 g Typtone, 10 g Yeast Extract, 5 g NaCl, 100 μg / mL kanamycin, 20% glucose, final concentration 1 mM arabinose, and the rest is water.

[0135] PBST (1 L): containing 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, 0.1% Tween-20, and the rest is water.

[0136] BoNT / F-Hc was used as an antigen, and the constructed nanobody phage library was used for solid-phase screening to obtain anti-F botulinum toxin specific nanobodies.

[0137] The constructed anti-F botulinum toxin specific nanobody phage library bacteria liquid was transferred to 2YT-GA medium (1 L) and cultured to logarithmic growth phase. M13KO7 helper phage was added at a ratio of MOI = 10, and the infection was carried out at room temperature for 30 min. Then, the culture was incubated at 37 °C and 150 rpm for 30 min. The deep well plate was centrifuged at 4000 rpm and room temperature for 15 min, and the supernatant was discarded. 2YT-KAA medium (1 L) was used for overnight presentation. The next day, the culture presentation supernatant was collected, and the phage was concentrated using a 20% PEG / NaCl solution (1 L solution containing 200 g PEG6000, 146.25 g NaCl) to obtain a high-titer antibody library presentation product for subsequent screening. The nanobody phage library was used to screen specific nanobodies. The BoNT / F-Hc protein was coated with 0.05 M NaHCO3 solution (pH = 9.6) to the immunization tube at 4 °C overnight. The next day, the immunization tube was washed with PBS twice for 3 min each, and then blocked with blocking solution (2% bovine serum albumin) at room temperature for 2 h. Then, the nanobody phage library solution was added and incubated at room temperature for 2 h. Then, it was shaken at 200 rpm for 20 min. Then, it was washed with PBST (1 L) and then with PBS for 5 times. After washing, 1 mL of elution solution (0.1 M Glycine-HCl, pH 2.2) was added, and the elution was carried out at 400 rpm for 20 min. The elution solution in the antigen immunization tube was removed, and 20-60 µL of neutralization solution (1 M Tris-HCl, pH 8.0) was added for neutralization. E. coli TG1 in logarithmic growth phase was infected, and the infection was carried out at room temperature for 30 min. Then, the culture was incubated at 37 °C and 150 rpm for 30 min. The phage was produced and purified for the next round of screening. The same screening process was repeated for 3 rounds, and the enrichment results are shown in Table 2.

[0138] Table 2, Anti-F botulinum toxin phage nanobody library screening enrichment degree analysis

[0139]

[0140] After the above-mentioned 3 rounds of screening, single colonies with obvious spacing and regular shape growing well on the plates were selected and inoculated into 96-well deep well plates containing 2YT-GA medium 250 μL per well, and the remaining 2 wells were not inoculated with clones or inoculated with other antibody clones as negative control wells; the bacterial solution was cultured at 37 °C until the logarithmic growth phase, and then M13KO7 helper phage (NEW ENGLAND BioLabs, N0315S) was added at a ratio of MOI≈10, that is, 100 μL of M13KO7 helper phage was added to each well of the deep well plate containing a single phage clone, and after 30 min of infection at room temperature, the deep well plate was cultured at 37 °C and 150 rpm for 30 min; the deep well plate was centrifuged at 2000 rpm and room temperature for 10 min, and the supernatant was discarded, and the phage particles displaying nanobodies were obtained by inducing expression with 1 mM arabinose at 28 °C and 220 rpm overnight.

[0141] Example 3, Phage-ELISA identification of F-type botulinum toxin specific nanobodies

[0142] The BoNT / F-Hc protein was used as an antigen to coat the enzyme-linked plate, and the antigen concentration was diluted to 2 ng / μL with 0.05 M NaHCO3 coating solution, and the amount was about 200 ng / well. The adjacent column of the antigen column was coated with 2% BSA antigen as a negative control, and the enzyme-linked plate was coated overnight at 4 °C; the next day, the enzyme-linked plate was taken out and washed 6 times on the plate washer with PBST, then blocked with blocking solution (30 g of skimmed milk powder was added to 1 L of PBS) at 200 μL / well, and blocked at 37 °C for 2 h; the single colony bacterial solution induced and cultured overnight was centrifuged at 4 °C and 3000 rpm for 10 min, and 125 μL of supernatant of each bacterial solution was taken to a 96-well deep well plate containing 125 μL of blocking solution, and pre-bound for 30 min; the blocked induced expression supernatant was added to the corresponding enzyme-linked plate coated with the target antigen and the control antigen at 100 μL / well, and incubated at 37 °C for 1.5 h.

[0143] After washing 6 times with PBST on the plate shaker, the HRP-labeled anti-M13 mouse monoclonal antibody (Sino Bioligical, 1973-MM05T-H) was diluted 4000 times with blocking solution, and 100 μL / well was added to the enzyme-linked plate, which was incubated at 37 °C for 45 min. After washing 6 times with PBST on the plate shaker, the color developing solution (10 mL of color developing solution containing 1 mL of 10×OPD, 9 mL of 0.2 M Na2HPO4 and 0.1 M citric acid mixed solution, and 10 μL of 30% hydrogen peroxide) was prepared, and 100 μL / well was added to the enzyme-linked plate, which was developed in the dark for 15-20 min. 2 M H2SO4 was added to terminate the reaction, and 50 μL / well was added to terminate the reaction. The enzyme-labeled instrument was used to read at dual wavelengths of 492 / 630 nm. The ratio of the light absorption values of the antigen group and the negative control group was greater than 5, and the monoclonal was determined to be positive. Some Phage-ELISA experimental results are shown in Table 1. Figure 1 Table 1: Phage-ELISA experimental results The positive clone corresponding to the bacterial liquid was sent to a biotechnology service company for sequence determination, and the DNA sequence of the inserted fragment was obtained, and finally a phage clone F13 capable of specifically binding to BoNT / B-Hc was obtained.

[0144] The amino acid sequence of the nanobody F13 is shown in SEQ ID No. 1: including the framework region (FR: FR1, FR2, FR3, FR4) and the complementarity determining region (CDR: CDR1, CDR2, CDR3), wherein the amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID No. 1 at positions 26-33, the amino acid sequence of CDR2 is shown in SEQ ID No. 1 at positions 51-58, and the amino acid sequence of CDR3 is shown in SEQ ID No. 1 at positions 97-120; the four parts of the framework region are sequentially recorded as SEQ ID No. 1 at positions 1-25, SEQ ID No. 1 at positions 34-50, SEQ ID No. 1 at positions 59-96, and SEQ ID No. 1 at positions 121-131.

[0145] SEQ ID No. 1:

[0146] QVQLQESGGGSVQAGGSLRLSCAASGYIYGSNYMGWFRQAPEKEREGIAAIYAGGGSTYYADSVKGRFTISLDNAKATLYLQMNNLKPEDTAMYYCAAVDDPGLVVADTEYILQALAFGSTGQGTQVTVSS.

[0147] The gene encoding the above-mentioned anti-F type botulinum toxin nanobody F13 has a nucleotide sequence shown in SEQ ID No. 2.

[0148] SEQ ID No. 2:

[0149] CAGGTACAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTACGGTAGCAACTACATGGGCTGGTTCCGCCAGGCTCCAGAGAAGGAGCGCGAGGGAATCGCAGCTATTTATGCTGGTGGTGGTAGCACATACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCTAGACAACGCCAAGGCCACATTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGTGGACGACCCCGGACTGGTGGTAGCGGATACTGAGTATATCCTCCAGGCCCTTGCTTTTGGTTCCACGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0150] Example 4, Preparation of Anti-F botulinum toxin Nanobody

[0151] 1. Construction of anti-F botulinum toxin Nanobody hFc fusion protein eukaryotic expression plasmid pTSE-F13-hFc

[0152] According to the gene sequence (SEQ ID No. 2) of the anti-F botulinum toxin Nanobody F13, the carboxyl terminal thereof is connected with the hFc segment of human immunoglobulin to constitute an anti-F botulinum toxin Nanobody hFc fusion protein. The coding nucleotide sequence of the fusion protein is shown in SEQ ID No. 3, and the amino acid sequence of the fusion protein is shown in SEQ ID No. 4.

[0153] SEQ ID No. 3:

[0154]

[0155] SEQ ID No. 4:

[0156] QVQLQESGGGSVQAGGSLRLSCAASGYIYGSNYMGWFRQAPEKEREGIAAIYAGGGSTYYADSVKGRFTISLDNAKATLYLQMNNLKPEDTAMYYCAAVDDPGLVVADTEYILQALAFGSTGQGTQVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0157] The obtained F13 gene sequence is cloned into pTSE-hFc expression vector by basic PCR amplification, enzyme digestion, ligation and other techniques, and a single clone is picked for sequencing verification. After successfully inserting the obtained VHH gene fragment into the corresponding vector, a eukaryotic expression plasmid is constructed, and the obtained recombinant plasmid is named pTSE-F13-hFc.

[0158] The structure of the recombinant vector pTSE-F13-hFc is described as follows: the DNA fragment with SEQ ID No. 2 is inserted between the recognition sites of restriction endonuclease Sal I and Nhe I of the pTSE-hFc vector, and the other sequences of the pTSE-hFc vector remain unchanged to obtain the recombinant vector. The recombinant vector pTSE-F13-hFc can express nanobody fusion protein F13-hFc.

[0159] 2. Expression and purification of anti-botulinum toxin nanobody fusion protein F13-hFc

[0160] The constructed pTSE-F13-hFc expression plasmid is transfected into FreeStyle 293F cells by using transfection reagent FectoPRO DNA Transfection Reagent (Polyplus, 116-001). TMHEK293-F cells (Invitrogen, R79007) were used to express the nanobody fusion protein F13-hFc. After 72 hours, the cell activity was monitored daily, and when the cell activity decreased from 95-100% to 80-85%, the cell supernatant was collected for purification to obtain the nanobody fusion protein F13-hFc.

[0161] SDS-PAGE electrophoresis was used to analyze the purified antibody, and the results are shown in Figure 2 : The molecular weight of the antibody F13-hFc was as expected, and the band size of the nanobody fusion protein F13-hFc under reducing conditions was about 40 kDa Figure 2 (middle lane 2), and the band size under non-reducing conditions was about 80 kDa Figure 2 (middle lane 1).

[0162] Example 5: Evaluation of the properties of the nanobody fusion protein F13-hFc

[0163] 1. The binding activity between the nanobody fusion protein F13-hFc and the BoNT / F-Hc protein was detected by ELISA experiment, and the experimental method was as follows:

[0164] The BoNT / F-Hc protein was diluted to a concentration of 2 μg / mL with carbonate coating buffer (pH 9.6) and added to a 96-well ELISA plate at 100 μL / well, coated overnight at 4 ℃; the coating solution was discarded, and the plate was washed 6 times on a plate washer with PBST (1 L PBS solution containing 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, 0.1% Tween-20), and the residual liquid in the plate was patted dry, and blocking solution (3% skim milk powder) was added at 200 µL / well, and blocked at 37 ℃ for 2 h; the antibody to be tested for binding activity was diluted by 2 times, and the initial dilution was 100 µg / mL, the blocking solution in the plate was discarded, and after washing 6 times with PBST, the residual liquid was patted dry, and the F13-hFc to be tested was added to the plate at 100 μL / well, and incubated at 37 ℃ for 1.5 h; the HRP-labeled goat anti-human IgG was diluted with blocking solution at a ratio of 1:4000, the primary antibody was discarded, and washed 6 times with PBST, and added to the plate at 100 μL / well, and incubated at 37 ℃ for 45 min; the secondary antibody was discarded, and washed 6 times with PBST, and peroxidase substrate developing solution was added at 100 μL / well, and developed in the dark for 15-20 min, and the developing effect was observed, and after complete development, 50 μL / well of 2 M sulfuric acid was added to terminate the reaction; the enzyme label instrument was used to measure the optical density value at 492 nm / 630 nm dual wavelength. The binding capacity between them was evaluated by calculating the concentration required for the antibody to bind 50% of the antigen protein (EC 50 ).

[0165] Results are shown in Figure 3 Figure 6, the half maximal effective concentration (EC 50 ) of the binding of the nanobody fusion protein F13-hFc to BoNT / F-Hc antigenic protein was 0.293 nM.

[0166] 2. Specificity of the nanobody was identified by ELISA experiment, and the experimental method was as follows:

[0167] BoNT / A-Hc, BoNT / B-Hc, BoNT / E-Hc, BoNT / F-Hc, BoNT / FL-HN, and TeNT-Hc were diluted to a concentration of 2 µg / mL with carbonate coating buffer, 100 μL / well was added to a 96-well ELISA plate, and the plate was coated at 4 ℃ overnight; the next day, the coating solution was discarded, and the 96-well plate coated overnight was washed 6 times with PBST (0.1% Tween-20) on a plate washing machine, then the residual liquid in the wells was patted dry, 200 μL / well of blocking solution (3% skimmed milk powder) was added, and the plate was blocked at 37 ℃ for 2 h; F13-hFc protein primary antibody solution was prepared, and the antibody was diluted to a concentration of 6 µg / mL, the blocking solution was discarded, and the plate was washed 6 times with PBST, then 100 μL / well of F13-hFc was added, and the plate was incubated at 37 ℃ for 1.5 h; HRP-labeled goat anti-human IgG was diluted with blocking solution at a ratio of 1:4000, the primary antibody was discarded, the plate was washed 6 times with PBST, 100 μL / well of the solution was added to the plate, and the plate was incubated at 37 ℃ for 45 min; the secondary antibody was discarded, the plate was washed 6 times with PBST, then 100 μL / well of peroxidase substrate color developing solution was added, color development was carried out in the dark for 15-20 min, the color development effect was observed, 2 M sulfuric acid was added to the plate at a volume of 50 μL / well to stop the reaction, and the optical density value was measured using an enzyme label instrument at 492 nm / 630 nm dual wavelength, and the data results were analyzed using GraphPad Prism 8 software.

[0168] Results are shown in Figure 4 The nanobody fusion protein F13-hFc specifically bound to BoNT / F-Hc antigen, and did not bind to other antigens or had very weak binding activity.

[0169] Example 6, Evaluation of the neutralization activity of the anti-botulinum toxin nanobody

[0170] 1. Sample preparation

[0171] Diluent: KH2PO40.7 g, Na2HPO4•12H2O 2.4 g, NaCl 6.8 g, gelatin 2 g, water to 1 L, high-pressure sterilization;

[0172] Botulinum toxin solution: F type botulinum toxin (purchased from China Institute for Food and Drug Control) was diluted to 100 LD 50 / mL with diluent.

[0173] F13-hFc solution: the solution was prepared by dissolving the fusion protein F13-hFc prepared in Example 4 with diluent.

[0174] Horse-derived botulinum antitoxin standard solution (BAT-F): the solution was prepared by dissolving horse-derived antitoxin serum (purchased from China Institute for Food and Drug Control) with diluent.

[0175] 2. Specific experimental grouping

[0176] The antibody neutralization activity was determined by mixing the antibody and the lethal dose of F type botulinum toxin in vitro and then injecting KM (purchased from Beijing Sibeifu Biotechnology Co., Ltd.) mice. KM mice, 4 in each group, weighed 18-20 g, and the health status and survival of the mice were monitored for 7 days. The groups were as follows:

[0177] (1) BoNT / F solution 20×LD 50 group: each KM mouse was injected intraperitoneally with 200 μL of botulinum toxin solution at 100×LD 50 / mL, so that the dose of toxin contained in the solution injected into each mouse was 20×LD 50 / mouse.

[0178] (2) BoNT / F 20×LD 50 + F13-hFc-0.016 μg group: the above botulinum toxin solution was mixed with F13-hFc solution, the volume of each group was supplemented to 2.5 mL with diluent, and then uniformly mixed and incubated at 37 ℃ for 30 min to obtain the BoNT / F + F13-hFc solution, which was injected into KM mice intraperitoneally, 500 μL per mouse, so that the dose of BoNT / F was 20×LD 50 / mouse, and the dose of F13-hFc in the solution injected into each mouse was 0.016 μg / mouse.

[0179] (3) BoNT / F 20×LD 50 + F13-hFc-0.008 μg group: the difference between this group and the BoNT / F + F13-hFc-0.016 μg group was that the dose of F13-hFc was 0.008 μg / mouse, and the rest of the operation was the same as that of the BoNT / F 20×LD 50 + F13-hFc-0.016 μg group.

[0180] (4) BoNT / F 20×LD 50+F13-hFc-0.004 μg group: the difference between this group and BoNT / F + F13-hFc-0.016 μg group is that the dose of F13-hFc is 0.004 μg per mouse, and the rest of the operation is the same as BoNT / F 20×LD 50 +F13-hFc-0.016 μg group.

[0181] (5) BoNT / F 20×LD 50 +F13-hFc-0.002 μg group: the difference between this group and BoNT / F + F13-hFc-0.016 μg group is that the dose of F13-hFc is 0.002 μg per mouse, and the rest of the operation is the same as BoNT / F 20×LD 50 +F13-hFc-0.016 μg group.

[0182] (6) BoNT / F 20×LD 50 +F13-hFc-0.001 μg group: the difference between this group and BoNT / F + F13-hFc-0.016 μg group is that the dose of F13-hFc is 0.001 μg per mouse, and the rest of the operation is the same as BoNT / F 20×LD 50 +F13-hFc-0.016 μg group.

[0183] (7) BoNT / F 20×LD 50 +BAT-F-0.2 IU group: the botulinum toxin solution was mixed with the horse-derived botulinum antitoxin standard solution, and the volume of each group was supplemented to 2.5 mL with the diluent, and after uniform mixing, it was incubated at 37 ℃ for 30 min to obtain a BoNT / F + BAT-F solution, which was injected into KM mice intraperitoneally, 500 μL per mouse, so that the dose of botulinum toxin was 20×LD 50 / mouse, and the dose of horse-derived botulinum antitoxin BAT-F was 0.2 IU per mouse.

[0184] The results are shown in Table 1. Figure 5 0.008 μg of F13-hFc can completely neutralize the lethal dose of 20×LD 50 BoNT / F.

[0185] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A camelid nanobody targeting botulinum toxin type F, characterized in that, The nanobody has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID No. 1 at positions 26-33, the amino acid sequence of the CDR2 is shown in SEQ ID No. 1 at positions 51-58, and the amino acid sequence of the CDR3 is shown in SEQ ID No. 1 at positions 97-120.

2. The Nanobody according to claim 1, characterized in that, The nanobody is any one of the following A1) or A2): A1) a nanobody having the amino acid sequence shown in SEQ ID No. 1; A2) a nanobody obtained by connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No.

1.

3. A biological material related to the nanobody of claim 1 or 2, which 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 of B1); B3) a recombinant vector containing the nucleic acid molecule of B1); B4) a recombinant microorganism containing the nucleic acid molecule of B1).

4. The biomaterial of claim 3, wherein, The nucleic acid molecule of B1) is a nucleic acid molecule encoding the nanobody of claim 1 or 2, wherein the gene encoding the CDR1 is the nucleotide sequence shown in SEQ ID No. 2 at positions 76-99, the gene encoding the CDR2 is the nucleotide sequence shown in SEQ ID No. 2 at positions 151-174, and the gene encoding the CDR3 is the nucleotide sequence shown in SEQ ID No. 2 at positions 289-360.

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

2.

6. A method for the production of a Nanobody according to claim 1 or 2, comprising the steps of: The nucleic acid molecule encoding the nanobody of claim 1 or 2 is introduced into a recipient cell to obtain a transgenic cell expressing the nanobody, and the transgenic cell is cultured to obtain the nanobody.

7. A Nanobody® fusion protein characterized in that, The nanobody fusion protein is a fusion of the nanobody of claim 1 or 2 with the Fc domain of human immunoglobulin.

8. The Nanobody fusion protein of claim 7, wherein, The fusion protein is any one of the following: M1) a fusion protein having the amino acid sequence shown in SEQ ID No. 4; M2) a protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No.

4.

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

10. Any one of the following uses: E1) use of the nanobody of claim 1 or 2 in the preparation of a product for detecting botulinum toxin type F; E2) use of the biological material of any one of claims 3-5 in the preparation of a product for detecting botulinum toxin type F; E3) use of the preparation method of claim 6 in the preparation of a product for detecting botulinum toxin type F; E4) use of the kit of claim 9 in the preparation of a product for detecting botulinum toxin type F; E5) use of the nanobody of claim 1 or 2 in the preparation of a medicament for preventing and / or treating infection with botulinum toxin type F.

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