CN118955703A - Anti-type F botulinum toxin neutralizing antibody F13 and its related biomaterials and applications - Google Patents

Abstract

The present invention discloses an anti-type F botulinum toxin neutralizing antibody F13 and its related biomaterials and applications. The present invention belongs to the field of immunotherapy, and specifically relates to an anti-type F botulinum toxin neutralizing antibody F13 and its related biomaterials and applications. The nanobody targeting type F botulinum toxin of the present invention or the antigen-binding fragment containing the nanobody has three complementary determinant clusters CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is the 26-33rd position of SEQ ID No.1, the amino acid sequence of CDR2 is the 51-58th position of SEQ ID No.1, and the amino acid sequence of CDR3 is the 97-120th position 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 type F botulinum toxin, and its ED ₅₀ against 20 LD ₅₀ is 0.0016nM.

Description

Anti-F botulinum toxin neutralizing antibody F13 and related biological material and application thereof

Technical Field

The invention belongs to the field of immunotherapy, and in particular relates to an anti-F botulinum toxin neutralizing antibody F13 and a related biological material and application thereof.

Background

Botulinum toxin is the most toxic natural protein known to date, and serotypes are largely divided into seven types a-G, A, B, E, F of which are the major types that are toxic to humans. Toxicity caused by botulinum toxin is a potentially fatal disease, and a serologically distinct class of neurotoxin can prevent the release of acetylcholine from neuromuscular junctions leading to paralysis. The onset of botulism is rapid, post-exposure vaccination is not useful, and neutralizing antibodies are urgently needed for preventive treatment, so research on botulism is multiplied worldwide.

Antibody therapy is a method for treating botulism, the only treatment method at present is antitoxin, horse serum antitoxin and human immunoglobulin are respectively allowed to treat adult botulism and infant botulism, but the application of the antitoxin and the human immunoglobulin has side effects such as serum diseases, hypersensitivity reactions including asystole and the like, and the application of the antitoxin and the human immunoglobulin in treatment and recovery after treatment are limited. In the current development of efficient antitoxin based on monoclonal antibody as an effective alternative, the drugs aiming at the botulinum antibody are mostly in the molecular discovery stage, and the drugs are not used in batches, so that the development of antibody drug-rich poisoning treatment means is urgently needed.

In the 90 s of the 20 th century, a unique class of "heavy chain only" antibodies was found in camelid serum that bound to antigen through a variable region, known as VHH or Nanobody (Nb). The therapeutic nano antibody is in an early development stage, and the special structure endows the nano antibody with the outstanding advantages of small volume, strong stability, strong antigen binding affinity and the like, so that the nano antibody becomes an ideal substitute for the traditional antibody, is widely applied to disease diagnosis and treatment, and has wide prospect.

Although botulism caused by botulinum neurotoxin type F is relatively rare, the gene encoding BoNT/F is highly differentiated by up to 25% and is the most diverse of the seven major serotypes. The lack of therapeutic methods associated with botulinum toxin type F has highlighted the need for treatment of botulinum toxin and there is an urgent need to develop antibodies with good therapeutic effects. Therefore, there is an urgent need in the art to develop efficient and safe neutralizing nanobodies against botulinum toxin type F for replacing commercially available serum products to meet the needs of humans using nanobody technology.

Disclosure of Invention

The invention aims to solve the technical problem of developing a specific nano antibody capable of efficiently neutralizing botulinum toxin, providing candidate antibodies for diagnosis and prevention of the botulinum toxin and enriching means for preventing and treating the botulinum toxin.

In order to solve the above problems, the present invention provides a nanobody targeting botulinum toxin type F or an antigen-binding fragment containing the same.

The invention provides a nano antibody targeting type F botulinum toxin or an antigen binding fragment containing the nano antibody, wherein the nano antibody has 3 complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in positions 26-33 of SEQ ID No.1, the amino acid sequence of the CDR2 is shown in positions 51-58 of SEQ ID No.1, and the amino acid sequence of the CDR3 is shown in positions 97-120 of SEQ ID No. 1.

The CDRs are sequences defined according to the IMGT system analysis results.

Nanobodies described herein generally include VHH consisting of 4 Framework Regions (FRs) and 3 Complementarity Determining Regions (CDRs), termed FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, and the antigen binding fragment comprises at least a portion of the nanobody sufficient to confer the fragment the ability to specifically bind botulinum toxin type F.

The 4 framework regions may be FR1, FR2, FR3 and FR4.

The amino acid sequence of the FR1 is the 1 st-25 th position of SEQ ID No. 1;

the amino acid sequence of the FR2 is 34 th-50 th site of SEQ ID No. 1;

the amino acid sequence of the FR3 is the 59 th-96 th position of SEQ ID No. 1;

the amino acid sequence of FR4 is 121-131 of SEQ ID No. 1.

In a specific embodiment, the SEQ ID No.1 may be as follows:

QVQLQESGGGSVQAGGSLRLSCAASGYIYGSNYMGWFRQAPEKEREGIAAIYAGGGSTYYADSVKGRFTISLDNAKATLYLQMNNLKPEDTAMYYCAAVDDPGLVVADTEYILQALAFGSTGQGTQVTVSS.

in the nanobody or antigen-binding fragment described above, the nanobody may be any of the following:

a1 A nano antibody with an amino acid sequence shown as SEQ ID No. 1;

a2 A protein label is connected to the N end and/or the C end of the amino acid sequence shown in SEQ ID No.1, and the nano antibody is obtained.

The protein tag refers to a polypeptide or protein which is expressed together with a target protein in a fusion way, so that the target protein can be expressed, detected, tracked and/or purified conveniently. The protein tag may be a His tag, flag tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, fc segment of immunoglobulin G, etc.

In the present invention, the protein tag is the Fc segment (hFc) of human immunoglobulin G.

In the above nanobody, the nanobody is composed of the complementarity determining region and a framework region.

The term "antibody" in the present invention is an iso-tetralin protein of about 150000 daltons having the same structural characteristics, consisting 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, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. 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 a plurality of constant regions. Each strip is provided with

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 the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

"Variable" in the context of the present invention means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding portion of the antibody (see Kabat et al NIH Publ, no.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

The term "single domain antibody (VHH)", "nanobody" (nanobody) in the present invention has the same meaning, and means that

Cloning the variable region of the antibody heavy chain, a single domain antibody (VHH) consisting of only one heavy chain variable region was constructed, which is the smallest antigen binding fragment with complete function.

The antigen binding fragments described above may be whole antibodies, fusion antibodies, antibody drug conjugates, fab fragments, fv fragments, fab 'fragments, F (ab') 2 fragments, single chain antibodies (ScFv), or Minimal Recognition Units (MRU) comprising the nanobody.

The term "Fab fragment" is a heterodimer formed by the disulfide bond between the heavy chain Fd and the intact light chain, containing only one antigen binding site. The heavy chain Fd mentioned above refers to about 1/2 of the H chain portion (about 225 amino acid residues including VH, CH1 and part of the hinge region) of the Fab.

The term "Fv fragment" refers to a functional Fv antibody that can be assembled by separately constructing vectors containing the VH and VL genes, co-transfecting the cells, and separately expressing them; a termination codon may be placed between the VH and VL in the vector to express two small molecule protein fragments, respectively, which are then non-covalently bound to form an Fv antibody (Fv fragment).

The term "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 and CH2 domains, whereby an inter-chain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form a F (ab ') 2 molecule.

The term "F (ab') 2 fragment" contains two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

In some embodiments, nanobodies of the invention may be truncated at the N-or C-terminus such that they comprise only a portion of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity.

In the present invention, the nanobody is named as nanobody F13.

The invention also provides biological materials related to the nanobody as described above, which can be any of the following:

b1 A nucleic acid molecule encoding a nanobody or antigen-binding fragment as described above;

B2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

B4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism containing the recombinant vector of B4).

In the above biological material, the nucleic acid molecule may be DNA such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, B2) the expression cassette refers to a DNA capable of expressing the nanobody in a host cell, and the DNA may include not only a promoter for initiating transcription of the nanobody-encoding gene, but also a terminator for terminating transcription of the nanobody-encoding gene. Further, the expression cassette may also include an enhancer sequence.

In the above biological material, the vector may be a plasmid, cosmid, phage or viral vector. Recombinant vectors containing the expression cassette can be constructed using existing expression vectors.

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

The vector pTSE-hFc is obtained by connecting the genes of the Fc domain of human immunoglobulin on a pCMV vector in an engineered way.

In the present invention, the recombinant vector may be recombinant vector pTSE-F13-hFc, and the structure of pTSE-F13-hFc is described as follows: and (3) inserting a DNA fragment with a sequence of 3 between recognition sites of the pTSE-hFc vector restriction enzymes Sal I and Nhe I, and keeping other sequences of the pTSE-hFc vector unchanged to obtain the recombinant vector.

In the above biological material, the host cell comprises a nucleic acid molecule or vector of a nanobody or antigen-binding fragment as described above. Such host cells include, but are not limited to, prokaryotic cells, such as E.coli cells, and eukaryotic cells, such as yeast cells, animal cells (e.g., mammalian cells, e.g., mouse cells, human cells), insect cells, plant cells, and the like.

In the present invention, the cell may be a FreeStyle ^TM HEK293-F cell.

In the above biological material, the nucleic acid molecule of B1) may be a nucleic acid molecule encoding the nanobody described above, where in the nucleic acid molecule, the coding gene of CDR1 is nucleotide 76-99 of SEQ ID No.2, the coding gene of CDR2 is nucleotide 151-174 of SEQ ID No.2, and the coding gene of CDR3 is nucleotide 289-360 of SEQ ID No. 2.

In the above biological material, the nucleic acid molecule of B1) may be any of the following:

c1 A DNA molecule with a nucleotide sequence shown as SEQ ID No. 2;

c2 A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in C1) and encodes the nanobody;

C3 A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in any one of C1) -C2) and encoding the nanobody.

In a specific embodiment, the SEQ ID No.2 may be as follows:

CAGGTACAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTACGGTAGCAACTACATGGGCTGGTTCCGCCAGGCTCCAGAGAAGGAGCGCGAGGGAATCGCAGCTATTTATGCTGGTGGTGGTAGCACATACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCTAGACAACGCCAAGGCCACATTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGTGGACGACCCCGGACTGGTGGTAGCGGATACTGAGTATATCCTCCAGGCCCTTGCTTTTGGTTCCACGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

Wherein the stringent conditions may be as follows: hybridization at 50℃in a mixed solution of 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃PO₄ and 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: hybridization at 50℃in a mixed solution of 7% SDS, 0.5M Na ₃PO₄ and 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: hybridization at 50℃in a mixed solution of 7% SDS, 0.5M Na ₃PO₄ and 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: hybridization at 50℃in a mixed solution of 7% SDS, 0.5M Na ₃PO₄ and 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: hybridization at 50℃in a mixed solution of 7% SDS, 0.5M Na ₃PO₄ and 1mM EDTA, rinsing at 65℃in 0.1 XSSC, 0.1% SDS; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

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

The invention also provides a preparation method of the nano antibody, which comprises the following steps: introducing nucleic acid molecules encoding the nanobody into receptor cells to obtain transgenic cells expressing the nanobody, and culturing the transgenic cells to obtain the nanobody.

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

In the above preparation method, the nucleotide sequence of the nucleic acid molecule encoding the nanobody described above may specifically be any one of the following:

C1 A DNA molecule with a nucleotide sequence shown as SEQ ID No. 1;

c2 A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in C1) and encodes the nanobody;

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

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

In a specific embodiment of the invention, the recipient cell may specifically be an E.coli TG1 or FreeStyle ^TM HEK293-F cell.

The present invention also provides a nanobody fusion protein in which the nanobody or antigen-binding fragment described above is fused to another molecule, which may include an Fc domain of an immunoglobulin, a fluorescent protein, or a VHH having different specificity.

In a specific embodiment of the invention, the further molecule is an Fc domain of a human immunoglobulin.

In a specific embodiment of the invention, the amino acid sequence of the Fc domain of the human immunoglobulin described above is at positions 367-594 of SEQ ID No. 4.

In a specific embodiment of the invention, the nanobody fusion protein described above may be any of the following:

m1) fusion protein with the amino acid sequence shown as SEQ ID No. 4;

m2) a fusion protein having 75% or more identity with the protein represented by M1) obtained by substitution and/or deletion and/or addition of an amino acid residue to the protein of M1);

m3) protein obtained by connecting protein tags to the N end and/or the C end of the amino acid sequence shown in SEQ ID No. 4.

In the nanobody fusion protein, the nucleic acid molecule encoding the fusion protein may be any of the following:

d1 A DNA molecule with a nucleotide sequence shown as SEQ ID No. 3;

D2 A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in D1) and which codes for said fusion protein;

D3 A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in D1) or D2) and encoding the fusion protein.

In a specific embodiment, the nanobody fusion protein is F13-hFc.

The invention also provides an ELISA detection kit for targeting the F-type botulinum toxin, wherein the kit comprises the nanobody, the biological material or the fusion protein.

The invention also provides any one of the following applications:

e1 Use of a nanobody or antigen-binding fragment as described hereinbefore for the preparation of a product for the detection of botulinum toxin type F;

e2 Use of a biomaterial as hereinbefore described in the manufacture of a product for the detection of botulinum toxin type F;

E3 Use of the preparation method described hereinbefore for the preparation of a product for the detection of botulinum toxin type F;

E4 Use of a kit as hereinbefore described for the preparation of a product for the detection of botulinum toxin type F;

E5 Use of a nanobody as hereinbefore described for the preparation of a product which binds botulinum toxin type F;

E6 Use of any of the biological materials described above for the preparation of a product that binds botulinum toxin type F;

e7 Use of the preparation method described hereinbefore for the preparation of a product to which botulinum toxin type F is bound;

E8 Use of a kit as hereinbefore described for the preparation of a product for binding to botulinum toxin type F;

E9 Use of a nanobody or antigen-binding fragment as described hereinbefore for the preparation of a botulinum toxin type F detection reagent;

E10 Use of a nanobody or antigen-binding fragment as described hereinbefore for the preparation of a diagnostic reagent for botulinum toxin type F;

e11 Use of a nanobody or antigen-binding fragment as described hereinbefore for the preparation of a medicament for the prophylaxis and/or treatment of botulinum toxin type F.

The above product can be a medicament.

The invention includes not only whole antibodies, but also fragments of said nanobody or fusion proteins of antibodies with other sequences having immunological activity. Thus, the invention also includes fragments, derivatives, analogs, and the like of the nanobody as polypeptides that retain the same biological function or activity as the antibody of the invention. The polypeptide may be as follows: d1 A single chain antibody comprising the nanobody described above; d2 Fab containing nanobodies as described above; d3 A whole antibody comprising the nanobody described above; d4 A fusion antibody comprising the nanobody described above; d5 Antibody drug conjugates comprising the nanobodies described herein before.

D4 The fusion antibody of the nano antibody can be a nano antibody fusion protein with an amino acid sequence shown as SEQ ID No. 4.

As known to those of skill in the art, the conjugate and fusion antibody expression products include: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the nanobodies or fragments thereof.

The invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.

According to the invention, the phage nanobody display library is prepared by immunizing a camel with antigen, and the nanobody F13-hFc capable of neutralizing the F-type botulinum toxin is obtained by screening, so that the nanobody has the advantages of quick preparation, simple structure and convenience in reconstruction into a multispecific antibody, and is expected to be developed into an efficient and safe neutralizing nanobody for resisting the F-type botulinum toxin, and is used for replacing serum products supplied in the market so as to meet the demands of human beings.

Drawings

FIG. 1 shows the binding of a portion of Phage clones to the antigen of interest or control antigen after the third round of screening by Phage-ELISA assay. Wherein the odd columns are antigens of interest; the even columns are control antigens.

FIG. 2 shows SDS-PAGE electrophoresis to detect the expression of purified anti-F botulinum toxin nanobody-hFc fusion protein F13-hFc. Wherein M is Marker band, lane 1 is F13-hFc non-reducing SDS-PAGE detection result, and lane 2 is F13-hFc reducing SDS-PAGE detection result.

FIG. 3 is a graph showing the detection of the binding activity of anti-F-type botulinum toxin nanobody-hFc fusion protein F13-hFc.

FIG. 4 shows the specific detection of anti-F-type botulinum toxin nanobody-hFc fusion protein F13-hFc. AHc, BHc, EHc, FHc of which are Hc antigens of the four serotypes of botulinum toxin A, B, E, F, respectively; THc is the Hc antigen of tetanus toxin; FL-HN is the L-HN antigen of botulinum toxin type F.

FIG. 5 is an evaluation of neutralizing activity of anti-F-type botulinum toxin nanobody-hFc fusion protein F13-hFc. Wherein BoNT/F is botulinum toxin type F, ab is Fc fusion protein F13-hFc of anti-botulinum toxin type F nano antibody F13 and human immunoglobulin, and BAT-F is horse source F type botulinum antitoxin standard solution.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

Botulinum toxin type F (BoNT/F) and horse-derived botulinum antitoxin standard solution (BAT-F) in the examples below were purchased from China food and drug testing institute.

NEN-SCFV in the following examples was constructed by ligating the genes of phage surface proteins pIII and the arabinose operon onto pET vectors. 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 Zand Zheng T (2023) A humanized antihuman adenovirus 55 monoclonal antibodywith good neutralization ability.Front. Immunol. 14:1132822. doi: 10.3389/fimmu.2023.1132822. public to obtain this biomaterial from the applicant, which was used only for repeated experiments of the invention and not as a further use.

PTSE-hFc in the following examples was engineered by ligating the gene of the Fc domain of human immunoglobulin onto pCMV vector, pTSE-hFc being described in: xie Qing, li Zhiying, zhang Wei et al screening and identification of antibodies against pestis protective antigen V J. J.China etiology journal of etiology, 2022,17 (03): 266-271. The public obtains the biological material from the applicant, which is used only for repeated experiments of the present invention, and is not used for other purposes.

The Hc antigen of tetanus toxin in the examples described below was 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. public to obtain this biomaterial from the applicant, which was used only for repeated experiments of the invention and was not used for other purposes.

The Hc antigens AHc, BHc, EHc, FHc of the A, B, E, F four serotypes of botulinum toxins in the examples described below were prepared by the present 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. public to the applicant as having access to this biomaterial which was used only in duplicate experiments of the invention and was not available for other uses.

The L-HN antigen of botulinum toxin type F in the examples described below was prepared by the present 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. public to obtain this biomaterial from the applicant, which was used only for repeated experiments of the present invention and was not used for other purposes.

The irrelevant control antibody (T23) in the following examples was prepared by the present laboratory as follows: tetanus TL-HN protein immune camel, separating camel peripheral blood lymphocytes, amplifying VHH gene fragments by nested PCR, constructing a specific nanobody phage library, screening to obtain nanobody molecules which specifically bind to TL-HN protein, and naming one antibody as T23. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

The following examples were run using GRAPHPAD PRISM software on data expressed as mean ± standard deviation, using One-way ANOVA test, P < 0.05 (x) indicated significant differences, P < 0.01 (x) indicated significant differences, and P < 0.001 (x) indicated significant differences.

EXAMPLE 1 construction of anti-F botulinum toxin nanobody library

1. Camel immunity

The BoNT/F-Hc antigen was mixed with an equal volume of freund's complete adjuvant (Sigma, F5881), emulsified by shaking, and after sufficient emulsification, a healthy adult llama was injected by subcutaneous multipoint injection, after which booster immunization was performed every two weeks, except for the first freund's complete adjuvant, and each immunization was performed with freund's incomplete adjuvant (Sigma, F5506).

2. Separation of camel peripheral blood lymphocytes

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

3. Nested PCR amplification of VHH gene fragments

Total RNA from PBMC was extracted using the OMEGA E.Z.N.A Total RNA kit I kit (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).

Round 1 PCR: PCR amplifying the antibody CH2 region sequence by using the synthesized cDNA as a template and using a designed IgG specific upstream primer CALL001 and a designed downstream primer CALLOO; and (3) performing a second round of PCR amplification by using the recovered first round PCR product as a template and the designed primers VHH-F and VHH-R to amplify the VHH fragment.

TABLE 1 primer sequences used for two rounds of PCR

4. Electrotransport ligation products

The vector NEN-SCFV and the VHH gene amplified in step 3 were digested with restriction enzymes NcoI and NotI. The ligation product was constructed using the T4 ligase ligation.

The ligation products were transformed into E.coli TG1 competent cells (WG 1220, vietnam Biotechnology Co., beijing Hua) using electric shock transformation techniques to construct an anti-F botulinum toxin specific phage antibody library, and the capacity and transformation efficiency were determined using a double dilution phage antibody library.

The library capacity of the antibody library reaches 4X 10 ⁸ pfu through identification. To test the library for accuracy, 48 clones were randomly selected for colony PCR, and sequence alignment showed that the VHH fragment insertion rate reached 100% and the sequences were different.

Example 2 screening of anti-F botulinum toxin specific nanobody phage library

The following media and solutions were prepared:

2YT-GA medium (1L): 16g Typtone, 10 g Yeast Extract, 5, g NaCl, 100 μg/mL ampicillin, 20% glucose, the remainder being water.

2YT-KAA medium (1L): 16 g Typtone, 10 g Yeast Extract, 5g Nacl, 100 μg/mL kanamycin, 20% glucose, a final concentration of 1mM arabinose, the balance being water.

PBST (1L): contains 8.0 g NaCl, 0.2 g KCl, 1.42 g Na ₂HPO₄、0.27 g KH₂PO₄, 0.1% Tween-20 and the balance water.

And (3) taking BoNT/F-Hc as an antigen, and performing solid-phase screening by using the constructed nanobody phage library to obtain the anti-F-type botulinum toxin specific nanobody.

Transferring the constructed anti-F-type botulinum toxin specific nano antibody phage stock solution to a 2YT-GA culture medium (1L) for culturing to logarithmic phase, adding M13KO7 auxiliary phage according to the ratio of MOI approximately equal to 10, standing at room temperature for infection of 30 min, and culturing at 37 ℃ under low speed 150 rpm for 30 min; the deep well plate was placed at 4000 rpm, centrifuged at 15: 15 min at room temperature and the supernatant was discarded and presented overnight using 2YT-KAA medium (1. 1L). The following day the culture presentation supernatant was collected and phages were concentrated with 20% PEG/NaCl solution (200 g PEG6000, 146.25 g NaCl in 1L solution) to obtain high titer antibody library presentation products for subsequent screening. Screening specific nanobody by using a nanobody phage library solid phase, coating BoNT/F-Hc protein with a coating solution (pH=9.6) of 0.05M NaHCO ₃ to an immune tube at 4 ℃ for incubation overnight, washing the immune tube 2 times and 3 min times the next day by using PBS, blocking 2h by using a blocking solution (2% bovine serum albumin) at normal temperature, adding a nanobody phage library solution, and incubating at room temperature for 2 h; then shake at 200 rpm and combine 20: 20 min; PBST (1L) was washed 5 times with PBS, after which 1 mL eluent (0.1M Glycine-HCl, pH 2.2) was added and the 20 min was eluted under shaking conditions 400 rpm; taking out eluent in the target antigen immune tube, adding 20-60 mu L of neutralizing solution (1M Tris-HCl, pH 8.0) for neutralization; after infecting E.coli TG1 in logarithmic growth phase, standing at room temperature for 30 min, culturing at 37 deg.C and rotation speed of 150 rpm for 30 min, and obtaining and purifying phage for the next round of screening, and repeating the same screening process for 3 rounds, wherein the enrichment results are shown in Table 2.

TABLE 2 screening enrichment degree analysis of anti-F botulinum toxin phage nanobody library

After 3 rounds of screening, selecting single clones with obvious intervals and regular shapes from a plate with good phage growth, and inoculating the single clones into 96-well deep hole plates containing 250 mu L of 2YT-GA culture medium in each hole, wherein 2 clones which are not inoculated with clones or are inoculated with other antibodies remain as negative control holes; after the bacterial liquid is cultivated to a logarithmic growth phase at 37 ℃, M13KO7 helper phage (NEW ENGLAND BioLabs, N0315S) is added according to the ratio of MOI approximately 10, namely 100 mu L of each well of a deep-hole plate for cultivating single phage clone is added, and after standing at room temperature for infection of 30min, the culture is carried out at 37 ℃ and low speed of 150 rpm for 30 min; the deep-well plate was placed at 2000 rpm, centrifuged at 10 min at room temperature, and the supernatant was discarded, induced to express using arabinose with a final concentration of 1mM, and cultured overnight at 28℃and 220rpm to obtain phage particles displaying nanobodies.

EXAMPLE 3 Phage-ELISA method for identification of botulinum toxin specific nanobodies F

The BoNT/F-Hc protein is taken as an antigen coating enzyme-linked plate, the antigen concentration is diluted to 2 ng/mu L by 0.05M NaHCO ₃ coating liquid, the dosage is about 200 ng/hole, 2% BSA antigen is coated in parallel on adjacent columns of an antigen column to be taken as a negative control, and the antigen column is coated overnight at 4 ℃; taking out the ELISA plate the next day, washing with PBST on a plate washer for 6 times, and sealing with a sealing liquid (30 g skimmed milk powder is added into 1L PBS) at 200 mu L/hole, and sealing at 37 ℃ for 2 h; centrifuging overnight induced and cultured monoclonal bacterial liquids at 4 ℃ and under the condition of 3000 rpm for 10min, taking 125 mu L supernatant of each bacterial liquid into a 96-hole deep hole plate added with 125 mu L sealing liquid, and pre-combining 30 min; the blocked induced expression supernatant was added to the corresponding enzyme-linked plates coated with the antigen of interest and the control antigen at 100 μl/well and incubated at 37 ℃ for 1.5 h.

After washing 6 times with PBST shaking on a plate washer, diluting HRP-labeled anti-M13 murine monoclonal antibody (Sino Bioligical,1973-MM 05T-H) 4000 times with a blocking solution, adding into an enzyme-linked plate according to 100 mu L/well, and incubating at 37 ℃ for 45 min; washing the plate washer with PBST for 6 times, preparing a color development liquid (wherein 10 mL color development liquid contains 1mL of 10 xOPD, 9 mL of 0.2M Na ₂HPO₄ and 0.1M citric acid mixed solution, and 10 [ mu ] L of 30% hydrogen peroxide), adding into the ELISA plate according to 100 [ mu ] L/hole, and developing for 15-20 minutes in dark place; adding 2M H ₂ SO4 stop solution and 50 mu L/hole stop reaction; and (3) reading under the condition of dual wavelengths 492/630 nm by using an enzyme-labeled instrument, judging the monoclonal antibody with the light absorption value ratio of the antigen group to the negative control group being greater than 5 as positive clone, wherein the results of partial Phage-ELISA experiments are shown in figure 1, the odd columns are target antigens, and the even columns are control antigens. And (3) taking the bacterial solution corresponding to the positive clone, and sending the bacterial solution to a biotechnology service company for sequence determination to obtain the DNA sequence of the insert fragment, so as to finally obtain the phage clone F13 capable of specifically combining with BoNT/B-Hc.

The amino acid sequence of the nano antibody F13 is shown as SEQ ID No. 1: comprises a framework region (FR: FR1, FR2, FR3, FR 4) and a complementarity determining region (CDR: CDR1, CDR2, CDR 3), wherein the amino acid sequence of CDR1 of the complementarity determining region is shown in positions 26-33 of SEQ ID No.1, the amino acid sequence of CDR2 is shown in positions 51-58 of SEQ ID No.1, and the amino acid sequence of CDR3 is shown in positions 97-120 of SEQ ID No. 1; the four parts of the framework region are marked as positions 1-25 of SEQ ID No.1, positions 34-50 of SEQ ID No.1, positions 59-96 of SEQ ID No.1 and positions 121-131 of SEQ ID No.1 in sequence.

Wherein SEQ ID No.1:

QVQLQESGGGSVQAGGSLRLSCAASGYIYGSNYMGWFRQAPEKEREGIAAIYAGGGSTYYADSVKGRFTISLDNAKATLYLQMNNLKPEDTAMYYCAAVDDPGLVVADTEYILQALAFGSTGQGTQVTVSS.

The nucleotide sequence of the gene for encoding the anti-F botulinum toxin nano antibody F13 is shown as SEQ ID No. 2.

SEQ ID No.2：

CAGGTACAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTACGGTAGCAACTACATGGGCTGGTTCCGCCAGGCTCCAGAGAAGGAGCGCGAGGGAATCGCAGCTATTTATGCTGGTGGTGGTAGCACATACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCTAGACAACGCCAAGGCCACATTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGTGGACGACCCCGGACTGGTGGTAGCGGATACTGAGTATATCCTCCAGGCCCTTGCTTTTGGTTCCACGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

EXAMPLE 4 preparation of anti-F botulinum toxin nanobody

1. Construction of eukaryotic expression plasmid pTSE-F13-hFc of anti-F-type botulinum toxin nanobody hFc fusion protein

According to the gene sequence (SEQ ID No. 2) of the anti-F-type botulinum toxin nano antibody F13, the carboxyl terminal of the anti-F-type botulinum toxin nano antibody is connected with the hFc segment of human immunoglobulin to form the anti-F-type botulinum toxin nano antibody hFc fusion protein, the coding nucleotide sequence of the fusion protein is shown as SEQ ID No.3, and the amino acid sequence of the fusion protein is shown as SEQ ID No. 4.

SEQ ID No.3：

CAGGTACAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTACGGTAGCAACTACATGGGCTGGTTCCGCCAGGCTCCAGAGAAGGAGCGCGAGGGAATCGCAGCTATTTATGCTGGTGGTGGTAGCACATACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCTAGACAACGCCAAGGCCACATTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGTGGACGACCCCGGACTGGTGGTAGCGGATACTGAGTATATCCTCCAGGCCCTTGCTTTTGGTTCCACGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCTAGCgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgCtgcatgaggctctgcacaGccactacacgcagaagagcctctccctgtccccgggtaaatga.

SEQ ID No.4：

QVQLQESGGGSVQAGGSLRLSCAASGYIYGSNYMGWFRQAPEKEREGIAAIYAGGGSTYYADSVKGRFTISLDNAKATLYLQMNNLKPEDTAMYYCAAVDDPGLVVADTEYILQALAFGSTGQGTQVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

Cloning the obtained F13 gene sequence onto pTSE-hFc expression vector by basic PCR amplification, enzyme digestion, ligation and other techniques, and selecting a single clone for sequencing verification. After the amplified VHH gene fragment is successfully inserted into a corresponding vector, a eukaryotic expression plasmid is constructed, and the obtained recombinant plasmid is named pTSE-F13-hFc.

The structure of recombinant vector pTSE-F13-hFc is described below: a DNA fragment with a sequence of SEQ ID No.2 is inserted between recognition sites of pTSE-hFc vector restriction enzymes Sal I and Nhe I, and the other sequences of the pTSE-hFc vector are kept unchanged to obtain the recombinant vector. Recombinant vector pTSE-F13-hFc can express nanobody fusion protein F13-hFc.

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

The constructed pTSE-F13-hFc expression plasmid was transfected into FreeStyle ^TM HEK293-F cells (Invitrogen, R79007) with a transfection reagent FectoPRO DNA Transfection Reagent (Polyplus, 116-001), and cell activity was monitored daily after 72 hours, and when the cell activity was reduced from 95-100% to 80-85%, cell supernatants were collected and purified to obtain nanobody fusion protein F13-hFc.

Purified antibodies were analyzed by SDS-PAGE electrophoresis, and the results are shown in FIG. 2: the molecular weight of antibody F13-hFc was as expected, and the band size under reducing conditions of nanobody fusion protein F13-hFc was about 40 kDa (lane 2 in FIG. 2) and the band size under non-reducing conditions was about 80 kDa (lane 1 in FIG. 2).

Example 5 evaluation of nanobody fusion protein F13-hFc Properties

1. The binding activity between the nanobody fusion protein F13-hFc and the BoNT/F-Hc protein was detected by ELISA experiments, which were performed as follows:

BoNT/F-Hc protein concentration was diluted to 2. Mu.g/mL with carbonate coating buffer (pH 9.6), added to 96-well ELISA plates at 100. Mu.L/well, and coated overnight at 4 ℃; the coating liquid is discarded, PBST (the solution 1L PBS contains 8.0 g NaCl, 0.2 g KCl, 1.42 g Na ₂HPO₄、0.27 g KH₂PO₄ and 0.1 percent Tween-20) is used for washing for 6 times, residual liquid in the plate is beaten to dryness, sealing liquid (3 percent skimmed milk powder) is added according to 200 mu L/hole, and the plate is sealed at 37 ℃ for 2 h; diluting an antibody for detecting the binding activity by using a blocking solution by a ratio of 2 times, wherein the initial dilution is 100 mu g/mL, discarding the blocking solution in the plate, cleaning the PBST for 6 times, then drying the residual liquid by beating, adding F13-hFc to be detected into the plate according to 100 mu L/hole, and incubating at 37 ℃ for 1.5 h; the blocking solution was used according to 1: dilution of HRP-labeled goat anti-human IgG at 4000 ratio, discarding primary antibody, PBST wash 6 times, addition to plates at 100 μl/well, incubation at 37 ℃ 45 min; discarding the secondary antibody, washing for 6 times by PBST, adding a peroxidase substrate color development liquid according to 100 mu L/hole, developing 15-20 min in dark, observing the color development effect, and adding 50 mu L/hole 2M sulfuric acid to terminate the reaction after the color development is completed; optical density values were measured using a microplate reader 492 nm/630 nm dual wavelength. The binding capacity between antibodies was assessed by calculating the concentration (EC ₅₀) required for the antibodies to bind 50% of the antigen protein.

As a result, as shown in FIG. 3, the half-effective concentration (EC ₅₀) of the nanobody fusion protein F13-hFc binding to the BoNT/F-Hc antigen protein was 0.293 nM.

2. The specificity of the nanobody was identified by ELISA experiments as follows:

The concentrations of BoNT/A-Hc, boNT/B-Hc, boNT/E-Hc, boNT/F-Hc, boNT/FL-HN, teNT-Hc were diluted to 2. Mu.g/mL with carbonate coating buffer, 100. Mu.L/Kong Jiazhi 96-well ELISA plates, and coated overnight at 4 ℃; the next day, the coating liquid is discarded, the 96-well plate is coated with PBST (0.1% Tween-20) on a plate washer for 6 times, residual liquid in the well is dried by beating, sealing liquid (3% skimmed milk powder) is added according to 200 mu L/well, and the plate washer is sealed at 37 ℃ for 2 h times; preparing F13-hFc protein primary antibody solution, diluting the antibody concentration to 6 mug/mL, discarding the sealing solution, cleaning PBST for 6 times, then beating to dry, adding F13-hFc into 100 mu L/hole, and incubating at 37 ℃ for 1.5 h; the blocking solution was used according to 1: dilution of HRP-labeled goat anti-human IgG at 4000 ratio, discarding primary antibody, PBST wash 6 times, addition to plates at 100 μl/well, incubation at 37 ℃ 45 min; discarding the secondary antibody, washing for 6 times by PBST, adding a peroxidase substrate color development liquid according to 100 mu L/hole, performing light-shielding color development for 15-20 min, observing a color development effect, and adding 50 mu L/hole 2M sulfuric acid to stop the reaction after the color development is completed; optical density values were measured using a microplate reader 492 nm/630 nm dual wavelength, and the data results were analyzed by GRAPHPAD PRISM software.

As shown in FIG. 4, the nanobody fusion protein F13-hFc specifically binds to the BoNT/F-Hc antigen, but does not bind to other antigens or has weak binding activity.

EXAMPLE 6 evaluation of neutralizing Activity of anti-botulinum toxin nanobody

1. Sample preparation

Dilution liquid: KH ₂PO₄0.7 g,Na₂HPO₄•12H₂ O2.4 g, naCl 6.8 g, gelatin 2g, water to 1L, and autoclaving;

Botulinum toxin solution: diluting the F-type botulinum toxin (purchased from China food and drug inspection institute) to 100 LD ₅₀/mL with a diluent;

f13—hfc solution: the fusion protein F13-hFc prepared in example 4 was dissolved in a diluent.

Horse-derived botulinum antitoxin standard solution (BAT-F): is a solution obtained by dissolving horse-derived anti-botulinum toxin serum (purchased from Chinese food and drug testing institute) in a diluent.

2. Specific experimental group

Antibody neutralization activity was determined by in vitro mixing of antibody and lethal dose of botulinum toxin type F, followed by injection of KM ((kunming), available from beijing Bei Fu biotechnology limited). The number of KM mice in each group is 4, the weight is 18-20 g, and the health condition and survival of the mice within 7 days are monitored. The grouping is as follows:

(1) BoNT/F solution 20 XLD ₅₀ group: each KM mouse was intraperitoneally injected with 200. Mu.L of a botulinum toxin solution of 100 XLD ₅₀/mL, such that the toxin dose contained in each injection solution was 20 XLD ₅₀/mouse.

(2) BoNT/F20 XLD ₅₀ +F13-hFc-0.016. Mu.g group: the botulinum toxin solution and the F13-hFc solution are mixed, the volume of each group is supplemented to 2.5 mL by a diluent, the mixture is incubated for 30min at 37 ℃ after uniform mixing, boNT/F+F13-hFc solution is obtained, and the BoNT/F+F13-hFc solution is intraperitoneally injected into KM mice, and each injection is 500 mu L, so that the dosage of BoNT/F is 20 times LD ₅₀/each injection, and the dosage of F13-hFc in each injection solution is 0.016 mu g/each injection.

(3) BoNT/F20 XLD ₅₀ +F13-hFc-0.008. Mu.g group: this group differed from the BoNT/F+F13-hFc-0.016. Mu.g group in that the dose of F13-hFc was 0.008. Mu.g/piece, and the rest of the procedure was the same as that of the BoNT/F20 XLD ₅₀ +F13-hFc-0.016. Mu.g group.

(4) BoNT/F20 XLD ₅₀ +F13-hFc-0.004. Mu.g group: this group differed from the BoNT/F+F13-hFc-0.016. Mu.g group in that the dose of F13-hFc was 0.004. Mu.g/g, and the rest of the procedure was the same as that of the BoNT/F20 XLD ₅₀ +F13-hFc-0.016. Mu.g group.

(5) BoNT/F20 XLD ₅₀ +F13-hFc-0.002 μg group: this group differed from the BoNT/F+F13-hFc-0.016. Mu.g group in that the dose of F13-hFc was 0.002. Mu.g/g, and the rest of the procedure was the same as that of the BoNT/F20 XLD ₅₀ +F13-hFc-0.016. Mu.g group.

(6) BoNT/F20 XLD ₅₀ +F13-hFc-0.001 μg group: this group differed from the BoNT/F+F13-hFc-0.016. Mu.g group in that the dose of F13-hFc was 0.001. Mu.g/g, and the rest of the procedure was the same as that of the BoNT/F20 XLD ₅₀ +F13-hFc-0.016. Mu.g group.

(7) BoNT/F20X ₅₀ +BAT-F-0.2 IU group: the botulinum toxin solution and the horse source botulinum antitoxin standard solution are mixed, the volume of each group is supplemented to 2.5 mL by a diluent, the mixture is uniformly mixed, and then incubated for 30 min at 37 ℃ to obtain BoNT/F+BAT-F solution, and the BoNT/F+BAT-F solution is intraperitoneally injected into KM mice, and 500 mu L of each Bont/F solution is injected, so that the dose of botulinum toxin is 20 times LD ₅₀/horse source botulinum antitoxin BAT-F is 0.2 IU/horse source botulinum antitoxin BAT-F.

As a result, as shown in FIG. 5, 0.008. Mu.g of F13-hFc could completely neutralize the lethal dose of 20 XLD ₅₀ BoNT/F.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (10)

1. A nanobody targeting botulinum toxin type F or an antigen-binding fragment containing the nanobody, characterized in that the nanobody has three complementary determining clusters CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in positions 26-33 of SEQ ID No.1, the amino acid sequence of the CDR2 is shown in positions 51-58 of SEQ ID No.1, and the amino acid sequence of the CDR3 is shown in positions 97-120 of SEQ ID No.1. 2. The nanobody or antigen-binding fragment according to claim 1, characterized in that the nanobody is the following A1) or A2): A1) a Nanobody with an amino acid sequence as 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 biomaterial associated with the Nanobody of claim 1 or 2, wherein the biomaterial is any of the following: B1) a nucleic acid molecule encoding the Nanobody or antigen-binding fragment 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 molecule described in B1); B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4). 4. The biomaterial according to claim 3, characterized in that the nucleic acid molecule B1) is a nucleic acid molecule encoding the nanobody according to claim 1 or 2, wherein the CDR1 encoding gene is nucleotides 76-99 of SEQ ID No. 2, the CDR2 encoding gene is such as nucleotides 151-174 of SEQ ID No. 2, and the CDR3 encoding gene is such as nucleotides 289-366 of SEQ ID No. 2. 5. A method for preparing the nanobody according to claim 1 or 2, comprising the following steps: introducing a nucleic acid molecule encoding the nanobody according to claim 1 or 2 into a recipient cell to obtain a transgenic cell expressing the nanobody, and culturing the transgenic cell to obtain the nanobody. 6. The biomaterial according to claim 3 or 4 or the preparation method according to claim 5, characterized in that the nucleic acid molecule in B1) is any one of the following: C1) a DNA molecule whose nucleotide sequence is shown in SEQ ID No. 2; C2) a DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in C1) and encodes said Nanobody; C3) A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more homology with the DNA sequence defined in C1) or C2) and encodes the Nanobody. 7. A nanobody fusion protein, characterized in that the nanobody fusion protein is a fusion of the nanobody or antigen-binding fragment according to claim 1 or 2 with another molecule, wherein the other molecule includes an Fc domain of an immunoglobulin, a fluorescent protein, or a VHH with different specificities. 8. The nanobody fusion protein according to claim 7, characterized in that the fusion protein is any one of the following: M1) a fusion protein with an amino acid sequence as 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 detection kit targeting botulinum toxin type F, characterized in that the kit comprises the nanobody according to claim 1 or 2, the biological material according to any one of claims 3-5, or the fusion protein according to claim 7 or 8. 10. Any of the following applications: E1) Use of the Nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a product for detecting botulinum toxin type F; E2) Use of the biological material according to any one of claims 3 to 5 in the preparation of a product for detecting botulinum toxin type F; E3) Use of the preparation method according to claim 6 or 7 in the preparation of a product for detecting botulinum toxin type F; E4) Use of the kit according to 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 product bound to botulinum toxin type F; E6) Use of the biomaterial according to any one of claims 3 to 5 in the preparation of a product combined with botulinum toxin type F; E7) Use of the preparation method according to claim 6 or 7 in the preparation of a product combined with botulinum toxin type F; E8) Use of the kit according to claim 9 in the preparation of a product combined with botulinum toxin type F; E9) Use of the nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a botulinum toxin type F detection reagent; E10) Use of the nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a botulinum toxin type F diagnostic reagent; E11) Use of the nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a medicament for preventing and/or treating botulinum toxin type F infection.