C-type botulinum toxin receptor binding domain nano antibody and application thereof

Abstract

The invention provides a nano antibody for resisting a C-type botulinum toxin receptor binding domain and application of the nano antibody. The C-type botulinum toxin receptor binding domain is used as an antibody to immunize alpaca, RNA is extracted from mononuclear cells of the alpaca, and the nano antibody resisting the C-type botulinum toxin receptor binding domain is screened from the mononuclear cells by using a phage display library technology. The nano antibody provided by the invention can be specifically bound with a C-type botulinum toxin receptor binding domain, has high affinity, can effectively inhibit specific binding of C-type botulinum toxin and a target cell membrane, and can neutralize the C-type botulinum toxin to play a protective role. The nano antibody provided by the invention is expected to be applied to preparation of C-type botulinum toxin poisoning antidotes. Meanwhile, the nano antibody provided by the invention can be applied to qualitative and quantitative analysis and detection of C-type botulinum toxin as a capture antibody and a detection antibody.

Description

Technical Field

[0001] This invention belongs to the fields of cellular immunology and genetic engineering, and specifically relates to a type C botulinum toxin nanobody that can be used as an effective antidote. Background Technology

[0002] Botulinum toxin is a highly lethal neurotoxin produced by Clostridium botulinum and is considered one of the most potent known biological and chemical toxins. Based on its antigenicity, botulinum toxin is classified into eight serotypes, AG and X, all composed of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) linked by disulfide bonds. The heavy chain consists of an N-terminal translocation domain (HCN) and a C-terminal receptor domain (HCR). The toxin enters the cell via endocytic vesicles formed by binding to specific receptors on the presynaptic membrane via the HCR. The lower pH within the endocytic vesicle causes the disulfide bond between HC and LC to break, while HCN binds to the cell membrane, releasing LC into the cytoplasm. The LC, being a protease domain, specifically cleaves SNARE proteins upon entering the cytoplasm, thereby blocking the fusion of synaptic vesicles with the presynaptic membrane, inhibiting neurotransmitter release, and causing a toxic reaction.

[0003] Botulinum toxin type C (BoNT / C) is one of the main causes of botulism in animals. Foodborne BoNT / C poisoning in animals leads to systemic muscle weakness; severe cases can result in respiratory distress and paralysis, typical clinical manifestations of botulism. It has a rapid onset, high mortality rate, and often occurs in clusters. Once poisoned, there is no effective antidote, causing significant economic losses to the livestock industry. Furthermore, if measures are not taken promptly, other carnivores may develop botulism symptoms or even die after consuming the carcasses of poisoned animals. This allows Clostridium botulinum to remain dormant and spread in nature, potentially causing wider and more severe botulism, and even threatening human food safety, becoming a public safety issue.

[0004] Studies have found that different types of botulinum toxin have varying durations of action. BoNT / C's duration of action is comparable to BoNT / A, which is widely used in aesthetic and medical fields, with less neuromuscular damage. BoNT / C shows promise as a good alternative to BoNT / A, addressing the issue of BoNT / A resistance. Furthermore, since BoNT / C is currently the only known botulinum toxin protein that simultaneously cleaves two SNARE substrate proteins (SNAP-25 and syntaxin), it suggests that, on the one hand, BoNT / C is suitable for patients who are naturally unresponsive to BoNT / A. On the other hand, studies show that syntaxin overexpression is seen in various hypersecretion diseases, suggesting that BoNT / C has the potential for use in the development of drugs for these diseases. In summary, BoNT / C has significant potential applications in the medical field. However, given that botulinum toxin is a highly toxic substance, it carries a high risk of iatrogenic poisoning.

[0005] To date, there is no effective antidote for BoNT / C poisoning. Prevention of botulism in animals relies solely on feed quality control and vaccination with type C botulinum toxin-like vaccines. However, these vaccines are complex to manufacture, have weak immunogenicity, and induce short-lived immune responses, requiring multiple vaccinations. Equine botulinum antitoxin is currently the only specific treatment for BoNT / C poisoning, but it only works for a very short time after poisoning, and due to species differences, it has a high rate of allergic reactions and is prone to serum sickness. In conclusion, BoNT / C is the main cause of botulism in animals, and its widespread clinical application increases the risk of iatrogenic toxin poisoning in humans. Therefore, there is an urgent need in the market for an effective antidote for BoNT / C botulism poisoning.

[0006] Binding of botulinum toxin to receptors on the presynaptic membrane is a crucial step in its entry into cells to exert its effects. Developing effective inhibitors targeting the receptor binding site of botulinum toxin to block its internalization into neurons and its ability to exert its effects can effectively reduce flaccid paralysis caused by the toxin. Nanobodies, derived from heavy-chain antibodies produced by camels or sharks, contain only the variable region fragment of the heavy chain compared to ordinary IgG antibodies. They can exist independently and stably in vitro and can act on antigens alone, possessing complete antigen recognition capabilities. Nanobodies are 4 nm long, 2.5 nm in diameter, and have a molecular weight of only about 15 kDa, making them the smallest known antigen-binding proteins. The CDR3 of nanobodies consists of 13-18 amino acids, longer than the CDR3 of traditional antibodies, enabling them to recognize deeply hidden antigenic epitopes that are difficult for traditional antibodies to recognize. Due to their unique structure, nanobodies possess superior characteristics such as low production cost, weak immunogenicity, and good tissue permeability, making them a hot research topic in many medical fields. Summary of the Invention

[0007] To address the lack of effective antidotes for botulinum toxin type C, this invention provides a nanobody that specifically binds to the BoNT / C receptor-binding domain. This nanobody, obtained through phage display library screening, effectively inhibits the specific binding of BoNT / C to its target cell membrane and neutralizes botulinum toxin type C. It exhibits protective effects in mouse BoNT / C lethality and gastrocnemius muscle paralysis experiments. The nanobody provided by this invention can be used as a capture antibody and detection antibody in the qualitative and quantitative analysis and detection of botulinum toxin type C. Furthermore, the nanobody provided by this invention shows promise for application in the preparation of antidotes for botulinum toxin type C poisoning.

[0008] Specifically, it includes the following:

[0009] In a first aspect, the present invention provides a type C botulinum toxin receptor-binding domain nanobody, wherein the sequence of the complementarity-determining region (CDR) of the nanobody is any one of the following (1)-(18):

[0010] (1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3;

[0011] (2) CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6;

[0012] (3) CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9;

[0013] (4) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, and CDR3 shown in SEQ ID NO.12;

[0014] (1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3;

[0015] (2) CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6;

[0016] (3) CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9;

[0017] (4) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, and CDR3 shown in SEQ ID NO.12;

[0018] (5) CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, and CDR3 shown in SEQ ID NO.15;

[0019] (6) CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, and CDR3 shown in SEQ ID NO.18;

[0020] (7) CDR1 shown in SEQ ID NO.19, CDR2 shown in SEQ ID NO.20, and CDR3 shown in SEQ ID NO.21;

[0021] (8) CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO.23, and CDR3 shown in SEQ ID NO.24;

[0022] (9) CDR1 shown in SEQ ID NO.25, CDR2 shown in SEQ ID NO.26, and CDR3 shown in SEQ ID NO.27;

[0023] (10) CDR1 shown in SEQ ID NO.28, CDR2 shown in SEQ ID NO.29, and CDR3 shown in SEQ ID NO.30;

[0024] (11) CDR1 shown in SEQ ID NO.31, CDR2 shown in SEQ ID NO.32, and CDR3 shown in SEQ ID NO.33;

[0025] (12) CDR1 shown in SEQ ID NO.34, CDR2 shown in SEQ ID NO.35, and CDR3 shown in SEQ ID NO.9;

[0026] (13) CDR1 shown in SEQ ID NO.36, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.37;

[0027] (14) CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9;

[0028] (15) CDR1 shown in SEQ ID NO.39, CDR2 shown in SEQ ID NO.40, and CDR3 shown in SEQ ID NO.41;

[0029] (16) CDR1 shown in SEQ ID NO.39, CDR2 shown in SEQ ID NO.40, and CDR3 shown in SEQ ID NO.42;

[0030] (17) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.43, and CDR3 shown in SEQ ID NO.44;

[0031] (18) CDR1 shown in SEQ ID NO.45, CDR2 shown in SEQ ID NO.43, and CDR3 shown in SEQ ID NO.44.

[0032] Preferably, the sequence of the nanobody is as shown in any one of SEQ ID NO.46-65.

[0033] In a second aspect, the present invention provides a nucleic acid molecule that encodes the type C botulinum toxin receptor-binding domain nanobody described in the first aspect above.

[0034] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect above.

[0035] Fourthly, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0036] Fifthly, the present invention provides the application of the C-type botulinum toxin receptor-binding domain nanobody described in the first aspect above in the detection of C-type botulinum toxin for non-disease diagnostic purposes.

[0037] In a sixth aspect, the present invention provides the application of the type C botulinum toxin receptor-binding domain nanobody described in the first aspect above in the preparation of products for detecting type C botulinum toxin.

[0038] In a seventh aspect, the present invention provides the use of the type C botulinum toxin receptor-binding domain nanobody described in the first aspect above in the preparation of a medicament for treating botulinum toxin poisoning.

[0039] Preferably, the botulism is caused by botulinum toxin type C.

[0040] Eighthly, the present invention provides a reagent for qualitative and quantitative detection of botulinum toxin type C content, the reagent comprising the botulinum toxin type C receptor-binding domain nanobody described in the first aspect above.

[0041] In a ninth aspect, the present invention provides a medicament for treating botulinum toxin poisoning caused by botulinum toxin type C, wherein the active ingredient of the medicament comprises the botulinum toxin type C receptor-binding domain nanobody described in the first aspect above.

[0042] Eighthly, the present invention provides a reagent for qualitative and quantitative detection of botulinum toxin type C content, the reagent comprising the nanobody described in the first aspect above.

[0043] In a ninth aspect, the present invention provides a medicament for treating botulinum toxin poisoning, wherein the active ingredient of the medicament comprises the nanobody described in the first aspect above.

[0044] The beneficial effects of this invention are as follows: First, this invention utilizes phage display library technology to screen and obtain a nanobody that can specifically bind to the BoNT / C receptor binding domain; second, the nanobody can effectively inhibit the specific binding of BoNT / C to its target cell membrane and can neutralize botulinum toxin type C, playing a protective role in mouse BoNT / C lethality experiments and mouse gastrocnemius muscle paralysis experiments; moreover, the nanobody can be used as a capture antibody and detection antibody in the qualitative and quantitative analysis and detection of botulinum toxin type C; simultaneously, the nanobody is expected to be used in the preparation of antidotes for botulinum toxin type C poisoning. Attached Figure Description

[0045] Figure 1 Results of induction, purification, and desalting of recombinant protein His6-BCHCR; A: Induction and purification of recombinant protein His6-BCHCR, lane 1: protein molecular weight standard; lane 2: uninduced; 3: 0.5 mM IPTG induction; 4: precipitate after centrifugation of lysed culture; 5: supernatant after centrifugation of lysed culture; 6: Ni column chromatography breakthrough; 7: washing with binding buffer; 8-11: elution with 250 mM imidazole gradient, fractional collection of 1-4; B: Desalting of recombinant protein His6-BCHCR, lane 1: protein molecular weight standard; 2: lane 9 in Figure A; 3-5: desalting with 20 mM PB and 100 mM NaCl, fractional collection of 1-3.

[0046] Figure 2 Results of induction, expression and purification of recombinant protein GST-BCHCR; Lane 1: Protein molecular weight standard; Lane 2: Uninduced; Lane 3: Induced by 0.5 mM IPTG; Lane 4: Precipitate from centrifugation of lysed culture; Lane 5: Supernatant from centrifugation of lysed culture; Lane 6: Ni column chromatography breakthrough; Lane 7: Washing with Binding buffer; Lanes 8-12: Eluent elution of reduced glutathione and fractional collection of 1-5.

[0047] Figure 3Phage-ELISA positive clone screening results; A: 92 clones in total (numbered 1-92); B: 92 clones in total (numbered 93-184).

[0048] Figure 4 Purification results of BoNT / C HCR nanobodies; Lane 1: Protein molecular weight standard; Lanes 2-13: Nanobodies A2, A3, B7, C6, D1, D12, E2, E5, E11, E12, F9, and G5, respectively.

[0049] Figure 5 Results of nanobody inhibition of BCHCR targeting binding to cell membrane; A: Laser confocal microscopy detection results of nanobody inhibition of BoNT / CHCR binding activity with target cells, fluorescence imaging results of 12 nanobodies co-incubated with BoNT / CHCR, with BSA protein set as a control group, scale bar size 5μm; B: Statistical results of average cell fluorescence intensity, the data shown are mean ± standard deviation (n=10); Compared with the BoNT / CHCR control group, **P<0.01, ***P<0.001, ns P>0.05.

[0050] Figure 6 Western blot analysis of the effect of nanobody A2 on the uptake of toxins by primary cultured neurons; A: Western blot analysis of the effect of A2 on the uptake of toxins by primary cultured neurons; B: Semi-quantitative analysis of the inhibitory effect of A2 on the activity of BoNT / C SNAP-25 cleavage, with gray values ​​as the quantitative index and the ordinate representing the proportion of uncleaved SNAP-25 in total SNAP-25 protein. Data are presented as mean ± standard deviation (n = 3). Compared with the BoNT / C group, ***P < 0.001.

[0051] Figure 7 Results of the neutralizing and protective effects of nanobodies on BoNT / C; A: Neutralizing effects of different nanobodies on BoNT / C, with A2 showing the best effect; B: Co-incubation of 0.1, 1, and 10 μg of nanobodies A2 with 125 pg of BoNT / C resulted in dose-dependent neutralization of BoNT / C, exerting a protective effect; C: Nanobodies A2 and different doses of BoNT / C were mixed at a ratio of 1:10. 6 Co-incubation significantly neutralizes BoNT / C, exerting a protective effect.

[0052] Figure 8 Results of the antidote effect of nanobodies on BoNT / C poisoning: A: Intraperitoneal injection of BoNT / C, tail vein injection of A2; B: Intramuscular injection of BoNT / C, intramuscular injection of A2 at the original site; C: Intramuscular injection of BoNT / C, tail vein injection of A2. Detailed Implementation

[0053] The present invention will be further illustrated below with reference to a detailed description of specific embodiments. However, the embodiments described below are merely illustrative of the technical solutions of the present invention and do not limit the technical solutions of the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, the bioinformatics software and products used are all commercially available, and the experimental processes and methods are also conventional methods known in the art. The source, trade name, and components of the materials used, if necessary, are indicated upon their first appearance. Unless otherwise specified, the same reagents used thereafter are the same as those initially indicated.

[0054] Furthermore, it should be noted that the site combinations and applications provided by this invention were achieved through the arduous creative labor and optimization work of the inventors of this application.

[0055] Example 1: Expression and purification of His6-BCHCR and GST-BCHCR proteins

[0056] 1. Experimental Materials

[0057] Kanamycin sulfate (Sangon Biotech Co., Ltd., A506636-0025), ampicillin sodium (Sangon Biotech Co., Ltd., A610028-0025), IPTG (Sangon Biotech Co., Ltd., A600168-0025), imidazole (Sangon Biotech Co., Ltd., A600277-0100), tryptone (Thermo Fisher Scientific, LP0042B), yeast extract (Thermo Fisher Scientific, LP0021), sodium chloride, sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, methanol, glacial acetic acid, and anhydrous ethanol were purchased from Tianjin Baishi Chemical Co., Ltd., SDS (Sangon Biotech Co., Ltd., A600485-0100), and glycine (Sangon Biotech Co., Ltd., A6...). 10235-0005), Tris (Sangon Biotech Co., Ltd., A610195-0005), Coomassie Brilliant Blue R-250 (Sangon Biotech Co., Ltd., A610037), Bromophenol Blue (Beijing Solarbio Science & Technology Co., Ltd., B8120), TEMED (Beijing Solarbio Science & Technology Co., Ltd., T8090), Ammonium Persulfate (Beijing Jinming Biotechnology Co., Ltd., 7727-54-0), Acrylamide (Beijing Jinming Biotechnology Co., Ltd., 79-06-1), N,N-Methylenebisacrylamide (Beijing Jinming Biotechnology Co., Ltd., 110-26-9), pET28a+ (Sangon Biotech Co., Ltd., B540183); pGEX-4T (Fenghui Biotechnology Co., Ltd., YH004); Ni Sepharose™ 6 Fast Flow (GE Healthcare, 17-5318-01), Sephadex G-25Resin chromatography media (Sangon Biotech Co., Ltd., C510060-0026), GST Sepharose 4FF (GST-Tag) (Sangon Biotech Co., Ltd., C600031), and glutathione-reduced GSH (purchased from Sangon Biotech Co., Ltd., A600229).

[0058] 2. Solution preparation

[0059] LB liquid medium: Weigh 5.0g tryptone, 2.5g yeast extract and 5.0g NaCl and dissolve in 400ml deionized water, then bring the volume up to 500ml. Autoclave at 121℃ for 20min and store at room temperature.

[0060] LB solid medium: Weigh 7.5g of agar powder into 500ml of unsterilized LB liquid medium, autoclave at 121℃ for 20min, and when the medium temperature cools to 50-60℃, add an appropriate amount of antibiotics as needed for the experiment, mix well, plate, and store at 4℃.

[0061] 1M IPTG: Weigh 11.915g of IPTG and dissolve it in 40ml of deionized water, then bring the volume up to 50ml. Filter the solution through a 0.22μm filter to sterilize it, and store it at -20℃.

[0062] 100μg / ml kanamycin sulfate: Weigh 5.0g of kanamycin sulfate, dissolve it in 40ml of deionized water, then bring the volume up to 50ml. Filter the solution through a 0.22μm filter to sterilize it, and store it at -20℃.

[0063] 10×PBS solution: Weigh 80g NaCl, 1g KCl, 35.8g Na2HPO4·12H2O, and 2.7g KH2PO4·3H2O, dissolve them in 800ml deionized water, and then bring the volume up to 1L. Store at room temperature.

[0064] PBST: Measure 100 mL of 10×PBS solution and mix it with 900 mL of deionized water. Add 250 μl of Tween-20 and store at room temperature.

[0065] 5×SDS-PAGE electrophoresis buffer: Weigh 15.1g Tris, 94g glycine, and 5g SDS, dissolve them in 800ml deionized water, and then bring the volume up to 1L. Store at room temperature.

[0066] 5×SDS-PAGE loading buffer: 2.5 ml of 1M Tris-HCl (pH 6.8), 1 g of SDS, 0.05 g of bromophenol blue, and 2.5 ml of glycerol, dissolved in 8 ml of deionized water, then brought to a final volume of 10 ml and stored at -20°C.

[0067] Coomassie Brilliant Blue R-250 staining solution: Weigh 1.0g of Coomassie Brilliant Blue R-250, add 250ml of isopropanol and 100ml of glacial acetic acid, and then bring the volume up to 1L. Store at room temperature.

[0068] Coomassie Brilliant Blue staining destaining solution: Measure 100 ml of glacial acetic acid and 50 ml of anhydrous ethanol, then make up to 1 liter and store at room temperature.

[0069] 3. Experimental Procedure

[0070] 3.1 His6-BCHCR protein induced expression

[0071] The BoNT / C HCR gene sequence was synthesized by Genewiz (SEQ ID NO. 66) after codon optimization, based on amino acid information from the UniProt database (P18640, BXC_CBCP, range 866-1291), and cloned at the NdeI and EcoRI sites of pET28a+. This clone was then transformed into E. coli BL21(DE3) and cryopreserved as a glycerol bacterium. 5 μl of the cryopreserved glycerol bacterium was inoculated into 30 ml of LB medium containing 80 μg / ml kanamycin sulfate and incubated at 37°C with shaking at 200 rpm for 14 h as the inoculum. 2 ml of the inoculum was then inoculated into 500 ml of LB medium containing 80 μg / ml kanamycin sulfate and incubated at 37°C with shaking at 200 rpm for 4-5 h until OD (dose expiratory volume). 600 After reaching a protein expression level of 0.8-1.2, 1 ml of bacterial culture was taken as the uninduced sample, and 0.5 mM IPTG was added. Protein expression was induced at 20°C for 16 h, and then another 1 ml of bacterial culture was taken as the induced sample. After the induction was complete, the bacterial pellet was collected by centrifugation at 8000g for 3 min and stored at -20°C. Simultaneously, the uninduced and induced samples were centrifuged to prepare SDS-PAGE samples to detect protein expression.

[0072] 3.2 Purification of His6-BCHCR protein

[0073] Add 30 times its weight volume of binding buffer (binding buffer: 20 mM BP, pH 7.4, 300 mM NaCl, 50 mM imidazole) to the centrifuged bacterial pellet for resuspending. Homogenize at 850 bar using an autoclave until the bacterial cells are clear. Centrifuge at 1000 rpm for 10 min to obtain the supernatant. Load the supernatant at a flow rate of 1.5 ml / min onto a Ni solution pre-equilibrated with binding buffer. 2+ The Sepharose™ 6 Fast Flow affinity chromatography column was then washed with binding buffer at a flow rate of 1.5 ml / min until the eluent reached OD. 280 Once the baseline was reached, the target protein was eluted with elution buffer (20 mM PB, pH 7.4, 300 mM NaCl, 250 mM imidazole) at a flow rate of 1.5 ml / min, and the eluent was collected. The collected eluent was then passed through a Sephadex G-25 Resin desalting column to remove imidazole, and stored in a buffer of 20 mM PB, pH 7.4, 100 mM NaCl, and 5% glycerol, and flash-frozen in liquid nitrogen at -80°C.

[0074] 3.3 Inducible expression of GST-BCHCR

[0075] The BoNT / C HCR gene sequence was obtained from the UniProt database (P18640, BXC_CBCP, range 866-1291), and after codon optimization, the entire gene was synthesized by Genewiz (SEQ ID NO. 66) and cloned into the BamHI and EcoRI sites of pGEX 4T-1. The clone was then transformed into E. coli DH5α and cryopreserved as a glycerol bacterium. The target protein expression was induced according to method 3.1. 5 ml of the bacterial culture was inoculated into 500 ml of LB medium containing 100 μg / ml ampicillin sodium and incubated at 37°C with shaking at 200 rpm for 3 h until OD was reached. 600 After reaching 0.4-0.6, take 1 ml of bacterial culture as the uninduced sample, add 0.5 mM IPTG, induce protein expression at 20℃ for 16 h, and then take another 1 ml of bacterial culture as the induced sample. Detect by SDS-PAGE electrophoresis.

[0076] 3.4 Purification of GST-BCHCR

[0077] Following procedure 3.2, the bacteria were lysed. The supernatant was loaded at a flow rate of 1.5 ml / min onto a GST affinity chromatography column pre-equilibrated with binding buffer (20 mM PB, pH 8.0, 150 mM NaCl), and eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 20 mM GSH) to obtain the target protein. SDS-PAGE electrophoresis was used for detection.

[0078] 4. Experimental Results

[0079] Utilizing the principle of competitive binding of imidazole to nickel ions by His-tagged proteins, the supernatant of the lysed culture was passed through a nickel affinity chromatography column. The SDS-PAGE results of the purified protein are shown below. Figure 1 As shown in Figure A, by comparing the uninduced bacterial culture sample in lane 2 and the bacterial culture sample induced with 0.5 mM IPTG in lane 3, the target protein band was observed at approximately 45 kDa. The recombinant protein His6-BoNT / C HCR with high concentration and purity was successfully obtained in the elution buffer. The obtained target sample was desalted using a desalting column, and the results are shown in Figure A. Figure 1 For protein B, desalting and solution replacement can also be performed using dialysis.

[0080] The His6-BoNT / C HCR obtained in this embodiment can be used as an antigen to immunize alpacas. Considering the subsequent antigen screening aims to filter out nanobodies targeting the purification tag, a GST-tagged BoNT / C HCR was purchased and constructed in this embodiment. The target protein was purified using a GSH affinity chromatography column, and the results are as follows. Figure 2 Collect the eluted target protein GST-BoNT / C HCR, aliquot it, and store it at -80°C for later use.

[0081] Example 2: Establishment of a phage library containing C-type botulinum toxin receptor-binding domain nanobodies

[0082] 1. Materials

[0083] Total RNA Extraction Reagent (Shanghai Yisheng Biotechnology Co., Ltd., 10606ES60); AEBSF (Beijing Solarbio Technology Co., Ltd., IA0110); (NEB Corporation, R3140V); (NEB, R3162V); pMES4 phage vector (Fenghui Biotechnology Co., Ltd., ZT642); TG1 Escherichia coli cell line (Ruichu Biotechnology Co., Ltd., R09003); VCSM13 helper phage (Apak Biotechnology Co., Ltd., P007); SanPrep column PCR product purification kit (Sangon Biotech Co., Ltd., B518141-0100); Stripwell™ Microplate (COSTAR, 42592).

[0084] 2. Experimental Methods

[0085] 2.1 RNA extraction from peripheral blood mononuclear cells (PBMCs) obtained from alpaca immunization

[0086] The purified His6-BCHCR, with a concentration of 1 mg / ml and a purity of over 90%, was sent as an antigen to Shanxi Nami Biotechnology Development Co., Ltd. for outsourced immunization of alpacas. After four immunizations, the alpaca antiserum passed quality testing, achieving a titer of 10. 5 At that time, peripheral blood mononuclear cells were isolated from alpaca blood, and total RNA was extracted.

[0087] (1) Take out the contents containing 1.2×10 8 Add 11.0 ml of Total RNA Extraction Reagent to a TRIzol solution containing 1 PBMC to ensure that each ml of Total RNA Extraction Reagent contains 1 × 10⁻⁶ PBMCs. 7 After mixing the cells by repeatedly pipetting them, the cells were divided into 1ml tubes (12 tubes in total) and allowed to stand at room temperature for 5 minutes to ensure complete dissociation of the ribosomes.

[0088] (2) Add 200 μl of chloroform to each tube, shake vigorously for about 15 minutes, and let stand at room temperature for 3 minutes.

[0089] (3) Centrifuge at 12000g for 15 minutes at 4℃. After centrifugation, there should be three layers in the centrifuge tube: the upper layer is a transparent aqueous phase in which RNA exists; the middle layer is a white precipitate of DNA; and the lower layer is a red organic phase. The volume of the upper layer is about 50-60% of the total amount of Total RNA Extraction Reagent added.

[0090] (4) Carefully aspirate the aqueous phase into a new centrifuge tube, add 500 μl of isopropanol, invert and mix well, and let stand at room temperature for 10 min.

[0091] (5) Centrifuge at 12000g for 15 min at 4℃, and a gelatinous precipitate will appear at the bottom of the test tube;

[0092] (6) Carefully discard the isopropanol, add 1 ml of 75% ethanol prepared with DEPC water to the tube, and vortex to wash the precipitate thoroughly.

[0093] (7) Centrifuge at 7500g for 5 minutes at 4℃, and carefully discard the supernatant;

[0094] (8) Repeat steps (6)-(7) to ensure that the precipitate is thoroughly washed;

[0095] (9) Allow the RNA to air dry at room temperature for 10 minutes, add 40 μl of DEPC water to dissolve the RNA, and after it is completely dissolved, take a small amount for RNA quality testing, and use the rest for reverse transcription experiments.

[0096] 2.2 RNA reverse transcription into cDNA

[0097] Using the extracted RNA as a template, cDNA was reverse transcribed into cDNA by reverse transcriptase. The reverse transcription reaction system (20 μl system) was prepared according to Table 1, and the reverse transcription reaction procedure was as shown in Table 2.

[0098] Table 1 Reverse transcription reaction system

[0099]

[0100] Table 2 Reverse Transcription Procedure

[0101] Temperature (°C) Time (min) 25 5 55 15 85 5

[0102] 2.3 Nanobody fragments were obtained from cDNA via nested PCR.

[0103] The cDNA obtained from reverse transcription needs to be specifically amplified into nanobody fragments using nested PCR. The primers required for the first step of nested PCR are shown in Table 3, the PCR system in Table 4, and the PCR procedure in Table 5. A 700bp target fragment is obtained. The target band is then recovered using a gel extraction kit according to the instructions. The recovered product is used as the template for the second step of nested PCR. The primers required for the second step of nested PCR are shown in Table 6, the PCR system in Table 7, and the PCR procedure in Table 8. The approximately 450bp nanobody fragment obtained from the amplification is then recovered using a gel extraction kit.

[0104] Table 3 Primers for the first step of nested PCR amplification

[0105] Primers sequence CALL001 Primer GTCCTGGCTGCTCTTCTACAAGG(SEQ ID NO.67) CALL002 Primer GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO.68)

[0106] Table 4. Reaction system for the first step of nested PCR

[0107]

[0108] Table 5. Reaction procedure for the first step of nested PCR.

[0109]

[0110] Table 6 Primers for the second step of nested PCR amplification

[0111] Primers sequence VHH-Back GATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO.69) VHH-For CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO.70)

[0112] Table 7. Reaction system for the second step of nested PCR

[0113]

[0114] Table 8. Reaction Procedure for the Second Step of Nested PCR

[0115]

[0116] 2.4 Construction of Phage Nanobody Display Library

[0117] The 450bp nanobody fragment amplified by nested PCR was digested with pMES4 phage vector using PstI and BstEII double enzyme digestion. The double enzyme digestion system is shown in Table 9.

[0118] Table 9 Double Enzyme Digestion System

[0119]

[0120] After digestion at 37℃ for 1 hour, the enzyme was digested overnight at 4℃. The digestion products were recovered by gel extraction and ligated according to a molar ratio of pMES4 to VHH of 1:3. The ligation system is shown in Table 10.

[0121] Table 10 Connection System

[0122] Components volume pMES4 15μl VHH 15μl 10×Buffer 5μl T4 DNA Ligase 5μl <![CDATA[H2O]]> 10μl

[0123] The ligation product was purified using the SanPrep column PCR product purification kit after overnight incubation at 20°C, and eluted with 30 μl of sterile water. Twenty electroporations were performed using TGI competent cells under the following conditions: Geminni X2 electroporator, 1 cm electroporation cuvette, 1.8 kV, 25 μF, and 200 ohms. Immediately after electroporation, 1 ml of SOC culture medium was added, and the mixture was incubated at 37°C with shaking at 200 rpm for 1 hour. The culture was then diluted 1000-fold and plated onto LB agar plates (containing 100 μg / ml ampicillin sodium) as a control plate for calculating library capacity. The remaining bacterial culture was plated onto LB agar plates (containing 100 μg / ml ampicillin sodium) for library construction. Both were incubated overnight at 37°C. The next day, the control plate showed 590 colonies, and the calculated library capacity was 1.18 × 10⁻⁶. 7 The colonies met the requirements for library construction. The colonies on the agar plates were then washed and scraped off, and glycerol was added to a final concentration of 20%, and the plates were frozen at -80°C. Fifteen single clones were randomly selected for bacterial culture PCR to verify the insertion rate of the nanobody fragments. The results showed that the insertion rate of this invention reached 100%. The primers for bacterial culture PCR verification are shown in Table 11, the PCR system is shown in Table 12, and the PCR procedure is shown in Table 13.

[0124] Table 11 Primers for bacterial culture PCR verification

[0125] Primers sequence MP57 TTATGCTTCCGGCTCGTATG(SEQ ID NO.71) GIII CCACAGACAGCCCTCATAG(SEQ ID NO.72)

[0126] Table 12 Validation system for bacterial culture PCR

[0127]

[0128] Table 13 PCR reaction procedure for bacterial culture

[0129]

[0130] 3. Experimental Results

[0131] To obtain nanobody gene sequences from PBMCs, this invention uses TRIzol reagent to extract 1.2 × 10⁻⁶ nanobody genes. 7 Total RNA from all cells was collected and dissolved in 40 μl of enzyme-free sterile water. Electrophoresis showed that the 28S, 18S, and 5S rRNA bands were clearly visible, consistent with theoretical results, indicating that the total RNA was intact and undegraded. Furthermore, the A260 / A280 ratio of high-quality RNA should be between 1.8 and 2.1. The RNA solution concentration, measured by a nucleic acid analyzer, was 1,100 ng / μl, and the A260 / A280 ratio was 1.95, indicating good purity of the total RNA, meeting the requirements for further experiments.

[0132] To amplify the nanobody gene sequence from extracted total RNA, the total RNA was reverse transcribed into cDNA, and the nanobody gene fragment was amplified from the cDNA using nested PCR to improve the specificity of the PCR reaction. Different amounts of cDNA were used for PCR amplification. The first round of amplification yielded a 700 bp target fragment containing the nanobody gene sequence. Additionally, a heavy chain fragment band of a conventional antibody was observed at 1000 bp. Using 1 μl of the aforementioned cDNA as a template, eight parallel replicates were performed. The PCR products from each replicate were recovered via gel electrophoresis, separating and recovering the 700 bp band. The recovered product concentration was approximately 20 ng / μl. Approximately 20 ng of the recovered DNA fragment was then used as a template for the second round of amplification. Nucleic acid electrophoresis results showed that the nanobody fragment band appeared at approximately 450 bp, with clear and high brightness. The concentration of the DNA fragment obtained through gel recovery was 26.508 ng / μl. This invention utilizes nested PCR for two rounds of amplification, successfully obtaining the nanobody gene sequence from the reverse-transcribed cDNA at an appropriate concentration for subsequent experiments.

[0133] pMES4 is a phage vector that combines the functions of filamentous phages and plasmid vectors, and is commonly used in phage display technology. To construct a pMES4-nanobody recombinant plasmid, this invention utilizes two restriction endonucleases, PstI and BstEII, to digest the pMES4 vector and the nanobody gene fragment amplified by nested PCR, respectively, to achieve complete vector linearization. Nucleic acid electrophoresis results showed that after digestion, the pMES4 vector exhibited only one uniform band, and the linearized plasmid migrated more slowly and at a higher position than the circular plasmid, sharing the same sticky ends as the nanobody gene fragment. Furthermore, DNA with the same sticky ends was ligated using T4 DNA ligase at a fragment-to-vector molar ratio of 1:3, and the recombinant plasmid was obtained by PCR product purification, with a concentration of 14.91 ng / μl. In summary, by digesting and ligating the pMES4 vector with the nanobody gene fragment, a recombinant plasmid was successfully constructed, which can be used for the construction of phage display libraries.

[0134] Escherichia coli TG1 is a commonly used host bacterium in phage display technology. It contains the F′ factor, which allows phage infection and repackaging, and it can suppress the amber stop codon, enabling downstream genes of the plasmid stop codon to continue expression, such as the PIII gene required for phage display. A phage display library was constructed by electroporating the pMES4-nanobody recombinant plasmid into TG1 competent cells. To improve the efficiency of library construction, an electroporation efficiency of 1.706 × 10⁻⁶ was achieved. 10CFU / μg TG1 competent cells; to ensure sequence diversity in the library, multiple parallel experimental groups were set up during nested PCR to reduce amplification bias. Simultaneously, the nanobody sequence insertion rate in the library reached 100%, minimizing the influence of empty vectors, and the library size was greater than 10. 7 Count of colonies.

[0135] Example 3: Amplification, enrichment, and screening of a phage library containing type C botulinum toxin receptor-binding domain nanobodies.

[0136] 1. Experimental Materials

[0137] PEG6000 (Shanghai Yisheng Biotechnology Co., Ltd., P8250), bovine serum albumin V (Beijing Solarbio Science & Technology Co., Ltd., A8020), trypsin, AEBSF (Beijing Solarbio Science & Technology Co., Ltd., IA0110); serine inhibitor (Beijing Solarbio Science & Technology Co., Ltd., IA0110); RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., R0010); PMSF protease inhibitor (Beijing Solarbio Science & Technology Co., Ltd., P0100).

[0138] 2. Solution preparation

[0139] 2×YT liquid culture medium: Weigh 8.0g of tryptone, 5.0g of yeast extract and 2.5g of sodium chloride and dissolve them in 480ml of deionized water, then bring the volume up to 500ml. Autoclave at 121℃ for 20min and store at room temperature.

[0140] 2×YT solid medium: Weigh 7.5g of agar powder into 500ml of unsterilized 2×YT liquid medium, autoclave at 121℃ for 20min, and when the medium temperature cools to 50-60℃, add an appropriate amount of antibiotics as needed for the experiment, mix well, and plate.

[0141] 20% (w / v) PEG6,000 / 2.5M NaCl solution: Weigh 200g of PEG6,000 and 146g of NaCl, stir and dissolve in 800ml of deionized water, then bring the volume to 1L, autoclave at 121℃ for 20min, and store at room temperature.

[0142] 3. Experimental Methods 3.1 Phage Amplification

[0143] 100 μl of the total phage library was inoculated into 50 ml of 2×YT medium (containing 2% glucose and 100 μg / ml ampicillin sodium) and incubated at 37°C and 200 rpm until the OD600 value was approximately 0.5. 20 ml of the culture was transferred to a new centrifuge tube, and 50 μl of 4×10⁻⁶ phage sodium solution was added. 10PFU helper phages were incubated at 37°C for 30 min, and after full infection, the precipitate was collected by centrifugation. The precipitate was resuspended in 2×YT culture medium (containing 25 μg / ml kanamycin and 100 μg / ml ampicillin sodium) and incubated overnight at 37°C and 200 rpm.

[0144] 3.2 Phage enrichment

[0145] Centrifuge the overnight cultured bacterial solution at 5000g, 4℃ for 15 min to collect the supernatant. Add 1 / 5 volume of pre-chilled PEG6000, incubate on ice for 30 min, centrifuge at 3200g, 4℃ for 10 min to collect the precipitate, add 1 ml of pre-chilled PBS, resuspend by pipetting, centrifuge at 20000g, 4℃ for 1 min to collect the supernatant, add 250 μl of PEG6000, mix by inversion, incubate on ice for 10 min, centrifuge at 4℃, 20000g for 15 min to collect the precipitate, resuspend by 1 ml of PBS, centrifuge at 4℃, 20000g for 1 min to collect the supernatant, and obtain the phage concentrate.

[0146] 3.3 Phage titer determination

[0147] Fresh colonies of TG1 cells were picked from the plate and inoculated into 30 ml of LB medium. The medium was incubated at 37°C and 200 rpm until the OD600 reached approximately 0.5-0.6, then placed on ice. 10 μl of the phage concentrate was diluted with PBS from 10 μL of LB medium. -1 Gradual dilution to 10 -16 10 μl of each dilution was transferred to a 200 μl EP tube containing 90 μl of TG1 cells, mixed, and incubated in a metal bath at 37°C for 15 min. Then, 5 μl of each infected TG1 cell was dropped into an LB culture plate (containing 100 μg / ml ampicillin sodium and 2% glucose). At the same time, 5 μl of uninfected TG1 cells were set up as a control. The cells were cultured overnight at 37°C.

[0148] 3.4 Phage Screening

[0149] Coat each microplate with 100 μl of 6 μg / ml BCHCR protein, and set up an irrelevant protein BSA as a control well. Incubate overnight at 4°C. Wash three times with PBST, then add 250 μl of 5% BSA to each well for blocking. Shake at 700 rpm for 2 h. Wash five times with PBST, then add 100 μl of incubated phage (50 μl phage concentrate mixed with 10 μl 5% BSA and 400 μl PBS, shake at 700 rpm for 0.5 h) to each well. Shake at 700 rpm for 2 h. Wash 15 times with PBST, then add 100 μl of 2.5 mg / ml trypsin to each well. Shake at 700 rpm for 0.5 h. Transfer the samples from the control and experimental wells to prepared tubes, and add 5 μl of AEBSF (protease inhibitor). Serially dilute 10 μl of the experimental and control samples to a final concentration of 10 μL. -8 The titer was then determined (method as described in 3.3). Simultaneously, 50 μl of each sample from the experimental group was added to 3 ml of OD solution. 600 TG1 cells with a pH of 0.5 were incubated at 37°C for 30 min. The volume was increased to 10 ml with LB medium, and 10 μl of ampicillin sodium (final concentration 100 μg / ml) and 2% glucose were added. The cells were cultured overnight at 37°C and 200 rpm, and then stored at -80°C with 20% glycerol as the first round of enrichment screening.

[0150] The bacterial strains obtained from the first round of screening were repeatedly amplified and enriched. The screening process yielded bacterial strains for the second round of phage screening. This process was repeated to complete the third and fourth rounds of phage screening. After the fourth round of screening, the titers of the experimental group and the control group differed by a factor of 100, indicating that specific phages were effectively enriched and could be screened for phage-ELISA clones.

[0151] 4. Experimental Results

[0152] To improve screening efficiency, this invention utilizes a high-adsorption ELISA plate to solid-phase-coat GST-BoNT / C HCR protein. A four-round "amplification-adsorption-elution" phage panning process is employed, recording the titer of the concentrated phage solution and the final phage titer after each round. A BSA-coated control group is also included to detect non-specifically adsorbed phages. Results show that the difference in phage titers between the experimental and control groups gradually increases with each screening round, indicating that specifically bound phages are effectively enriched during the screening process. Specifically, in the fourth round of screening, the phage titer at enrichment was 3 × 10⁻⁶. 13 pfu / ml, after elution, the phage titer in the control group was 1.4 × 10⁻⁶. 8 The pfu / ml level was lower than that of the experimental group, which had a phage titer as high as 1.4 × 10⁻⁶ pfu / ml. 10pfu / ml. The phage titer in the experimental group was significantly higher than that in the control group, by a factor of 100, indicating that after four rounds of screening, phages bound to the target protein were highly enriched (Table 14).

[0153] Table 14. Phage enrichment levels in four rounds of screening

[0154]

[0155] Example 4: Identification of Phage-ELISA Positive Clones

[0156] 1. Experimental Materials

[0157] High-efficiency RIPA tissue / cell rapid lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., R0010), TMB single-component chromogenic solution (Beijing Solarbio Science & Technology Co., Ltd., PR1200), Anti-His6 Tag Rabbit pAb (Wuhan Saive Biotechnology Co., Ltd., GB111251), HRP×Goat Anti Rabbit IgG (H+L) (Aibosen Biotechnology Co., Ltd., ASG032N).

[0158] 2. Experimental Methods 2.1 Preparation of Nanoparticle Supernatant

[0159] 10 μl of bacterial culture was taken from the fourth round of phage panning library and diluted to 10 μL with LB. -4 and 10 -5 The culture was spread on LB medium containing 100 μg / ml ampicillin sodium and 2% glucose, and incubated overnight at 37°C. Two hundred single colonies were picked from the plate and placed in multiple 96-well plates (100 μl 2×YT, 100 μg / ml ampicillin sodium, 2% glucose, 10% glycerol), and incubated overnight at 37°C. Then, 10 μl of the bacterial culture from each well was added to 1 ml of 2×YT (100 μg / ml ampicillin sodium, 2% glucose) culture medium, and incubated at 37°C, 200 rpm for 6 hours. Afterward, 1 μl of 1M IPTG was added to each tube, and the plates were incubated overnight at 37°C, 200 rpm. The plates were then centrifuged at 3200g for 10 minutes at room temperature, the supernatant was discarded, and 250 μl of RIPA lysis buffer was added to each tube. The plates were vortexed to mix, and the plates were placed on ice and shaken for 1 hour. Centrifuge at 4℃ and 10000rpm for 5 minutes, and collect 200μl of the supernatant, which is the supernatant of the nanoparticles.

[0160] 2.2 Screening of Phage-ELISA-positive clones

[0161] 100 μl of 0.5 μg / ml GST-BCHCR protein was used to coat an ELISA plate, with BSA (an irrelevant protein) as a control well. Coating was performed overnight at 4°C. The plate was washed three times with PBST, and each well was blocked with 250 μl of 5% BSA (dissolved in PBST, w / v). The plate was shaken at 700 rpm for 2 h. After washing five times with PBST, 50 μl of buffer (5% BSA:PBST = 1:20) was added to each well. 50 μl of one type of nanobody was added to each experimental well, and 50 μl of RIPA lysis buffer was added to four control wells. The plate was shaken at 700 rpm for 1 h. After washing five times with PBST, the Anti-6×His6 Tag Rabbit pAb antibody was diluted 2000-fold with buffer (5% BSA:PBST = 1:20), 100 μl per well, and shaken at 700 rpm for 1 h. Wash 5 times with PBST. Dilute the HRP×Goat AntiRabbit IgG (H+L) secondary antibody 10000-fold with buffer (5% BSA:PBST = 1:20), add 100 μl / well, and shake at 700 rpm for 1 h. Wash 10 times with PBST. Add 100 μl / well of TMB chromogenic solution and incubate in the dark for 30 min. Add 50 μl / well of 2M H2SO4 to stop the chromogenic reaction and measure the absorbance at 450 nm.

[0162] 2.3 Sequencing

[0163] The 200 clones were sequentially screened using ELISA to obtain OD. 450 Forty-four monoclonal antibodies with high nucleic acid values ​​were obtained by outsourcing nucleic acid sequencing to Genewiz, yielding 20 nanobody protein sequences.

[0164] 3. Experimental Results

[0165] To rapidly screen high-affinity nanobody monoclonals from the sublime library obtained from the fourth round of phage panning, this invention utilizes the specific binding of antigen and antibody, employing an ELISA method to compare the affinity of 184 selected nanobodies. Experimental results are as follows: Figure 3 As shown, the OD of the negative control group 450 The value was 0.1062, with the negative control group OD 450 A value greater than 2.1 times the effective threshold indicates that the OD values ​​of the vast majority of experimental groups are above the threshold. 450 All values ​​meet the requirements. However, in order to select nanobodies with higher affinity, the OD values ​​need to be carefully chosen. 450 Gene sequencing was performed on 49 monoclonal antibodies with a value greater than 1.

[0166] This invention sequenced 49 nanobodies, identifying 20 nanobodies with unique amino acid sequences, named A2, A3, A10, A11, B1, B2, B5, B7, B8, C6, C12, D1, D12, E2, E5, E11, E12, F9, G5, and G11; the FR regions showed high homology. The specific sequences are as follows:

[0167] A2: LQESGGGLVQAGGSLRLSCAASGSTFRIDVMGWYRQAPGKRRELVAVLGSGGRTDYADSVKGRFSILGDNAKNTVYLQMNSLKPEDTAVYYCNAEEPEHEYWGQGTQVT (SEQ ID NO.46); where CDR1: GSTFRIDVM (SEQ ID NO.1); CDR2: VLGSGGRTDYA (SEQ ID NO.2); CDR3: NAEEPEHEY (SEQ ID NO.3);

[0168] A3: LQESGGGLVQAGGSLRLACAASGSTFGIDVMGWYRQAPGKQRELVAVIGSGGRTDYGDSVKGRFSISGDNAKNTVYLQMNSLKPEDTAVYYCNAEEPEYEYWGQGTQVT (SEQ ID NO.47); where CDR1: GSTFGIDVM (SEQ ID NO.4); CDR2: VIGSGGRTDYG (SEQ ID NO.5); CDR3: NAEEPEYEY (SEQ ID NO.6);

[0169] A10: LQESGGGLVQAGGSLRLSCAASGTTFNIDVMGWYRQAPGKQRDMVAVMNTGGRTDYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCNADGPSYDYWGQGTQVT (SEQ ID NO.48); where CDR1: GTTFNIDVM (SEQ ID NO.7); CDR2: VMNTGGRTDYA (SEQ ID NO.8); CDR3: NADGPSYDY (SEQ ID NO.9);

[0170] A11: LQESGGGLVQAGGSLRLSCAASEDTFSITMGWYRQAPGKQRELVATINIQNNTNYAESLKGRVTINRGNAKNTVYLQISSLKPEDTAVYYCYQRTMVSTYWGQGTQVT (as shown in SEQ ID NO.49); where, CDR1: EDTFSITM (as shown in SEQ ID NO.10); CDR2: TINIQNNTNYA (as shown in SEQ ID NO.11); CDR3: YQRTMVSTY (as shown in SEQ ID NO.12);

[0171] B1: LQESGGGLVQPGGSLGLSCAASGSIYSINAMGWYRQAPGKQRELVATISGPERTSWTNYANSVKGRFTISRDYGRYTVYLQMNSLNPEDTAVYYCYADVVVSDPHGDGDYWGQGTQVT (SEQ ID NO. 50); where CDR1: GSIYSINAM (SEQ ID NO. 13); CDR2: TISGPERTSWTNYA (SEQ ID NO. 14); CDR3: YADVVVSDPHGDGDY (SEQ ID NO. 15);

[0172] B2: LQESGGGLVQPGGSLRLSCAASGRIFSINAMGWYRQAPGNQREFVAGISRGGNIVYADSVQGRSTISIDNAKNTMYLQMNSLKPEDTAVYWCNAGGWTDSGRYVMGNYWGQGTQVT (SEQ ID NO. 51); wherein, CDR1: GRIFSINAM (SEQ ID NO. 16); CDR2: GISRGGNIVYA (SEQ ID NO. 17); CDR3: NAGGWTDSGRYVMGNY (SEQ ID NO. 18);

[0173] B5: LQESGGGLVQAGGSLRLSCAASGSTLIIDVMGWYRQAPGKEREMVAVMNTGGRTDFADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCNADGPTYDYWGQGTQVT (SEQ ID NO.52); where CDR1: GSTLIIDVM (SEQ ID NO.19); CDR2: VMNTGGRTDFA (SEQ ID NO.20); CDR3: NADGPTYDY (SEQ ID NO.21);

[0174] B7: LQESGGGLVQAGGSLRLSCAASGTTFNIDVMGWYRQAPGKQRDMVAVMNTGGRTDYADSVKGRFTISRDNGKNTVYLQMNNLKPEDTAVYYCNADGPSYDYWGQGTQVT (shown in SEQ ID NO.53); among them, CDR1: GTTFNIDVM (shown in SEQ ID NO.7); CDR2: VMNTGGRTDYA (SEQ ID NO.8 shown); CDR3: NADGPSYDY (SEQ ID NO.9 shown);

[0175] B8: LQESGGGLVQAGGSLRLSCAASGSTSSRNPMGWYRQAPGKQRELVATISTQGTITNYADSVKGRFTISRDNTQNTVYLQMSSLKPEDTAVYYCNAINPPSGSWGQGTQVT (SEQ ID NO. 54); where CDR1: GSTSSRNPM (SEQ ID NO. 22); CDR2: TISTQGTITNYA (SEQ ID NO. 23); CDR3: NAINPPSGS (SEQ ID NO. 24);

[0176] C6: LQESGGGLVQAGGSLRLSCAASGDTLSISRMGWYRQAPGKLRELVAQIMMGGSAIYGDSVKGRFTISKDSANNIVYLQMNSLKPEDTAVYYCNARGAWSHQNYWGQGTQVT (shown in SEQ ID NO.55); among them, CDR1: GDTLSISRM (shown in SEQ ID NO.25); CDR2: QIMMGGSAIYG (SEQ ID NO.26); CDR3: NARGAWSHQNY (SEQ ID NO.27);

[0177] C12: LQESGGGLVQAGGSLTLSCAASGFAFSNAPMGWYRQAPGKRRELVATVSSLGGTTNYADSVKGRFTISRDNAKNTVYMQMNNLKPEDTAVYYCNTINGYLVGRNYWGQGTQVT (SEQ ID NO. 56); where CDR1: GFAFSNAPM (SEQ ID NO. 28); CDR2: TVSSLGGTTNYA (SEQ ID NO. 29); CDR3: NTINGYLVGRNY (SEQ ID NO. 30);

[0178] D1: LQESGGGLVQAGGSLRLSCAASGITFSIDVLGWYRQAPGKQRELVAVLNSGGTTDYADSVKGGFTISTDNANNTVYLQMNSLKPEDAAVYYCNAHGPSYDYWGQGTQVT (SEQ ID NO. 57); where, CDR1: GITFSIDVL (SEQ ID NO. 31); CDR2: VLNSGGTTDYA (SEQ ID NO. 32); CDR3: NAHGPSYDY (SEQ ID NO. 33);

[0179] D12: LQESGGGLVQAGGSLRLSCTASGIVDTIDTMGWYRQAPGKQREMVAVRSISGNTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADGPSYDYWGQGTQVT (SEQ ID NO.58); where CDR1: GIVDTIDTM (SEQ ID NO.34); CDR2: VRSISGNTDYA (SEQ ID NO.35); CDR3: NADGPSYDY (SEQ ID NO.9);

[0180] E2: LQESGGGLVQAGGSLRLSCAASGITFDIDVMGWYRQAPGKQREMVAVMNTGGRTDYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCNADGLSYDYWGQGTQVT (SEQ ID NO. 59); where CDR1: GITFDIDVM (SEQ ID NO. 36); CDR2: VMNTGGRTDYA (SEQ ID NO. 8); CDR3: NADGLSYDY (SEQ ID NO. 37);

[0181] E5: LQESGGGLVQAGGSLRLSCTASGSTFSIDVMGWYRQAPGKQRELVAVMNTGGRTDYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNADGPSYDYWGQGTQVT (SEQ ID NO. 60); where CDR1: GSTFSIDVM (SEQ ID NO. 38); CDR2: VMNTGGRTDYA (SEQ ID NO. 8); CDR3: NADGPSYDY (SEQ ID NO. 9);

[0182] E11: LQESGGGLVQAGGSLRLSCAASGTTLGIDVMGWYRQAPGKQRELVAVISSSGNTDFADFVKGRFTIDGDFAKNTVHLQMNSLKPEDTAVYYCNAETPTSEYWGQGTQVT (as shown in SEQ ID NO. 61); where CDR1: GTTLGIDVM (as shown in SEQ ID NO. 39); CDR2: VISSSGNTDFA (as shown in SEQ ID NO. 40); CDR3: NAETPTSEY (as shown in SEQ ID NO. 41);

[0183] E12: LQESGGGLVQAGGSLRLSCAASGTTLGIDVMGWYRQAPGKQRDLVAVISSSGNTDFADSVKGRFTIDGDFAKNTVHLQMNSLKPEDTAVYYCNAETVTSEYWGQGTQVT (SEQ ID NO. 62); where CDR1: GTTLGIDVM (SEQ ID NO. 39); CDR2: VISSSGNTDFA (SEQ ID NO. 40); CDR3: NAETVTSEY (SEQ ID NO. 42);

[0184] F9: LQESGGGLVQPGGSLRLSCAASGSTFRIDVMGWYRQAPGKQREMVAVIGSGGRTDYADSVKGRFSISGDNAKNTVYLQMNSLKPEDTAVYYCNAEEPESEYWGQGTQVT (SEQ ID NO. 63); where CDR1: GSTFRIDVM (SEQ ID NO. 1); CDR2: VIGSGGRTDYA (SEQ ID NO. 43); CDR3: NAEEPESEY (SEQ ID NO. 44);

[0185] G5: LQESGGGLVQAGGSLRLSCAASGSIFRIDVMGWYRQAPGKQREMVAVIGSGGRTDYADSVKGRFSISGDNAKNIVYLQMNSLKPEDTAVYYCNAEEPESEYWGQGTQVT (SEQ ID NO. 64); where CDR1: GSIFRIDVM (SEQ ID NO. 45); CDR2: VIGSGGRTDYA (SEQ ID NO. 43); CDR3: NAEEPESEY (SEQ ID NO. 44);

[0186] G11: LQESGGGLVQAGGSLRLSCAASGTTLGIDVMGWYRQAPGKQRELVAVISSSGNTDFADSVKGRFTIDGDFAKNTVHLQMNSLKPEDTAVYYCNAETPTSEYWGQGTQVT (SEQ ID NO. 65); where CDR1: GTTLGIDVM (SEQ ID NO. 39); CDR2: VISSSGNTDFA (SEQ ID NO. 40); CDR3: NAETPTSEY (SEQ ID NO. 41).

[0187] Example 5: Expression and purification of nanobodies

[0188] 1. Experimental Materials

[0189] Same as implementation list 1.

[0190] 2. Experimental Methods

[0191] 2.1 Induced Expression of Nanobodies

[0192] Twenty nanobody bacterial cultures with distinct protein sequences were obtained after testing. 5 μl of each culture was inoculated into 30 ml of LB medium containing 100 μg / ml ampicillin sodium and incubated at 37°C with shaking at 200 rpm for 14 hours as inoculum. Then, 5 ml of each inoculum was inoculated into 500 ml of LB medium containing 100 μg / ml ampicillin sodium and incubated at 37°C with shaking at 200 rpm for 3 hours until OD (Organic Depth) was reached. 600 Once the protein expression threshold (Pt) reaches 0.4-0.8, 1 ml of bacterial culture is taken as the uninduced sample, and 0.5 mM IPTG is added. Protein expression is induced at 20°C for 16 h, and then another 1 ml of bacterial culture is taken as the induced sample. After complete induction, the bacterial cell pellet is collected by centrifugation at 8000g for 3 min and stored at -20°C. Simultaneously, the uninduced and induced samples are centrifuged to prepare SDS-PAGE samples to detect protein expression.

[0193] 2.2 Purification of Nanobodies

[0194] Add 20 times its weight volume of binding buffer (20 mM PB, pH 7.4, 20 mM NaCl, 100 mM imidazole) to the centrifuged bacterial pellet for resuspending. Homogenize at 850 bar using an autoclave until the bacterial cells are clear. Centrifuge at 1000 rpm for 10 min to obtain the supernatant. Load the supernatant at a flow rate of 1.5 ml / min onto a Ni solution pre-equilibrated with binding buffer. 2+ The Sepharose™ 6 Fast Flow affinity chromatography column was then washed with binding buffer at a flow rate of 1.5 ml / min until the eluent reached OD. 280 Once the baseline was reached, the target protein was eluted with elution buffer (20mM PB, pH 7.4, 20mM NaCl, 500mM imidazole) at a flow rate of 1.5 ml / min. The effluent was collected, and the purified nanobody was analyzed by SDS-PAGE.

[0195] 3. Experimental Results

[0196] This invention employs nickel column chromatography to purify nanobody proteins, and the purification results are as follows: Figure 4 As shown, 12 nanobody protein solutions were purified from 20 nanobody sequences with unique sequences, namely A2, A3, B7, C6, D1, D12, E2, E5, E11, E12, F9, and G5. The remaining 8 could not be effectively induced to express, and the band positions were slightly different due to different sequences, but all were around 15 kDa.

[0197] Example 6: Nanobody inhibits the specific binding of BCHCR to the target cell membrane

[0198] 1. Experimental Materials

[0199] Neuro-2a (CL0243, Fenghui Biotechnology); Sephadex G25 medium (C510060, Sangon Biotech (Shanghai) Co., Ltd.); SF-680 fluorescent dye (1035, Beijing Fubai Biotechnology Co., Ltd.).

[0200] 2. Experimental Methods

[0201] 2.1 SF-680 fluorescent dye labeling of BCHCR protein:

[0202] 1) BCHCR protein treatment: Replace the BCHCR protein solution with 0.1M NaHCO3 using a 10kDa ultrafiltration tube.

[0203] 2) Dye dissolution: The solid dye powder was dissolved in DMSO to a concentration of 1 mg / ml.

[0204] 3) Labeling: Add 15 μl of dye to 0.8 ml of 0.8 mg / mL BCHCR protein solution, shake on a shaker at 150 rpm at 25 °C for 1 h in the dark, and let stand overnight at 4 °C in the dark.

[0205] 4) Desalting and Purification: Prepare a Sephadex G25 medium gravity desalting column. Wash the packing material with 5 column volumes of deionized water, then equilibrate with 5 column volumes of PBS buffer. Add the labeled protein, and once the solution has completely entered the packing material, add PBS for desalting. Collect fluorescent protein samples based on the UV detector reading. 2.2 Nanobody Inhibits BCHCR's Targeted Binding to the Cell Membrane

[0206] 1) Cell resuscitation: Neuro-2a mouse neuroblastoma cells were passaged and plated: The cell cryopreservation tubes were taken out from the liquid nitrogen tank and quickly transferred to a water bath preheated to 37°C to thaw. After thawing, 1 ml of cell culture medium was added and transferred to a 5 ml centrifuge tube. The tubes were centrifuged at 1000g for 3 min and the supernatant was discarded. 1 ml of culture medium was added and the cells were gently dispersed and seeded into culture dishes. The cells were then placed in a CO2 incubator at 37°C for incubation.

[0207] 2) Cell passage: When the cell density reaches 90%, discard the culture medium, add a small amount of PBS along the side of the cell culture dish to wash the cells, discard the PBS, add 1 ml of trypsin, place in an incubator to digest for 1 min, add 1 ml of culture medium to stop digestion, gently pipette the bottom of the flask and the cells to fully suspend them, and transfer them to a centrifuge tube. Centrifuge at 1000g for 3 min, discard the supernatant, add 1 ml of culture medium again and mix well. Take 25% of the cell suspension and transfer it to a culture dish containing 8 ml of culture medium, gently shake back and forth to mix, and place in a 37℃ CO2 incubator to continue culturing.

[0208] 3) Cell slides: Wash cell slides successively with 0.2M HCl and 75% ethanol, and then place them in 12-well cell culture plates. Add approximately 1.5 × 10⁻⁶ cells / well to each well. 5 Prepare a cell suspension and add 1 mL of culture medium, then mix well. Incubate at 37°C in a 5% CO2 incubator.

[0209] 4) Cell cryopreservation: When the Neuro-2a cell density is about 90%, after digestion and centrifugation, 2 mL of cell cryopreservation solution is added to the cells, mixed thoroughly, and then dispensed into two cryopreservation tubes. The tubes are placed in a programmed cooling box and stored overnight at -80°C. Then they are transferred to a liquid nitrogen tank for long-term storage.

[0210] 5) Verification of the cell targeting ability of recombinant protein BoNT / C HCR: BoNT / C HCR protein labeled with Super-Flour 680 fluorescent dye was added to a final concentration of 1 μM in 12-well cell culture plates containing Neuro-2a cell smears and incubated for 2 h. A control group was set up with fluorescently labeled BSA. The culture medium containing BoNT / C HCR was discarded, and 500 μl of 4% tissue cell fixative was added. After standing for 15 min, the fixative was discarded, and the cells were washed with PBS. The cell smears were carefully removed and placed on a glass slide containing anti-fluorescence attenuation mounting medium, and allowed to air dry in the dark. The binding of BoNT / C HCR protein to the Neuro-2a cell membrane was observed using a laser confocal imaging microscope.

[0211] 3. Experimental Results

[0212] Neuro-2a cells were used as the research subject to investigate whether nanobodies could inhibit the binding of BoNT / C HCR to the cell membrane. Nanobodies and fluorescently labeled BoNT / C HCR protein were mixed at a molar ratio of 10:1 and incubated in cell culture medium for 2 hours. The mean fluorescence intensity of cells was observed using laser confocal imaging and statistically analyzed using ImageJ. One-way ANOVA and Dunnett's test were used to compare the differences in BoNT / C HCR values ​​between the treatment group and the control group. *P < 0.05 was considered statistically significant. Experimental results are as follows: Figure 5 As shown, the mean fluorescence intensity of cells in the experimental group co-incubated with nanobody A2 and BoNT / C HCR was 40.654±1.77, significantly lower than that of the control group with only BoNT / C HCR (mean fluorescence intensity 50.36±2.71, ***P<0.001). BSA protein did not affect the binding of BoNT / C HCR to the cell membrane (mean fluorescence intensity 51.85±2.77, nsP>0.05). Therefore, A2 was chosen for subsequent activity verification.

[0213] Example 7 Detection of the effect of nanobody on the uptake of BoNT / C target cells by Western blot

[0214] 1. Experimental materials

[0215] SNAP-25 rabbit polyclonal antibody (GB11678-100, Wuhan Sevier Biotechnology Co., Ltd.); Anti-betaactin mouse mAb (GB12001, Wuhan Sevier Biotechnology Co., Ltd.); Goat anti mouse IgG (H+L) (HRP) (RS0001, Immunoway); Goat anti Rabbit IgG (H+L) (HRP) (RS0002, Immunoway); Syntaxin 1 antibody (HPC-1) (sc-12736, Santa Cruz Biotechnology); HEPES (1112GR025, Guangzhou Saiguo Biotechnology Co., Ltd.); ECL chemiluminescence kit (36208ES60, Shanghai Yisheng Biotechnology Co., Ltd.); The full-length BoNT / C was prepared using Escherichia coli as the receptor bacterium according to the method disclosed in the literature (Zanetti G et al., PLoS Pathog. 2017; 13(8):e1006567. doi:10.1371 / journal.ppat.1006567). SD pregnant mice about 18 days pregnant were purchased from the Experimental Animal Center of Lanzhou University, with the license number SCXK(Gan)2023-0003.

[0216] 2. Solution preparation

[0217] Neuronal cell medium: Add 2 ml of B-27 TM Additive (50×) and 1 ml of L-glutamine to 100 ml of Neurobasal TM Plus medium, mix well, and store at 4°C.

[0218] 10×D-Hanks: Weigh 8.0 g of NaCl, 0.4 g of KCl, 0.126 g of Na2HPO4·12H2O, 0.35 g of NaHCO3 and 0.06 g of KH2PO4, dissolve them in 8 ml of deionized water, adjust the pH to 7.0, and then make up the volume to 10 ml, store at 4°C.

[0219] 1M HEPES: Weigh 11.92 g of HEPES, dissolve it in 40 ml of deionized water, then make up the volume to 50 ml, filter and sterilize with a 0.22 μm filter head, and store at -20°C.

[0220] Dissection solution: Measure 1 ml of 2M glucose, 1 ml of 1M HEPES, 10 ml of 10×D-Hanks, add 88 ml of deionized water, mix well, and store at 4℃.

[0221] 10× Transfer Buffer: Weigh 30.3g Tris and 151.5g glycine, dissolve in 800ml deionized water, and then bring the volume up to 1L. Store at room temperature.

[0222] Western blot membrane transfer buffer: 100 ml of 10× transfer buffer, 200 ml of methanol, bring to a final volume of 1 L, and store at room temperature.

[0223] 10×TBS solution: Weigh 24g of Tris and 88g of sodium chloride, dissolve them in 800ml of deionized water, adjust the pH to 7.6 with hydrochloric acid, and then bring the volume to 1L. Store at room temperature.

[0224] TBST: Measure 100 ml of 10×TBS solution and mix it with 900 ml of deionized water. Add 500 μl of Tween-20 and store at room temperature.

[0225] 3. Experimental Methods

[0226] 3.1 Extraction of hippocampal neurons

[0227] 1) Add 200 μl of 0.1 mg / ml poly-L-lysine to each well of a 24-well cell culture plate and coat the plate overnight at 4°C. Aspirate the poly-L-lysine, wash three times with sterile water, and set aside.

[0228] 2) Use high concentrations of CO2 to euthanize pregnant mice that are about 18 days pregnant, quickly remove the fetus from the pregnant mouse's body, and cut off the head and place it in pre-cooled dissection fluid.

[0229] 3) Add 12 ml of dissection fluid to a 15 ml centrifuge tube and place it on ice. Observe the dissected fetal mouse brain under a microscope in a clean bench, carefully remove the adhered cortical tissue and blood vessels, and take out the hippocampal tissue and place it into a centrifuge tube.

[0230] 4) Carefully discard the dissection fluid, add 1 ml of preheated trypsin digestion solution to a centrifuge tube, and incubate at 37°C for digestion. Shake the centrifuge tube every 10 minutes to ensure complete digestion.

[0231] 5) After digestion for approximately 30-40 minutes, discard the pancreatic enzyme digestion solution. Add 10 ml of the dissection fluid to a centrifuge tube, invert and mix to wash away any remaining pancreatic enzyme digestion solution. Repeat three times until the pancreatic enzyme is completely removed.

[0232] 6) Transfer the cells from the tube to a new 1.5ml centrifuge tube and add 2ml of neuronal cell culture medium, thoroughly resuspending the cells until no obvious cell clumps remain. Add an appropriate amount of cell suspension to a 24-well cell culture plate treated with poly-L-lysine, add 500μl of culture medium, and mix gently. Incubate at 37℃ in a 5% CO2 incubator for 12-14 days to allow the neuronal cells to mature.

[0233] 3.2 Nanobodies and BoNT / C co-incubated with neuronal cells

[0234] Nanobody A2 at concentrations of 5 nM, 45 nM, and 150 nM was mixed with 1 nM BoNT / C and shaken at 700 rpm for 45 min in a 37°C metal bath to ensure complete binding of nanobody A2 and BoNT / C. The mixture was then filtered and added to neuronal cell culture medium, and co-incubated with the cells for 48 h. A buffer control group was also included.

[0235] 3.3 Extraction of total cell protein using RIPA lysis buffer

[0236] Discard the culture medium and add an appropriate amount of PBS buffer to the wells to wash away any residual culture medium on the cell surface. Discard the PBS, then add 100 μl of RIPA lysis buffer (containing 1 μl of PMSF protease inhibitor) to each well. Gently scrape the cells with a pipette tip and lyse on ice for 30 min. Transfer the cell suspension to a 1.5 ml centrifuge tube. Centrifuge at 10,000 rpm for 10 min at 4 °C. Transfer the supernatant to a new 1.5 ml centrifuge tube, add an appropriate amount of 5×SDS-PAGE loading buffer, and denature in a metal bath at 100 °C for 10 min.

[0237] 3.4 Western blot detection method

[0238] 1) SDS-PAGE electrophoresis: Proteins of different molecular weights are separated by SDS-PAGE.

[0239] 2) Transfer: Cut off the gel region containing the target proteins β-actin, Syntaxin, and SNAP-25, and cut the PDVF membrane to size according to the gel block. Activate the membrane in methanol for 30 seconds. Assemble the membrane in the following order: cellulose pad, filter paper, gel, PVDF membrane, filter paper, cellulose pad, ensuring tight adhesion between layers. Place the membrane in the transfer tank with the gel facing the negative electrode and the PVDF membrane facing the positive electrode, and add 1 L of transfer buffer. Transfer at 300 mA for 2 hours in an ice-water bath.

[0240] 3) Sealing: Add 5% skim milk powder (dissolved in TBST, w / v) and soak the PVDF membrane at room temperature for 2 hours.

[0241] 4) Incubation with primary antibodies: Rabbit polyclonal antibodies against β-actin, Syntaxin, and SNAP-25 were diluted 5,000-fold using TBST solution. PVDF membranes were further cut according to the molecular weight differences of β-actin, Syntaxin, and SNAP-25 proteins. The cut PVDF membranes were then placed in the corresponding antibody dilution solutions and incubated at room temperature for 2 hours.

[0242] 5) Membrane washing: Wash the membrane three times with TBST solution at room temperature, 10 minutes each time.

[0243] 6) Incubation of secondary antibodies: Dilute the two antibodies, Goat anti mouse IgG(H+L)(HRP) and Goat anti rabbit IgG(H+L)(HRP), at a ratio of 1:5,000. Place the PVDF membrane containing β-actin and Syntaxin into the Goat anti mouse IgG(H+L)(HRP) antibody dilution solution, and place the PVDF membrane containing SNAP-25 into the Goat anti rabbit IgG(H+L)(HRP) antibody dilution solution. Incubate at room temperature for 1 hour.

[0244] 7) Washing the membrane: Same as step 5).

[0245] 8) Imaging: Add an appropriate amount of ECL chemiluminescent solution to the PVDF membrane for development, and place it in a gel imaging system to take pictures and record the chemiluminescent signal.

[0246] 4. Experimental Results

[0247] This embodiment assesses whether the nanobody significantly blocks the enzymatic cleavage activity of neuronal uptake of full-length BoNT / C against its substrate proteins Syntaxin and SNAP-25 by detecting the enzymatic cleavage of BoNT / C against these substrates. Western blot results are as follows: Figure 6 As shown, serially diluted nanobody A2 was mixed with BoNT / C and incubated with neuronal cells. Nanobody A2 dose-dependently inhibited the cleavage of the substrate proteins Syntaxin and SNAP-25 by BoNT / C. When the molar ratio of nanobody A2 to BoNT / C was 45:1, nanobody A2 completely blocked the uptake of toxins by neurons.

[0248] Example 8: In vivo neutralizing activity and efficacy of nanobody, and evaluation of its antidote effect on BoNT / C poisoning.

[0249] 1. Experimental Materials

[0250] Female Kunming mice (18-22g) were purchased from the Experimental Animal Center of Lanzhou University, license number SCXK(Gan)2023-0003.

[0251] 2. Experimental Methods

[0252] The in vivo bioactivity and efficacy of nanobodies were evaluated by detecting the neutralizing activity of nanobodies against BoNT / C and the delaying effect on BoNT / C-induced paralysis using the intraperitoneal injection lethal method and the toe abduction DAS score method in mice, respectively.

[0253] 2.1 Evaluation of the protective effect of nanobody against BoNT / C toxicity using the intraperitoneal injection lethal method in mice

[0254] Mice were randomly divided into four groups of four. The nanobody was serially diluted to 0.005 mg / kg, 0.05 mg / kg, and 0.5 mg / kg, and mixed with a lethal dose of 125 pg / mouse of BoNT / C. The mixture was incubated in a 37°C metal bath with shaking at 700 rpm for 45 min to ensure complete binding of the nanobody to BoNT / C. 100 μl of the mixture was injected intraperitoneally into each mouse. A BoNT / C control group was also included. Mice survival was monitored every 12 hours.

[0255] 2.2 Evaluation of the efficacy of nanobody in delaying paw paralysis in BoNT / C mice using the DAS scoring method

[0256] Mice were randomly divided into 8 groups, with 4 mice in each group. BoNT / C was diluted to 5 pg / μl, 7.5 pg / μl, 10 pg / μl, and 12.5 pg / μl, and then mixed with the nanobody at a molar ratio of 1:10. 6 When mixed, the corresponding concentrations of the nanobodies are 0.5 μg / μl, 0.75 μg / μl, 1 μg / μl, and...

[0257] 1.25 μg / μl. The mixture was incubated in a 37℃ metal bath with shaking at 700 rpm for 45 min to allow the nanobody to fully bind to BoNT / C. 10 μl of the mixture was injected into the gastrocnemius muscle of the left hind limb of mice, and the degree of gastrocnemius muscle paralysis was observed and recorded every 24 h.

[0258] To further verify whether nanobodies can rescue mice with systemic poisoning and local muscle paralysis, tests were conducted using the intraperitoneal injection method and the DAS (Do Not Disturbance Assay) score for toe abduction.

[0259] 2.3 Evaluation of the rescue effect of nanobodies on BoNT / C poisoning in mice using the intraperitoneal injection lethal method

[0260] Mice were randomly divided into 4 groups of 4 mice each. Each mouse was given a lethal dose of BoNT / C via intraperitoneal injection, followed by a tail vein injection of 5 mg / kg of nanobody at 1 h, 3 h, and 6 h. The survival of the mice was observed at regular intervals.

[0261] 2.4 Evaluation of the rescue effect of nanobodies on BoNT / C poisoning in mice using the DAS scoring method:

[0262] Mice were randomly divided into 8 groups of 8 mice each. Each mouse was injected intramuscularly with 10 μl of 100 pg / mouse BoNT / C into the gastrocnemius muscle of its left hind limb. Then, at 1 h, 3 h, and 6 h, mice were injected intramuscularly with 0.5 mg / kg of nanobody or intravenously via the tail vein with 5 mg / kg of nanobody. The degree of gastrocnemius paralysis was observed at regular intervals.

[0263] 3. Experimental Results

[0264] Nanobodies capable of blocking the binding of BoNT / C HCR to the cell membrane were selected to verify their in vivo neutralization effect against intact toxin activity. 10 pg of nanobodies were co-incubated with 125 pg of recombinant full-length BoNT / C, and then administered to mice via intraperitoneal or gastrocnemius injection. Results showed that nanobodies B7 and E12 failed to neutralize BoNT / C toxicity, but A2 completely neutralized the toxin, resulting in a 100% survival rate in mice. Figure 7 (As shown in Figure A). Furthermore, the neutralizing effect of nanobody A2 on the full-length BoNT / C by both intraperitoneal and gastrocnemius injection was dose-dependent. Figure 7 (As shown in B and C).

[0265] Furthermore, mice were first given different doses of BoNT / C via intraperitoneal or gastrocnemius injection, followed by injection of nanobody A2 at 1, 3, and 6 hours post-injection to treat toxin poisoning. The results showed that both intramuscular and intravenous injection of nanobody A2 had significant antidote effects, with the best antidote effect observed at 1 hour post-injection. Figure 8 (As shown).

[0266] Among the nanobodies obtained through screening, nanobodies A2 showed the most significant neutralizing effect and rescue effect on BoNT / C poisoning, indicating their great potential application in the preparation of antidotes for BoNT / C poisoning. Furthermore, the 20 nanobodies screened in this invention all exhibited significant specific binding to BoNT / C HCR at the molecular level. Figure 3 At the cellular level, A2, A3, B7, C6, D1, E2, E5, E11, E12, and G5 significantly inhibited the binding of BoNT / C HCR to target cells. Figure 5Therefore, the nanobodies provided by this invention can be used as capture antibodies and detection antibodies in the qualitative and quantitative analysis and detection of botulinum toxin type C. They can themselves be used as capture antibodies or detection antibodies in the assembly of BoNT / C content and activity detection kits, as will be understood by those skilled in the art.

[0267] BoNT / A, widely used clinically, often suffers from diminished therapeutic efficacy due to repeated injections. BoNT / C, however, offers similar therapeutic effects and duration of action to BoNT / A, treating dystonia and blepharospasm without exhibiting resistance or adverse reactions, making it a promising alternative to BoNT / A. Therefore, developing a detoxification companion product to combat the risk of iatrogenic toxin poisoning in humans is imperative. On the other hand, BoNT / C is one of the main causes of botulism in animals. Currently, there are no effective treatments for BoNT / C poisoning; multivalent antitoxins are prone to causing allergic reactions and are costly and limited in supply. Therefore, this invention uses phage display technology to screen for nanobodies targeting BoNT / C, aiming to block the binding of BoNT / C to its receptor, preventing it from entering cells and exerting its toxic effects, thus providing a new treatment method for BoNT / C poisoning.

Claims

1. A type C botulinum toxin receptor-binding domain nanobody, characterized in that, The sequence of the complementarity-determining region (CDR) of the nanobody is any one of the following (1)-(18): (1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3; (2) CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6; (3) CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9; (4) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, and CDR3 shown in SEQ ID NO.12; (5) CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, and CDR3 shown in SEQ ID NO.15; (6) CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, and CDR3 shown in SEQ ID NO.18; (7) CDR1 shown in SEQ ID NO.19, CDR2 shown in SEQ ID NO.20, and CDR3 shown in SEQ ID NO.21; (8) CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO.23, and CDR3 shown in SEQ ID NO.24; (9) CDR1 shown in SEQ ID NO.25, CDR2 shown in SEQ ID NO.26, and CDR3 shown in SEQ ID NO.27; (10) CDR1 shown in SEQ ID NO.28, CDR2 shown in SEQ ID NO.29, and CDR3 shown in SEQ ID NO.30; (11) CDR1 shown in SEQ ID NO.31, CDR2 shown in SEQ ID NO.32, and CDR3 shown in SEQ ID NO.33; (12) CDR1 shown in SEQ ID NO.34, CDR2 shown in SEQ ID NO.35, and CDR3 shown in SEQ ID NO.9; (13) CDR1 shown in SEQ ID NO.36, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.37; (14) CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9; (15) CDR1 shown in SEQ ID NO.39, CDR2 shown in SEQ ID NO.40, and CDR3 shown in SEQ ID NO.41; (16) CDR1 shown in SEQ ID NO.39, CDR2 shown in SEQ ID NO.40, and CDR3 shown in SEQ ID NO.42; (17) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.43, and CDR3 shown in SEQ ID NO.44; (18) CDR1 shown in SEQ ID NO.45, CDR2 shown in SEQ ID NO.43, and CDR3 shown in SEQ ID NO.

44.

2. The C-type botulinum toxin receptor-binding domain nanobody as described in claim 1, characterized in that, The sequence of the nanobody is shown in any of SEQ ID NO.46-65.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the type C botulinum toxin receptor-binding domain nanobody as described in claim 1 or 2.

4. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 3.

5. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 3 or the recombinant vector of claim 4.

6. The application of the C-type botulinum toxin receptor-binding domain nanobody as described in claim 1 or 2 in the detection of C-type botulinum toxin for non-disease diagnostic purposes.

7. The application of the C-type botulinum toxin receptor-binding domain nanobody as described in claim 1 or 2 in the preparation of products for detecting C-type botulinum toxin.

8. The use of the C-type botulinum toxin receptor-binding domain nanobody as described in claim 1 or 2 in the preparation of a medicament for treating botulism caused by C-type botulinum toxin.

9. A reagent for qualitative and quantitative detection of botulinum toxin type C content, characterized in that, The reagent includes the type C botulinum toxin receptor-binding domain nanobody as described in claim 1 or 2.

10. A drug for treating botulism caused by botulinum toxin type C, characterized in that, The active ingredient of the drug includes the C-type botulinum toxin receptor-binding domain nanobody as described in claim 1 or 2.

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