Method and product for detecting binding capacity of clostridium neurotoxin and receptor

Abstract

The invention discloses a method and a product for detecting the binding capacity of clostridium neurotoxin and a receptor. The research discovers that the synaptic binding protein Syt1, a Syt1 mutant or Syt2 can be used for promoting glycosylation modification and membrane localization of the synaptic vesicle protein SV2, so that the binding capacity of the synaptic vesicle protein SV2 and botulinum neurotoxin or tetanus toxin is remarkably enhanced. The synaptic binding protein Syt1, Syt1 mutant or Syt2 is prepared into a product for detecting the clostridium neurotoxin, so that the synaptic vesicle protein SV2 can retain natural conformation and post-translational modification sites, and the physiological expression state is effectively simulated, thereby realizing accurate analysis of the binding capacity of the clostridium neurotoxin and the synaptic vesicle protein SV2.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of neurotoxin detection, in particular, to a method and product for detecting the binding ability of Clostridial neurotoxins to receptors. BACKGROUND

[0002] Clostridial neurotoxins (CNTs), including Botulinum neurotoxins (BoNTs, serotypes A-G) and Tetanus neurotoxin (TeNT), are the most toxic proteinaceous neurotoxins known, whose pathogenesis begins with high-affinity binding of the toxin molecule to specific receptors on the neuronal surface (e.g., synaptic vesicle proteins SV2, synaptobrevin Syt, and glycan complex), followed by triggering of internalization and intracellular toxic effects. Accurate quantification of the binding ability between CNTs and receptors is crucial for understanding their pathogenesis, developing neutralizing therapies, and evaluating the activity of new biological agents (e.g., recombinant toxin drugs).

[0003] Studies have shown that the glycosylation modification state and membrane localization conformation of SV2 are key factors determining the binding efficiency of CNTs. However, existing analysis methods have fundamental defects in simulating the microenvironment of natural receptors and lack high-throughput and accurate detection: (1) Deviation of in vitro environment of biophysical techniques: ITC, BLI, and SPR techniques can monitor the molecular binding process in real time through thermal, optical, or electromagnetic signals and obtain binding kinetic parameters, but they rely on the purification of recombinant receptor proteins. SV2, as a partial CNTs protein receptor, when expressed in prokaryotic systems, can only express the extracellular domain, and cannot retain the glycosylation modification and transmembrane conformation of natural membrane proteins. Studies have confirmed that the binding affinity (K D) compared to native membrane proteins. Moreover, these techniques perform experiments in vitro solution or solid interface, which cannot reproduce the complex membrane lipid environment and receptor clustering state in cells, leading to significant deviations in binding parameters from physiological conditions. At the same time, the high cost of equipment purchase and maintenance limits the wide application of the technology. (2) Physiological relevance defects of in vitro screening technology: BACTH assay and Phage Display technology can be used to quickly screen a large number of mutants or libraries in vitro, which are suitable for locating key binding sites. However, both technologies cannot completely simulate the post-translational modifications (such as glycosylation, phosphorylation) of host cells and the membrane microenvironment, which may lead to deviations in binding capacity evaluation; non-specific protein interaction of BACTH and abnormal folding of phage surface proteins may lead to high false positive and false negative rates; in addition, the molecular weight of CNTs receptor binding domain is usually about 50 kDa, and the molecular weight of SV2 is greater than 80 kDa, which is close to the upper limit of the capacity of the carrier to carry and display proteins for BACTH and Phage Display technology, and the fault tolerance space is extremely limited. Due to the restriction of technical parameters and expression efficiency, only the local binding region of ligand-receptor can be studied in the experimental design, and it is difficult to achieve systematic analysis of the binding characteristics of the full-length receptor. (3) Bottlenecks of biochemistry detection technology in throughput and accuracy: ELISA, Co-IP and radioactive ligand binding experiments also face the problem of insufficient simulation of the microenvironment of natural receptors, and most of them are targeted at the binding region of the receptor, which cannot realize the analysis of the binding capacity of the full-length receptor. Although the radioactive ligand binding experiment has the advantage of quantification, it is complex to operate, has radioactive hazards, and is difficult to realize high-throughput detection; immunoprecipitation (Co-IP) relies on the specificity of antibodies, and the operation steps are relatively cumbersome and the quantitative repeatability is poor, which is not suitable for large-scale sample screening. (4) Application limitations of existing cell models: Although the primary neuron model can highly simulate the physiological environment, it is difficult to obtain, has a long cultivation period, high batch variability, and strict ethical review, which makes it difficult to establish a standardized detection process. Due to the differences in species and origin, the expression profiles of receptors in PC12 and Neuro2A cell lines are different from those in humans; although SH-SY5Y cells are human-derived, they have low differentiation efficiency, insufficient SV2 expression, and tumor cell characteristics that interfere with the accuracy of CNTs-SV2 receptor binding analysis. In addition, most existing cell-based methods focus on the end-point detection of toxin internalization or enzyme activity (substrate cleavage analysis), and lack a direct and efficient quantitative platform for the initial binding step.

[0004] Therefore, it is urgent to develop an engineered cell line-based, standardized cell-based method that can simulate physiological receptor expression and has high sensitivity and throughput to accurately analyze CNTs-receptor interactions. SUMMARY

[0005] To overcome the above-mentioned defects and deficiencies in the prior art, the present application provides a method and product for detecting the binding ability of Clostridium neurotoxin to receptors.

[0006] The first object of the present application is to provide the use of a glycosylation modification facilitator of synaptic vesicle protein SV2 in the preparation of a product for detecting Clostridium neurotoxin.

[0007] The second object of the present application is to provide the use of a recombinant cell in the preparation of a product for detecting Clostridium neurotoxin.

[0008] The third object of the present application is to provide the use of an overexpression system in the preparation of a product for detecting Clostridium neurotoxin.

[0009] The fourth object of the present application is to provide a product for detecting Clostridium neurotoxin.

[0010] The present application claims the following: The use of a glycosylation modification facilitator of synaptic vesicle protein SV2 in the preparation of a product for detecting Clostridium neurotoxin, the glycosylation modification facilitator comprising any one of the following proteins: (1) Synaptotagmin Syt1; (2) Synaptotagmin Syt1 mutant: the Clostridium neurotoxin is botulinum neurotoxin, the synaptotagmin Syt1 mutant comprises amino acid residues 46-421 in the sequence shown in NP_001028852.2, and there is at least one mutation in the sequence consisting of amino acid residues 1-45; the Clostridium neurotoxin is tetanus toxin, the synaptotagmin Syt1 mutant comprises amino acid residues 34-421 in the sequence shown in NP_001028852.2, and there is at least one mutation in the sequence consisting of amino acid residues 1-33; (3) Synaptotagmin Syt2.

[0011] Preferably, the mutation is substitution and / or deletion.

[0012] The use of a recombinant cell in the preparation of a product for detecting Clostridium neurotoxin, the recombinant cell being recombined with synaptic vesicle protein SV2, and further recombined with any one of the following proteins: (1) Synaptotagmin Syt1; (2) Synaptotagmin Syt1 mutant: the Clostridial neurotoxin is botulinum neurotoxin, the Synaptotagmin Syt1 mutant comprises amino acid residues 46-421 in the sequence shown in NP_001028852.2, and there is at least one mutation in the sequence consisting of amino acid residues 1-45; the Clostridial neurotoxin is tetanus toxin, the Synaptotagmin Syt1 mutant comprises amino acid residues 34-421 in the sequence shown in NP_001028852.2, and there is at least one mutation in the sequence consisting of amino acid residues 1-33; (3) Synaptotagmin Syt2.

[0013] Preferably, the mutation is substitution and / or deletion.

[0014] Preferably, the recombinant cell further comprises green fluorescent protein.

[0015] Use of a overexpression system in the preparation of a product for detecting Clostridial neurotoxin, the overexpression system overexpresses Synaptic vesicle protein SV2, and the overexpression system further overexpresses any one of the following proteins: (1) Synaptotagmin Syt1; (2) Synaptotagmin Syt1 mutant: the Clostridial neurotoxin is botulinum neurotoxin, the Synaptotagmin Syt1 mutant comprises amino acid residues 46-421 in the sequence shown in NP_001028852.2, and there is at least one mutation in the sequence consisting of amino acid residues 1-45; the Clostridial neurotoxin is tetanus toxin, the Synaptotagmin Syt1 mutant comprises amino acid residues 34-421 in the sequence shown in NP_001028852.2, and there is at least one mutation in the sequence consisting of amino acid residues 1-33; (3) Synaptotagmin Syt2.

[0016] Preferably, the mutation is substitution and / or deletion.

[0017] Preferably, the botulinum neurotoxin is botulinum neurotoxin type A.

[0018] Preferably, the Synaptic vesicle protein SV2 comprises SV2A protein and / or SV2B protein.

[0019] Preferably, the Clostridial neurotoxin is a botulinum neurotoxin, and the synaptotagmin Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN45 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant, or a Syt1-NQT mutant; The Clostridial neurotoxin is a tetanus toxin, and the synaptotagmin Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant, or a Syt1-NQT mutant; The Syt1ΔN28 mutant is a mutant in which the 1st-28th amino acid residues in the sequence shown in NP_001028852.2 are deleted; The Syt1ΔN33 mutant is a mutant in which the 1st-33rd amino acid residues in the sequence shown in NP_001028852.2 are deleted; The Syt1ΔN45 mutant is a mutant in which the 1st-45th amino acid residues in the sequence shown in NP_001028852.2 are deleted; The Syt1-NQ mutant is a mutant in which the asparagine at the 24th position in the sequence shown in NP_001028852.2 is mutated to glutamine; The Syt1-NQT mutant is a mutant in which the threonine at the 15th position is mutated to alanine, the threonine at the 16th position is mutated to alanine, and the asparagine at the 24th position in the sequence shown in NP_001028852.2 is mutated to glutamine.

[0020] The synergistic mechanism of Syt1 or Syt2 with SV2 includes: (1) glycosylation regulation: Syt1 or Syt2 interacts with the intracellular loop region of SV2 through the intracellular domain (such as the C2 domain), recruits endoplasmic reticulum molecular chaperones (Calnexin / Calreticulin), accelerates the conversion of the N-glycan of SV2 from high mannose type to complex type, and improves the conformational maturity; (2) membrane localization driving: the membrane anchoring property of Syt1 or Syt2 drives SV2 to be transported to the cell membrane through the Rab11-mediated recycling endosome pathway, reduces endoplasmic reticulum retention, and makes the membrane localization proportion of SV2 increase by 2.66 times.

[0021] The Syt1ΔN28, Syt1ΔN33, or Syt1ΔN45 mutant, after deleting the N-terminal redundant sequence, improves the binding affinity of Syt1 to SV2, while retaining the Ca 2+ dependent vesicle transport function, further enhancing the membrane localization efficiency of SV2; The Syt1-NQ or Syt1-NQT mutant eliminates the N-glycosylation (N24) and O-glycosylation (T15 / 16) sites of Syt1 itself, removes the spatial hindrance of the sugar chain to protein interaction, promotes the membrane expression of SV2, and significantly enhances the binding activity of SV2 to Clostridium botulinum neurotoxin.

[0022] Preferably, the product is used for detecting the binding of Clostridium botulinum neurotoxin to synaptic vesicle protein SV2. The synaptic binding protein Syt1, the synaptic binding protein Syt1 mutant or the synaptic binding protein Syt2 enhances the binding efficiency of SV2 to Clostridium botulinum neurotoxin by promoting the glycosylation modification (especially the formation of complex N-glycan) and membrane localization of synaptic vesicle protein SV2.

[0023] A product for detecting Clostridium botulinum neurotoxin, the product comprising a system overexpressing synaptic vesicle protein SV2, and the system further overexpressing any one of the following proteins: (1) a synaptic binding protein Syt1; (2) a synaptic binding protein Syt1 mutant: the Clostridium botulinum neurotoxin is botulinum neurotoxin, the synaptic binding protein Syt1 mutant comprises amino acid residues 46-421 in the sequence shown in NP_001028852.2, and at least one mutation exists in the sequence consisting of amino acid residues 1-45; the Clostridium botulinum neurotoxin is tetanus toxin, and the synaptic binding protein Syt1 mutant comprises amino acid residues 34-421 in the sequence shown in NP_001028852.2, and at least one mutation exists in the sequence consisting of amino acid residues 1-33; (3) a synaptic binding protein Syt2.

[0024] Preferably, the mutation is substitution and / or deletion.

[0025] Preferably, the Clostridium botulinum neurotoxin is botulinum neurotoxin, and the synaptic binding protein Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN45 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant or a Syt1-NQT mutant; the Clostridium botulinum neurotoxin is tetanus toxin, and the synaptic binding protein Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant or a Syt1-NQT mutant; the Syt1ΔN28 mutant is a mutant in which amino acid residues 1-28 in the sequence shown in NP_001028852.2 are deleted; The Syt1AN33 mutant is a mutant in which amino acid residues 1-33 in the sequence shown in NP_001028852.2 are deleted; The Syt1AN45 mutant is a mutant in which amino acid residues 1-45 in the sequence shown in NP_001028852.2 are deleted; The Syt1-NQ mutant is a mutant in which the asparagine at position 24 in the sequence shown in NP_001028852.2 is mutated into glutamine; The Syt1-NQT mutant is a mutant in which the threonine at position 15 in the sequence shown in NP_001028852.2 is mutated into alanine, the threonine at position 16 is mutated into alanine, and the asparagine at position 24 is mutated into glutamine.

[0026] Preferably, the system is a recombinant cell.

[0027] More preferably, the recombinant cell further comprises a green fluorescent protein.

[0028] Further preferably, the recombinant cell is a recombinant HEK293T cell. The HEK293T cell has a highly efficient protein post-translational modification system (such as glycosyltransferase, acyltransferase), and the Golgi glycosylation enzyme spectrum of the cell is similar to that of human primary neurons, with a similarity of 76%, which can correctly modify the transmembrane domain of SV2; at the same time, the proliferation characteristics of the cell line are suitable for high-throughput requirements: the doubling time is 18 h, the positive rate of liposome transfection is >70%, and a single experiment can process samples for detection in the scale of a 96-well plate.

[0029] A method for detecting Clostridium neurotoxin, using any of the above products for detection. The method is for the purpose of non-disease treatment diagnosis. The method is a high-throughput detection method based on green fluorescent protein GFP internal reference and fluorescence quantitative analysis, and the specific evaluation of the binding ability of Clostridium neurotoxin and synaptic vesicle protein SV2 is realized through non-permeable immunofluorescence. Among them, through the co-transfection of GFP to label positive cells, the analysis region (ROI) can be limited, and the background interference of non-transfected cells is excluded; through non-permeable staining, only the toxins bound to the cell membrane surface can be detected, avoiding non-specific binding signals in the cells, so that the signal-to-noise ratio of detection is improved.

[0030] Compared with the prior art, the present application has the following beneficial effects: The application discloses a method and product for detecting the binding ability of clostridial neurotoxin to receptors.

[0031] In addition, the product of the application can also be extended to other application fields: ① analyzing the binding differences between clostridial neurotoxin and different species SV2, identifying key residues affecting binding and screening high-affinity mutations; ② evaluating the binding ability of clostridial neurotoxin variants to human SV2, guiding targeted enhancement design; ③ performing competitive binding analysis of clostridial neurotoxin and vaccine candidates, and screening high-efficiency antagonists. The application can significantly improve the efficiency of neurotoxin engineering, promote the research and development of a new generation of high-efficiency, low-toxicity and small-side-effect therapeutic neurotoxin and its delivery tool, and is widely applied in the fields of treatment of neurological diseases and regulation of neural functions. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure 1 is an immunofluorescence staining result graph in Example 1. Scale bar: 20 μm.

[0033] Figure 2 Figure 2 is an immunofluorescence staining result graph in Example 2; A: only expressing SV2B (top) and co-expressing SV2B + Syt1 (bottom), immunofluorescence analysis of the distribution pattern change of SV2B (red), primary antibody: rabbit anti-SV2B antibody (Synaptic Systems, item number 119 111); secondary antibody: Alexa Fluor TM 546 labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific, item number A-11035); B: only expressing Syt1 (top) and co-expressing Syt1 + SV2B (bottom), immunofluorescence analysis of the distribution pattern change of Syt1 (red), primary antibody: mouse anti-Syt1 antibody (Synaptic Systems, item number 105 011); secondary antibody: Alexa Fluor TM 546 labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific, item number A-11035); B: only expressing Syt1 (top) and co-expressing Syt1 + SV2B (bottom), immunofluorescence analysis of the distribution pattern change of Syt1 (red), primary antibody: mouse anti-Syt1 antibody (Synaptic Systems, item number 105 011); secondary antibody: Alexa Fluor

[0034] Figure 3 Figure for immunofluorescence staining result of Example 3; A: immunofluorescence co-localization analysis of Syt1 expressed alone with endoplasmic reticulum protein marker Calnexin; red; primary antibody: mouse anti-Syt1 antibody, Synaptic Systems, Cat. No. 105011; secondary antibody: Alexa Fluor TM 546-labeled goat anti-mouse IgG antibody, Thermo Fisher Scientific, Cat. No. A-11030; green; primary antibody: rabbit anti-Calnexin antibody, Cell Signaling, Cat. No. 2679S; secondary antibody: Alexa Fluor TM 488-labeled goat anti-rabbit IgG antibody, Thermo Fisher Scientific, Cat. No. A-11008; B: immunofluorescence co-localization analysis of SV2B expressed alone with endoplasmic reticulum protein marker Calnexin; red; primary antibody: rabbit anti-SV2B antibody, Synaptic Systems, Cat. No. 119 111; secondary antibody: Alexa Fluor TM 546-labeled goat anti-rabbit IgG antibody, Thermo Fisher Scientific, Cat. No. A-11035; green; primary antibody: mouse anti-Calnexin antibody, Abeam, Cat. No. ab13505; secondary antibody: Alexa Fluor™ 488-labeled goat anti-mouse IgG antibody, Thermo Fisher Scientific, Cat. No. A-11001; C: immunofluorescence co-localization analysis of SV2B and Syt1 co-expressed; green; primary antibody: rabbit anti-SV2B antibody, Synaptic Systems, Cat. No. 119 111; secondary antibody: Alexa Fluor TM 488-labeled goat anti-rabbit IgG antibody, Thermo Fisher Scientific, Cat. No. A-11008; red; primary antibody: mouse anti-Syt1 antibody, Synaptic Systems, Cat. No. 105011; secondary antibody: Alexa Fluor™ 546-labeled goat anti-mouse IgG antibody, Thermo Fisher Scientific, Cat. No. A-11030; D: co-expression of SV2B and Syt1, co-localization analysis of endoplasmic reticulum marker Calnexin and SV2B by immunofluorescence; green; primary antibody: mouse anti-Calnexin antibody, Abeam, Cat# ab13505; secondary antibody: Alexa Fluor™ 488 goat anti-mouse IgG antibody, Thermo Fisher Scientific, Cat# A-11001; red; primary antibody: rabbit anti-SV2B antibody, Synaptic Systems, Cat# 119 111; secondary antibody: Alexa Fluor™ 546 goat anti-rabbit IgG antibody, Thermo Fisher Scientific, Cat# A-11035. Scale bar: 20 μm.

[0035] Figure 4 Figure for immunofluorescence staining result of Example 4; A: WB analysis of the effect of co-transfection of Syt1 or Syt2 on the total protein expression (left panel) and membrane expression (right panel) of SV2B in HEK293T cells, with Actin as the internal reference for protein loading amount; B: immunofluorescence analysis of the effect of co-transfection of Syt1 or Syt2 on the expression and localization of GFP-SV2A in CHO cells (left panel); the relative expression amount of total protein of SV2A was characterized by the green fluorescence intensity of GFP, and the membrane localization level of SV2A was characterized by the red fluorescence intensity of non-permeable immunofluorescence analysis with GFP antibody, and the efficiency of co-expression of Syt1 or Syt2 to enhance the membrane expression level of GFP-SV2A was quantitatively analyzed (right panel). Scale bar: 20 μm.

[0036] Figure 5 Figure for immunofluorescence staining result of Example 5; A: the effect of different Syt1 deletion mutants on the binding ability of SV2B to different Clostridium neurotoxins; B: the left panel is the effect of co-transfection of different Syt1 mutants on the total protein expression of SV2B; the right panel is the effect of co-transfection of different Syt1 mutants on the membrane localization expression amount of SV2B.

[0037] Figure 6 Figure for immunofluorescence staining result of Example 6; A: the effect of different Syt1 N-terminal glycosylation deletion mutants on the toxin binding ability of SV2B; B: quantitative analysis of the toxin binding ability in A. DETAILED DESCRIPTION

[0038] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] pEGFP-N1 plasmid: encoding green fluorescent protein GFP; pcDNA3.1-SV2B plasmid: containing human full-length SV2B gene, GenBank accession number NM_014848.7; pcDNA3.1-Syt1: containing rat full-length Syt1 gene, GenBank accession number NM_001033680, the NCBI accession number of the expressed protein Syt1 is NP_001028852.2; pcDNA3.1-Syt2: containing rat full-length Syt2 gene, GenBank accession number NM_012665.3, the NCBI accession number of the expressed protein Syt2 is NP_036797.2; pcDNA3.1-SV2A plasmid: containing human full-length SV2A gene, GenBank accession number NM_014849.5.

[0041] Example 1 GFP co-transfection labeling expression of SV2B cells I. Experimental methods HEK293T cells were seeded at a density of 2.5 x 10 5 cells / well in a 24-well plate in a sterile glass slide, and DMEM high glucose medium containing 10% fetal bovine serum (v / v), 1% double-antibiotic (penicillin-streptomycin, v / v) was added, and incubated at 37°C, 5% CO2 incubator for 24 h, when the cell density reached 60%-80%, transfection was performed. Using Lipofectamine 3000 transfection reagent, 0.25 μg pEGFP-N1 plasmid was mixed with 0.75 μg pcDNA3.1-SV2B plasmid before transfecting the cells, and 3 biological replicates were set for each group.

[0042] After 48 h of transfection, the culture medium was discarded, and the cells were washed once with pre-cooled PBS, and 4% paraformaldehyde solution (w / v) was added for room temperature fixation for 20 min. After permeation, SV2B immunofluorescence staining was performed, primary antibody: rabbit anti-SV2B antibody (Synaptic Systems, item number 119 111); secondary antibody: Alexa Fluor 546 labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific, item number A-11035). Image acquisition was performed using a Leica TCS SP8 laser confocal microscope with a 40x oil objective. TM

[0043] II. Experimental results Figure 1 ​The results showed that 48 hours after transfection, the cell density was close to 100%, and the transfection efficiency reached 50-70% (the transfection efficiency is the percentage of cells transfected with GFP to the total number of cells, Figure 1 GFP fluorescence signal is shown in the figure); cells expressing GFP all co-express SV2B, and the fluorescence signal of SV2B is only present in cells expressing GFP ( Figure 1 (shown as the fluorescence overlay results in the middle), indicating that GFP can be used as an internal control to mark cells expressing a specific target protein (SV2B).

[0044] The above results indicate that the co-transfection strategy of pEGFP-N1 and pcDNA3.1-SV2B in HEK293T cells can efficiently label SV2B-positive cells through GFP fluorescence signal, and GFP expression does not affect the membrane localization and protein stability of SV2B.

[0045] Example 2 Effect of co-expression of SV2B and Syt1 on the cellular distribution pattern of SV2B 1. Experimental Methods HEK293T cells were cultured at 2.5 × 10 5 Each well was inoculated with sterile glass slides in a 24-well plate and divided into three groups: Group 1: transfection with 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid (expressing SV2B alone); Group 2: transfection with 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1 plasmid + 0.375 μg pcDNA3.1-Syt1 plasmid (expressing Syt1 alone); Group 3: transfection with 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1 plasmid (co-expressing SV2B and Syt1); Lipofectamine 3000 was used for transfection, with three biological replicates per group. The cells were incubated at 37°C in a 5% CO2 incubator for 48 hours. Cells were permeabilized 48 hours after transfection, and immunofluorescence analysis of SV2B and Syt1 expression was performed. Images were acquired using a Leica TCS SP8 laser confocal microscope with a 40x oil objective.

[0046] 2. Experimental Results Immunofluorescence analysis results showed that compared with the expression of SV2B alone, co-expression of Syt1 significantly reduced the expression of SV2B and showed a trend of increased membrane localization ( Figure 2A); when Syt1 was expressed alone, Syt1 showed typical cell membrane distribution characteristics, and after co-expression of SV2B, Syt1 showed intracellular distribution characteristics (B). Figure 2

[0047] The above results show that SV2B and Syt1 have a synergistic localization effect in HEK293T cells: Syt1 promotes the membrane transport of SV2B, thereby increasing the proportion of cell membrane distribution of SV2B by more than 75%, and SV2B guides Syt1 to redistribute from the cell membrane to intracellular vesicles.

[0048] Example 3: Co-localization analysis of SV2B and Syt1 co-expression in CHO cells I. Experimental methods CHO cells were seeded at 2 x 10 5 cells / well in 24-well plates (with sterile coverslips) and divided into three groups: Group 1: transfected with 0.5 μg pcDNA3.1 plasmid + 0.5 μg pcDNA3.1-Syt1 plasmid (Syt1 expressed alone); Group 2: transfected with 0.5 μg pcDNA3.1 plasmid + 0.5 μg pcDNA3.1-SV2B plasmid (SV2B expressed alone); Group 3: co-transfected with 0.5 μg pcDNA3.1-SV2B plasmid + 0.5 μg pcDNA3.1-Syt1 plasmid (SV2B and Syt1 co-expressed); Lipofectamine 3000 transfection reagent was used, with 3 biological replicates per group, and incubated at 37°C, 5% CO2 incubator for 48 h. After 48 h of transfection, cell permeation was performed, and immunofluorescence was used to analyze the expression and endoplasmic reticulum protein marker Calnexin co-localization distribution changes of SV2B and Syt1. Image acquisition was performed using a Leica TCS SP8 laser confocal microscope with a 40x oil objective.

[0049] II. Experimental results The results show that in CHO cells, Syt1 expressed alone, Syt1 showed cell membrane expression characteristics, and had no significant co-localization with endoplasmic reticulum protein marker Calnexin (A); SV2B expressed alone showed intracellular distribution pattern, and partially co-localized with endoplasmic reticulum protein marker Calnexin (B); co-expression of SV2B and Syt1 showed intracellular distribution pattern, and had high consistency with SV2B localization (C), wherein the co-localization proportion of SV2B and endoplasmic reticulum protein marker Calnexin decreased (D). Figure 3 Figure 3 Figure 3 The above results show that SV2B and Syt1 have a synergistic localization effect in HEK293T cells: Syt1 promotes the membrane transport of SV2B, thereby increasing the proportion of cell membrane distribution of SV2B by more than 75%, and SV2B guides Syt1 to redistribute from the cell membrane to intracellular vesicles.

[0048] Example 3: Co-localization analysis of SV2B and Syt1 co-expression in CHO cells I. Experimental methods CHO cells were seeded at 2 x 10 5 cells / well in 24-well plates (with sterile coverslips) and divided into three groups: Group 1: transfected with 0.5 μg pcDNA3.1 plasmid + 0.5 μg pcDNA3.1-Syt1 plasmid (Syt1 expressed alone); Group 2: transfected with 0.5 μg pcDNA3.1 plasmid + 0.5 μg pcDNA3.1-SV2B plasmid (SV2B expressed alone); Group 3: co-transfected with 0.5 μg pcDNA3.1-SV2B plasmid + 0.5 μg pcDNA3.1-Syt1 plasmid (SV2B and Syt1 co-expressed); Lipofectamine 3000 transfection reagent was used, with 3 biological replicates per group, and incubated at 37°C, 5% CO2 incubator for 48 h. After 48 h of transfection, cell permeation was performed, and immunofluorescence was used to analyze the expression and endoplasmic reticulum protein marker Calnexin co-localization distribution changes of SV2B and Syt1. Image acquisition was performed using a Leica TCS SP8 laser confocal microscope with a 40x oil objective.

[0049] II. Experimental results The results show that in CHO cells, Syt1 expressed alone, Syt1 showed cell membrane expression characteristics, and had no significant co-localization with endoplasmic reticulum protein marker Calnexin (A); SV2B expressed alone showed intracellular distribution pattern, and partially co-localized with endoplasmic reticulum protein marker Calnexin (B); co-expression of SV2B and Syt1 showed intracellular distribution pattern, and had high consistency with SV2B localization (C), wherein the co-localization proportion of SV2B and endoplasmic reticulum protein marker Calnexin decreased (D). Figure 3 Figure 3 Figure 3 The above results show that SV2B and Syt1 have a synergistic localization effect in HEK293T cells: Syt1 promotes the membrane transport of SV2B, thereby increasing the proportion of cell membrane distribution of SV2B by more than 75%, and SV2B guides Syt1 to redistribute from the cell membrane to intracellular vesicles.

[0048] Example 3: Co-localization analysis of SV2B and Syt1 co-expression in CHO cells I. Experimental methods CHO cells were seeded at 2 x 10Figure 3 The results above show that SV2B and Syt1 have significant interaction in CHO cells, and the co-expression changes the distribution pattern of each other in the cells and both show highly consistent co-localization.

[0050] As a transmembrane glycoprotein, about 40-50% of SV2B molecules remain in the endoplasmic reticulum due to incomplete post-translational modification and show a dynamic distribution of “endoplasmic reticulum retention-cell membrane transport”. In the co-expression system, Syt1 interacts directly with SV2B through the intracellular domain (such as the C2 domain of Syt1 and the intracellular loop region of SV2B), and the inherent membrane tropism of Syt1 (containing a membrane anchor motif) dominates the transport of the complex along the Rab GTPase-regulated vesicle pathway (such as the Rab11-mediated recycling endosome), promotes the conformational maturation of SV2B, and increases the proportion of SV2B membrane localization. This interaction accurately reproduces the natural dynamic process of “receptor post-translational modification-subcellular compartment sorting-functional membrane localization” at the presynaptic terminal of neurons.

[0051] Example 4 Syt1 or Syt2 enhances cell membrane localization of SV2 by regulating SV2 glycosylation modification I. Experimental Methods Group transfection experiments were performed in HEK293T cells: Control group: transfection of pcDNA3.1-SV2B plasmid and pcDNA3.1 empty vector; Syt1 experimental group: transfection of pcDNA3.1-SV2B plasmid and pcDNA3.1-Syt1 plasmid; Syt2 experimental group: transfection of pcDNA3.1-SV2B plasmid and pcDNA3.1-Syt2 plasmid.

[0052] The experiment was divided into two detection parts: first, after collecting and lysing the cells, the total protein expression level of SV2B was analyzed by Western blot; Second, the membrane protein biotinylation technique was used: the cell membrane proteins were labeled with EZ-Link Sulfo-NHS-LC-Biotin, and the biotinylated membrane protein components were captured with NeutrAvidin agarose beads after cell lysis, and the membrane localization expression level of SV2B was detected by Western blot.

[0053] To analyze the change of SV2 membrane localization level after co-expression of Syt1 and SV2 protein, pcDNA3.1-GFP-SV2A recombinant plasmid was constructed, and GFP gene was inserted into the N-terminal of L4 region of SV2A. This design not only ensures that the GFP fluorescence signal is linearly related to the total protein expression level of SV2A, but also allows direct analysis of the SV2A protein level on the cell membrane by non-permeable immunofluorescence staining of GFP.

[0054] CHO cells were seeded at 2 x 10 5 The experiment was divided into three groups: Group 1: transfected 0.5 μg pcDNA3.1 plasmid + 0.5 μg pcDNA3.1-GFP-SV2A plasmid; Group 2: transfected 0.5 μg pcDNA3.1-GFP-SV2A plasmid + 0.5 μg pcDNA3.1-Syt1 plasmid; Group 3: co-transfected 0.5 μg pcDNA3.1-GFP-SV2A plasmid + 0.5 μg pcDNA3.1-Syt2 plasmid; Lipofectamine 3000 transfection reagent was used, and 3 biological replicates were set for each group. Incubation was performed at 37°C in a 5% CO2 incubator for 48 h. After 48 h of transfection, chicken anti-GFP antibody was used to label SV2A protein on the cell membrane, and Alexa 546-labeled secondary antibody was used for color development. Images were collected by Leica TCS SP8 laser confocal microscope 40x oil lens, and the relative expression of SV2A total protein was represented by the intensity of GFP green fluorescence. The red fluorescence intensity of non-permeable immunofluorescence analysis of GFP antibody represented the membrane localization level of SV2A, and then the relative membrane localization ratio of SV2A was calculated.

[0055] II. Experimental results Compared with the control group, the N-glycosylation modification of SV2B was significantly enhanced in the experimental group co-transfected with Syt1 or Syt2 (represented by the increase in the intensity of the high molecular weight glycosylation band in the WB pattern, as indicated by the A arrow in Figure 4 ImageJ quantitative analysis results showed that the glycosylation level in the region between the two arrows increased by more than 3 times. Verification by PNGase F enzyme treatment showed that the band was caused by N-glycan modification.

[0056] In CHO cells, co-expression of Syt1 or Syt2 can significantly improve the membrane localization efficiency of SV2A, which is 2.66 times higher than that of the GFP-SV2A molecule alone (B in Figure 4 Combined with the results of A in Figure 4 , it is suggested that the enhancement of SV2 glycosylation modification is positively correlated with the membrane localization efficiency of SV2.

[0057] Syt1 or Syt2 interacts with the intracellular domain of SV2B, promotes its Calnexin / Calreticulin quality control cycle in the endoplasmic reticulum, and accelerates the conversion of N-glycan from high mannose type to complex type; the membrane anchoring property of Syt1 or Syt2 drives SV2B to transport to the cell membrane through the Rab11-mediated recycling endosome pathway, reduces its retention in the intermediate zone between the endoplasmic reticulum and the Golgi apparatus, and thus increases the proportion of SV2B molecules localized on the membrane.

[0058] As a receptor protein molecule of TeNT, BoNT / A, BoNT / D and BoNT / E, to detect its binding ability with these CNTs, the membrane expression level and glycosylation level of SV2B in the functional model determine the sensitivity and accuracy of the detection. Based on the high-efficiency recombinant expression characteristics of HEK293T cells, combined with Syt1 co-expression strategy, the two purposes of increasing the membrane expression amount of SV2B and improving the protein maturity can be achieved, which breaks through the technical bottleneck of primary neuron culture, and provides a standardized platform with sensitivity and convenient operation for CNTs neutralizing antibody screening, toxin subtype and toxin mutant affinity identification, etc.

[0059] Example 5 Effect of Syt1 N-terminal deletion mutant on SV2B glycosylation modification and on SV2B binding to toxin protein I. Experimental method Syt1 N-terminal deletion mutants: Syt1ΔN28 (deletion of N-terminal 1-28 amino acids) and Syt1ΔN45 (deletion of N-terminal 1-45 amino acids) were constructed by reverse PCR, and after sequencing verification, they were cloned into pcDNA3.1 vector to obtain pcDNA3.1-Syt1ΔN28 plasmid and pcDNA3.1-Syt1ΔN45 plasmid.

[0060] The coding regions encoding BoNT / A HcR (872-1296 amino acids) and TeNT HcR (865-1315 amino acids) were amplified by PCR and cloned into the prokaryotic expression vector pET28a, with the N-terminal fusion of HA tag (YPYDVPDYA) and 6His tag. The recombinant proteins were expressed and purified in Escherichia coli BL21 (DE3) at low temperature. SDS-PAGE detection showed that the purity of the recombinant proteins was greater than 95%, and the protein was quantified by BCA method, thereby obtaining HA-BoNT / A-HcR recombinant protein and HA-TeNT-HcR recombinant protein.

[0061] HEK293T cells were seeded at 2.5×10 5 Each well of a 24-well plate was seeded with 2.5×10 Group 1: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid (expressing SV2B alone); Group 2: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1 plasmid; Group 3: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1ΔN28 plasmid; Group 4: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1ΔN45 plasmid; Lipofectamine 3000 transfection reagent was used, and 3 biological replicates were set for each group. Incubation was carried out at 37°C in a 5% CO2 incubator for 48 h. After 48 h of transfection, the transfected cells were washed with PBS, and 100 nM HA-BoNT / A-HcR recombinant protein or HA-TeNT-HcR recombinant protein was added, and incubation was carried out at 37°C for 30 min; cold PBS was washed 3 times, and fixation was carried out with 4% paraformaldehyde solution (w / v), non-permeable immunofluorescence analysis was carried out for the HA tag, and color development was carried out with Alexa 546-labeled secondary antibody. According to the red fluorescence intensity, the effects of Syt1, Syt1ΔN28 and Syt1ΔN45 on the binding level of HA-BoNT / A-HcR recombinant protein or HA-TeNT-HcR recombinant protein to SV2B were compared. Images were collected using a Leica TCS SP8 laser confocal microscope with a 40x oil objective.

[0062] Grouping transfection experiments were carried out in HEK293T cells: Control group: transfection of pcDNA3.1-SV2B plasmid and pcDNA3.1 empty vector; Syt1 experimental group: transfection of pcDNA3.1-SV2B and pcDNA3.1-Syt1; Syt1ΔN28 experimental group: transfection of pcDNA3.1-SV2B and pcDNA3.1-Syt1ΔN28; Syt1ΔN45 experimental group: transfection of pcDNA3.1-SV2B and pcDNA3.1-Syt1ΔN45.

[0063] The experiment was divided into two detection parts: one, after collecting and lysing the cells, the total protein expression level of SV2B was analyzed by Western blot; the other, the membrane protein biotinylation technology was used: the cell membrane protein was labeled with EZ-Link Sulfo-NHS-LC-Biotin, and after cell lysis, the biotinylated membrane protein components were captured by NeutrAvidin agarose beads, and then the membrane localization expression amount of SV2B was detected by Western blot.

[0064] II. Experimental results The experimental results show that, compared with the expression of SV2B alone, the co-expression of Syt1, Syt1ΔN28 and Syt1ΔN45 with SV2B can significantly improve the binding ability of SV2B with HA-BoNT / A-HcR recombinant protein; For HA-TeNT-HcR recombinant protein, the binding of HA-TeNT-HcR recombinant protein cannot be detected by expressing SV2B alone; the co-expression of Syt1 can make the binding of SV2B with HA-TeNT-HcR recombinant protein be detected by microscope, the co-expression of Syt1ΔN28 can significantly improve the binding of SV2B with HA-TeNT-HcR recombinant protein, and the co-expression of Syt1ΔN45 cannot detect the binding of HA-TeNT-HcR recombinant protein (A in the figure). Figure 5

[0065] The above results show that the sensitivity of SV2B-mediated binding with toxin protein is significantly insufficient when expressed alone; the co-expression of Syt1 can significantly improve the SV2B-mediated binding with toxin protein, and the N-terminal 28 amino acid mutant of Syt1 can further improve the SV2B-mediated binding with toxin protein, which expands the applicable range of detection of the binding ability with different toxin proteins.

[0066] In HEK293T, the co-expression of different Syt1 mutants can improve the glycosylation modification of SV2B, and Syt1ΔN28 is more significant in the high glycosylation modification of SV2B (B arrow in the figure). Figure 5

[0067] Therefore, the N-terminal 28 amino acid mutant of Syt1 can enhance the glycosylation modification of SV2B, thereby improving the binding ability of SV2B with toxin protein, and is more suitable for efficient analysis of the binding ability of Clostridium neurotoxin with receptors.

[0068] Example 6 Effect of N-terminal glycosylation mutant of Syt1 on SV2B binding with toxin protein I. Experimental method ​​Syt1ΔN33 (deletion of N-terminal 33 amino acids), Syt1-NQ (N24Q mutation to eliminate N-glycosylation modification at this site), and Syt1-NQT (introduction of T15A, T16A mutations on the basis of N24Q mutation to eliminate O-glycosylation modification at this site) were constructed by reverse PCR and point mutation methods and cloned into pcDNA3.1 expression vectors. All the mutants were verified by sequencing and confirmed by immunoblotting to obtain pcDNA3.1-Syt1ΔN33 plasmid, pcDNA3.1-Syt1-NQ plasmid, and pcDNA3.1-Syt1-NQT plasmid.

[0069] HEK293T cells were seeded at 2.5 x 10 5 into sterile glass slides in 24-well plates, and the experiment was divided into 5 groups: Group 1: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid; Group 2: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1 plasmid; Group 3: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1ΔN33 plasmid; Group 4: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1-NQ plasmid; Group 5: transfection of 0.25 μg pEGFP-N1 plasmid + 0.375 μg pcDNA3.1-SV2B plasmid + 0.375 μg pcDNA3.1-Syt1-NQT plasmid.

[0070] Lipofectamine 3000 transfection reagent was used, with three biological replicates per group, and cells were incubated at 37°C, 5% CO2 for 48 hours. Forty-eight hours after transfection, the transfected cells were washed with PBS and then added with 100 nM of the HA-BoNT / A-HcR recombinant protein or HA-TeNT-HcR recombinant protein prepared in Example 5. The cells were incubated at 37°C for 30 minutes. The cells were washed three times with cold PBS and fixed with 4% PFA. Non-permeabilized immunofluorescence analysis of the HA tag was performed using an Alexa Fluor 546-labeled secondary antibody for visualization. The effects of Syt1, Syt1ΔN33, Syt1-NQ, and Syt1-NQT on the binding of HA-BoNT / A-HcR or HA-TeNT-HcR recombinant proteins to SV2B were compared based on red fluorescence intensity. Images were acquired using a Leica TCS SP8 laser confocal microscope with a 40x oil immersion lens.

[0071] ImageJ was used for fluorescence quantitative analysis. Five fields of view were selected and regions of interest (ROIs) were delineated based on the GFP fluorescence signal. The GFP fluorescence signal area and the integrated density of the red fluorescence signal within the ROI were calculated. The integrated density of the red fluorescence was divided by the corresponding GFP fluorescence signal area to obtain the average fluorescence brightness value of the HA tag, which represents the binding ability of the recombinant protein. 2. Experimental Results Figure 6 Figure A shows that compared with the wild-type Syt1, the Syt1 N-terminal glycosylation deletion mutants (Syt1-NQ and Syt1-NQT) can significantly enhance the binding ability of SV2B to HA-BoNT / A-HcR recombinant protein and HA-TeNT-HcR recombinant protein; Syt1ΔN33, similar to Syt1ΔN28, can significantly enhance the binding ability of SV2B to TeNT-HcR recombinant protein.

[0072] The results of fluorescence quantitative analysis showed that ( Figure 6 Figure (B): For the group expressing SV2B alone, after co-expression of wild-type Syt1, the fluorescence intensities of bound HA-BoNT / A-HcR and HA-TeNT-HcR were 3.52-fold and 3.03-fold, respectively, compared with the group expressing SV2B alone. Compared with wild-type Syt1, the Syt1ΔN33, Syt1-NQ, and Syt1-NQT mutants increased the fluorescence intensity of HA-BoNT / A-HcR histone bound to SV2B by 1.19-fold, 1.68-fold, and 1.99-fold, respectively, and increased the fluorescence intensity of HA-TeNT-HcR recombinant protein bound to SV2B by 6.80-fold, 6.30-fold, and 5.23-fold, respectively (p<0.01).

[0073] The above results show that the Syt1 N-terminal glycosylation deletion mutant does not affect the ability of Syt1 to localize on vesicles and membranes, and the mutant is stronger than wild-type Syt1 in staying on the cell membrane surface, and the space hindrance of protein interaction by removing the glycosylation modification of Syt1 is thus significantly enhanced, so that the toxin binding capacity of SV2B is significantly enhanced: the Syt1-NQT mutant and the SV2B co-expression system can significantly enhance the detection capacity of HA-BoNT / A-HcR and HA-TeNT-HcR, and is suitable for high-sensitivity screening of Clostridium sordellii neurotoxin, and provides a standardized model for antitoxin drug research and development and toxin subtype identification.

[0074] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. Application of a glycosylation modification promoter of synaptic vesicle protein SV2 in the preparation of a product for detecting Clostridium difficile neurotoxins, characterized in that: The glycosylation modification promoter comprises any one of the following proteins: (1) Synaptotagmin Syt1; (2) Synaptotagmin Syt1 mutant: The Clostridium neurotoxin is a botulinum neurotoxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 46 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 45; The Clostridium neurotoxin is tetanus toxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 34 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 33; (3) Synaptotagmin Syt2.

2. Use of a recombinant cell in the preparation of a product for detecting Clostridium neurotoxin, characterized in that: The recombinant cell is recombined with synaptic vesicle protein SV2 and any one of the following proteins: (1) Synaptotagmin Syt1; (2) Synaptotagmin Syt1 mutant: The Clostridium neurotoxin is a botulinum neurotoxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 46 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 45; The Clostridium neurotoxin is tetanus toxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 34 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 33; (3) Synaptotagmin Syt2.

3. Use of an overexpression system in the preparation of a product for detecting Clostridium neurotoxins, characterized in that: The overexpression system overexpresses synaptic vesicle protein SV2, and the overexpression system also overexpresses any one of the following proteins: (1) Synaptotagmin Syt1; (2) Synaptotagmin Syt1 mutant: The Clostridium neurotoxin is a botulinum neurotoxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 46 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 45; The Clostridium neurotoxin is tetanus toxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 34 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 33; (3) Synaptotagmin Syt2.

4. The use according to any one of claims 1 to 3, characterized in that: The botulinum neurotoxin is botulinum neurotoxin type A.

5. The use according to any one of claims 1 to 3, characterized in that: The synaptic vesicle protein SV2 comprises SV2A protein and / or SV2B protein.

6. The use according to any one of claims 1 to 3, characterized in that: The Clostridium neurotoxin is a botulinum neurotoxin, and the synaptotagmin Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN45 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant, or a Syt1-NQT mutant; The Clostridium neurotoxin is tetanus toxin, and the synaptotagmin Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant, or a Syt1-NQT mutant; The Syt1ΔN28 mutant is a mutant lacking amino acid residues 1 to 28 in the sequence shown in NP_001028852.2; The Syt1ΔN33 mutant is a mutant lacking amino acid residues 1 to 33 in the sequence shown in NP_001028852.2; The Syt1ΔN45 mutant is a mutant lacking amino acid residues 1 to 45 in the sequence shown in NP_001028852.2; The Syt1-NQ mutant is a mutant in which the asparagine at position 24 in the sequence shown in NP_001028852.2 is mutated to glutamine; The Syt1-NQT mutant is a mutant in which the threonine at position 15 is mutated to alanine, the threonine at position 16 is mutated to alanine, and the asparagine at position 24 is mutated to glutamine in the sequence shown in NP_001028852.

2.

7. The use according to any one of claims 1 to 3, characterized in that: The product is used to detect the binding of Clostridium difficile neurotoxin to synaptic vesicle protein SV2.

8. A product for detecting Clostridium neurotoxin, characterized in that: The product comprises a system that overexpresses synaptic vesicle protein SV2, and the system also overexpresses any of the following proteins: (1) Synaptotagmin Syt1; (2) Synaptotagmin Syt1 mutant: The Clostridium neurotoxin is a botulinum neurotoxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 46 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 45; The Clostridium neurotoxin is tetanus toxin, and the synaptotagmin Syt1 mutant comprises amino acid residues 34 to 421 of the sequence shown in NP_001028852.2, and has at least one mutation in the sequence consisting of amino acid residues 1 to 33; (3) Synaptotagmin Syt2.

9. The product according to claim 8, characterized in that The Clostridium neurotoxin is a botulinum neurotoxin, and the synaptotagmin Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN45 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant, or a Syt1-NQT mutant; The Clostridium neurotoxin is tetanus toxin, and the synaptotagmin Syt1 mutant comprises a Syt1ΔN28 mutant, a Syt1ΔN33 mutant, a Syt1-NQ mutant, or a Syt1-NQT mutant; The Syt1ΔN28 mutant is a mutant lacking amino acid residues 1 to 28 in the sequence shown in NP_001028852.2; The Syt1ΔN33 mutant is a mutant lacking amino acid residues 1 to 33 in the sequence shown in NP_001028852.2; The Syt1ΔN45 mutant is a mutant lacking amino acid residues 1 to 45 in the sequence shown in NP_001028852.2; The Syt1-NQ mutant is a mutant in which the asparagine at position 24 in the sequence shown in NP_001028852.2 is mutated to glutamine; The Syt1-NQT mutant is a mutant in which the threonine at position 15 is mutated to alanine, the threonine at position 16 is mutated to alanine, and the asparagine at position 24 is mutated to glutamine in the sequence shown in NP_001028852.

2.

10. The product according to claim 8, characterized in that The system is a recombinant cell.

Images