A fret-based botulinum neurotoxin type a and type e detection cell sensor and application thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a FRET-based cell sensor for detecting botulinum toxin types A and E and its applications. The invention unexpectedly discovered the use of EGFP / tDimer2 as the donor and acceptor fluorophores, and SNAP-25 as the linker, to fuse and express EGFP-SANP-25-tDimer2. A cell sensor was constructed by transiently transfecting Neuro-2a cells with a plasmid expressing EGFP-SANP-25-tDimer2. This cell sensor can be applied to assess the bioactivity of materials containing BoNT / A and BoNT / E in the environment and during pharmaceutical manufacturing. It can also meet the in vitro screening requirements for botulinum toxin inhibitors and can be used for the diagnosis of BoNT / A and BoNT / E botulinum toxin poisoning.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and provides a FRET-based botulinum toxin type A and type E detection cell sensor and application thereof. The cell sensor can be applied in BoNT / A or BoNT / E botulinum toxin poisoning diagnosis, BoNT / A or BoNT / E inhibitor screening, or BoNT / A or BoNT / E product content and potency determination. BACKGROUND

[0002] Botulinum neurotoxins (BoNTs) are the most toxic natural toxins known to humans, mainly produced by gram-positive Clostridium botulinum in anaerobic environment, and the oral lethal dose is 1 μg / kg, and the parenteral human lethal dose is 0.1-1 ng / kg, which is listed as one of the six A class war agents by the US CDC (Centers for Disease Control and Prevention). BoNTs are divided into seven serotypes (A-G) based on immunological differences, and all BoNTs are composed of a heavy chain (HC) (~100 kDa) and a light chain (LC) (~50 kDa) connected by a disulfide bond. The C-terminal domain of HC binds to specific protein receptors and gangliosides on the surface of neuronal cells, allowing BoNTs to be internalized into acid vesicles through receptor-mediated endocytosis. The acid environment in the acid vesicles causes HC to insert into the vesicle membrane and LC to unfold, and HC helps LC to transfer to the cytoplasm of the cell, and then the disulfide bond is reduced to release LC and refold in the cytoplasm of the cell. The LC of BoNTs is a zinc endopeptidase that targets and cleaves soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are core proteins involved in the transport and release of synaptic vesicles containing neurotransmitters. Each BoNT serotype has a specific SNARE protein substrate and cleavage site, and BoNT / A and E cleave SNAP-25 at different sites, while BoNT / B, D, G, and F cleave vesicle-associated membrane protein 2 (VAMP-2) at different sites. BoNT / C cleaves SNAP-25 and syntaxin. Cleavage of SNARE proteins leads to disruption of the exocytosis mechanism, thus blocking the release of neurotransmitters, resulting in the characteristic flaccid paralysis of botulinum toxin poisoning, and severe poisoning can cause respiratory failure and death.

[0003] Botulinum toxin infection leads to botulism, initially manifesting as blurred vision, oral dehydration, difficulty speaking and swallowing, followed by rapid onset of flaccid paralysis, ultimately resulting in death in severe cases. The most common forms of poisoning are foodborne botulism, infant botulism, and wound botulism. In humans, botulism is primarily caused by toxin serotypes A, B, E, and F. According to a report from the US CDC, there were 673 laboratory-confirmed cases of botulism in the United States between 2012 and 2015, with BoNT / A accounting for the largest proportion (72%), followed by BoNT / E (20%) and BoNT / B (8%).

[0004] BoNT / A and BoNT / B can be used to treat a variety of medical conditions, including blepharospasm, cervical dystonia, chronic migraine, excessive sweating, strabismus, post-stroke upper limb spasticity, urinary incontinence, hemifacial spasm, and depression. BoNTs are also used in cosmetic medicine to treat frown lines and crow's feet, as well as for facial and leg slimming; approximately 50% of BoNT medical products are used in cosmetic medicine. To date, five BoNTs, all type A botulinum toxins, have been approved by the National Medical Products Administration (NMPA) of my country.

[0005] BoNTs are currently known to be the most toxic substances, with BoNT / A exhibiting the strongest toxicity among the seven known serotypes and accounting for the largest proportion of human botulism poisoning. Furthermore, of the seven serotypes of BoNTs, only BoNT / A and BoNT / B are used in medical and cosmetic applications, while BoNT / A has the widest range of applications. Therefore, developing bioactivity detection methods for BoNT / A is of great significance for the diagnosis of botulism, inhibitor screening, and potency determination.

[0006] The mouse bioassay is the "gold standard" for BoNT / A detection, with a sensitivity of up to 20 pg / mL. However, this method is time-consuming, costly, and faces ethical concerns related to animal welfare. Cell-dependent ELISA is the second FDA-approved method for BoNT / A detection. This method involves treating SiMa / hiPSCs with the sample, followed by cell lysis. The classic ELISA method is then used to detect the lysis of the substrate by BoNT / A. The activity of the toxin sample is directly proportional to the degree of substrate lysis. This method mimics the entire process of BoNT / A action and offers high sensitivity and short detection time. However, it faces issues such as SiMa cell ownership disputes and the time-consuming process of inducing differentiation in hiPSC cells.

[0007] FRET-based BoNT / A detection is a promising alternative. Chapman and Dong disclosed in a 2016 patent the construction of a CFP-SNAP-25(1-206)-YFP sensor for BoNT / A cell viability detection using a CFP / YFP fluorescent pair. However, the CFP / YFP pair suffers from drawbacks such as low dynamic range, UV excitation-induced phototoxicity, autofluorescence, and significant spectral crosstalk. Furthermore, the UV exciter required for CFP excitation is expensive, and most laser confocal excitation systems do not utilize it. EGFP / RFP fluorescent pairs, such as EGFP / DsRED, EGFP / mRuby, EGFP / mCherry, and EGFP / tDimer2, offer greater spectral separation and do not require single-fluorescent controls of EGFP and RFP fluorescent proteins, simplifying the FRET quantification process. EGFP excitation light is in the visible light range, thus eliminating the need for expensive UV exciters, significantly reducing detection costs and improving the applicability of the detection method.

[0008] US Patent 20220390461A1 discloses a recombinant substrate using a green fluorescent protein / red fluorescent protein pair coupled with VAMP (27-94) and SNAP-25 (141-206) as linkers for the detection of botulinum toxin in clinical samples and food. Intracellularly, SNAP-25 is anchored to the cytoplasmic side of the cell membrane via palmitoylation at its four Cys positions (85, 88, 90, and 92). After BoNT / A is taken up by the recipient cell, its LC active portion is released and transported to the inner side of the recipient cell membrane, thus efficiently cleaving SNAP-25. However, the VAMP (27-94) and SNAP-25 (141-206) linkers disclosed in US Patent 20220390461A1 lack palmitoylation anchoring sites and can only diffusely exist in the cytoplasm, therefore exhibiting low sensitivity in the detection of botulinum toxin type A.

[0009] This invention provides a cell sensor for in vitro detection of BoNT / A or BoNT / E activity or content, constructed by transiently transfecting Neuro-2a cells with SNAP-25 as a linker, fused with EGFP and tDimer2 expression at both ends. The sensor is capable of detecting the entire process of BoNT / A or BoNT / E poisoning, and is characterized by low cost and short detection time. The FRET-based cell sensor provided by this invention can be used for the determination of BoNT / A or BoNT / E product potency, the diagnosis of BoNT / A or BoNT / E botulinum toxin poisoning, or the screening of BoNT / A or BoNT / E inhibitors. Summary of the Invention

[0010] Based on the aforementioned technical problems, this invention unexpectedly discovered that using EGFP / tDimer2 as the donor and acceptor fluorophores and SNAP-25 as the linker, an expression vector for the EGFP-SNAP-25-tDimer2 fusion protein was constructed. After transiently transfecting this expression vector into Neuro-2a cells, a FRET-based BoNT / A or BoNT / E detection cell sensor was obtained, enabling the detection of BoNT / A or BoNT / E bioactivity or content in live cells. Specifically, this includes the following:

[0011] In a first aspect, the present invention provides a FRET-based BoNT / A or BoNT / E detection cell sensor, wherein the cell sensor is obtained by transfecting cells with a plasmid expressing the sensor molecule; the cell sensor contains an adaptor peptide, a donor fluorophore, and a receptor fluorophore, wherein the adaptor peptide is SNAP-25 protein; the donor fluorophore is EGFP; and the receptor fluorophore is tDimer2.

[0012] Preferably, the cells are Neuro-2a cells.

[0013] Preferably, the amino acid sequence of the EGFP is shown in SEQ ID No. 1, the amino acid sequence of the tDimer2 is shown in SEQ ID No. 3, and the sequence of the adaptor peptide is shown in SEQ ID No. 2.

[0014] Preferably, the cell sensor is composed of EGFP-SNAP-25-tDimer2.

[0015] Preferably, the sequence of the EGFP-SNAP-25-tDimer2 is shown in SEQ ID No. 4.

[0016] Secondly, the present invention provides the application of the cell sensor described in the first aspect above in the preparation of detection reagents for BoNT / A or BoNT / E.

[0017] Thirdly, the present invention provides the application of the cell sensor described in the first aspect above in the preparation of reagents for screening BoNT / A or BoNT / E inhibitors.

[0018] Fourthly, the present invention provides a method for preparing the cell sensor described in the first aspect above, the method comprising the following steps:

[0019] (1) Construct a recombinant plasmid expressing the cell sensor EGFP-SNAP-25-tDimer2;

[0020] (2) Transfect Neuro-2a cells with the recombinant plasmid described in step (1) to obtain cell sensors.

[0021] Fifthly, the present invention provides a FRET-based BoNT / A or BoNT / E detection kit, the kit containing the cell sensor described in the first aspect above.

[0022] Sixthly, the present invention provides a method for using the detection kit described in the fifth aspect above, the method comprising:

[0023] (1) Detection system: The cell sensor described in the first aspect above is placed in the sample to be tested;

[0024] (2) Results analysis: The FRET signal of the cell sensor before and after exposure to the sample was detected and compared. The decrease in FRET signal indicated the presence of BoNT / A or BoNT / E in the sample.

[0025] The beneficial effects of this invention are:

[0026] (1) In this invention, SNAP-25 is used as a linker, and EGFP and tDimer2 fluorescent proteins are fused to its two ends to construct an EGFP-SNAP-25-tDimer2 expression plasmid. This plasmid is then transiently transfected into Neuro-2a cells to obtain a cell sensor for BoNT / A detection based on FRET, which can realize the detection of BoNT / A biological activity.

[0027] (2) Based on the FRET cell sensor, the detection of BoNT / A or BoNT / E cell bioactivity covers the entire process of BoNT / A exerting its activity, including binding, internalization, translocation, and enzyme activity. The system uses laser confocal microscopy for automatic scanning imaging and data processing to meet the requirements of high-throughput detection. It can detect a large number of samples in a short time and can be applied to the detection of bioactivity of recombinant BoNT / A and traditionally extracted BoNT / A during production. It can also meet the requirements of in vitro screening of botulinum toxin inhibitors and diagnosis of botulinum toxin BoNT / A or BoNT / E poisoning.

[0028] (3) The FRET-based cell sensor used in this invention uses SNAP-25 as the linker between FRET pairs. Theoretically, in addition to detecting BoNT / A, it can also be used for detecting the bioactivity of BoNT / E. Furthermore, by replacing the linker, such as VAMP2 or Syntaxin, the bioactivity of other serum BoNT types can be detected.

[0029] (4) Compared with SNAP-25 (141-206aa), SNAP-25 can be used as a linker to anchor on the cell membrane, thus enabling the fabrication of a highly sensitive cell sensor.

[0030] (5) The molecular sensor prepared by the present invention using EGFP / DsRED as the fluorescent pair and SNAP-25 (141-206aa) as the linker has FRET stability, but when it is used to prepare a cell sensor, it cannot be stabilized by the after-ripening process; while the EGFP-SNAP-25-tDimer2 recombinant vector prepared by using EGFP / tDimer2 as the fluorescent pair and SNAP-25 as the linker was transiently transfected into cells to obtain a fluorescently stable cell sensor.

[0031] (6) It is known in the art that PC12 cells are more sensitive to BoNT / A than Neuro-2a. However, the present invention unexpectedly discovered that after transiently transfecting the EGFP-SNAP-25-tDimer2 recombinant plasmid into Neuro-2a, the cell sensor obtained has a wider response range to BoNT / A, is more sensitive to BoNT / A detection, and has more advantages, with unexpected technical effects. Attached Figure Description

[0032] Figure 1 The confocal image of BoNT / A detection in Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2; Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 were obtained by liposome transfection. After co-incubation with BoNT / A, the fluorescence of the EGFP channel was enhanced and the fluorescence of the FRET channel disappeared, indicating that BoNT / A was active.

[0033] Figure 2 The image shows the results of Western blot analysis of the sensitivity of PC12(a) and Neuro-2a(b) cells to BoNT / A. Lane 1 is the negative control, and lanes 2-6 are the results of Western blot analysis after co-incubating PC12(a) and Neuro-2a(b) cells with 3.125, 6.25, 12.5, 25 and 50 nM BoNT / A for 48 hours.

[0034] Figure 3The FRET / EGFP-Log[BoNT / A](nM) curves for detecting BoNT / A activity in Neuro-2a and PC12 cells transiently expressing EGFP-SNAP-25-tDimer2 were plotted. Neuro-2a and PC12 cells transiently expressing EGFP-SNAP-25-tDimer2 were incubated with BoNT / A in 96-well plates at 37°C for 48 hours. Whole-well scanning imaging of the EGFP channel (excitation, 488 nm; emission, 507 nm), FRET channel (excitation, 488 nm; emission, 579 nm), and tDimer2 channel (excitation, 557 nm; emission, 579 nm) was performed using a laser confocal microscope. Simultaneously, automated data processing was performed to obtain fluorescence images and intensities of the EGFP and FRET channels in the whole-well cells. FRET / EGFP was calculated, and the logarithmic value of the BoNT / A concentration was used. GraphPad was then used for the analysis. Based on Prism statistics, a FRET / EGFP-Log[BoNT / A](nM) curve was generated.

[0035] Figure 4 A standard curve for detecting BoNT / A in Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 was generated. Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 and BoNT / A were co-incubated in 96-well plates at 37°C for 48 hours. Whole-well scanning imaging of the EGFP channel (excitation, 488 nm; emission, 507 nm), FRET channel (excitation, 488 nm; emission, 579 nm), and tDimer2 channel (excitation, 557 nm; emission, 579 nm) was performed using a laser confocal microscope. Simultaneously, automated data processing was used to obtain fluorescence images and intensities of the EGFP and FRET channels in the whole-well cells. FRET / EGFP was calculated, and the logarithmic value of the BoNT / A concentration was used. Statistical analysis was performed using GraphPad Prism to generate a FRET / EGFP-Log[BoNT / A](nM) curve, which was then plotted as a standard curve. Detailed Implementation

[0036] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0037] Example 1: Construction of Lenti-EGFP-SNAP-25-tDimer2 plasmid

[0038] 1.1 Biomaterials

[0039] EGFP sequence (OK067273.1, synthesized by Genewiz); SNAP-25 sequence (NM_001322903.2, synthesized by Genewiz); pCDNA3.1-tDimer2 (Ubibio); Top10 competent cells (Sangon Biotech (Shanghai) Co., Ltd.).

[0040] 1.2 Reagents

[0041] Gold PCR Master Mix high-fidelity enzyme premix (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10149ES03); 2×M5 homologous recombinase (Polymerex, catalog number 2011A); SpeedyCut XbaI, SpeedyCut BamHI, and SpeedyCut EcoRI were purchased from Sangon Biotech Co., Ltd.; all other reagents were of analytical grade.

[0042] 1.3 Implementation Steps

[0043] 1.3.1 Primer Design and Synthesis

[0044] Based on the EGFP sequence, SNAP-25 sequence, and purchased vector sequence, appropriate restriction endonucleases were selected, restriction sites were introduced, and suitable upstream and downstream primers were designed and synthesized as shown in Table 1.

[0045] Table 1 Primer sequences for constructing pCDNA3.1-EGFP-SNAP-25-tDimer2 plasmid

[0046]

[0047]

[0048] 1.3.2 Plasmid Construction

[0049] (1) PCR amplification of EGFP and SNAP-25 fragments

[0050] The amplification systems are shown in Tables 2 and 4, and the amplification programs are shown in Tables 3 and 5, respectively.

[0051] Table 2 EGFP fragment PCR amplification system

[0052]

[0053] Table 3. PCR amplification program for EGFP fragments

[0054]

[0055] Table 4 SNAP-25 fragment PCR amplification system

[0056]

[0057] Table 5. SNAP-25 fragment PCR amplification program

[0058]

[0059] (2) Gel recovery of EGFP and SNAP-25 PCR amplification products: Follow the instructions in the gel recovery kit.

[0060] (3) EGFP-SNAP-25 fragment overlap PCR amplification

[0061] The recovered EGFP and SNAP-25 fragments were fused together using overlap PCR to obtain the EGFP-SNAP-25 fragment. The amplification system and amplification program are shown in Tables 6 and 7, respectively.

[0062] Table 6. EGFP-SNAP-25 fragment overlap PCR amplification system

[0063]

[0064] Table 7. EGFP-SNAP-25 fragment overlap PCR amplification program

[0065] Reaction step Reaction temperature (°C) Reaction time (S) Cycle number Pre-denaturation 98 180 1 Denaturation 98 30 10 / 30 Annealing 60 30 10 / 30 Extension 72 60 10 / 30 Final extension 72 600 1

[0066] Without adding primers, perform an overlap PCR amplification program with 10 denaturation, annealing, and extension cycles. After the program is complete, add primers and perform 30 denaturation, annealing, and extension cycles.

[0067] (4) Gel extraction of EGFP-SNAP-25 fragment overlap PCR amplification products. Follow the instructions in the gel extraction kit.

[0068] (5) pCDNA3.1-tDimer2 plasmid was digested with enzymes, and XbaI and EcoRI were selected as the enzyme digestion sites. The enzyme digestion system is shown in Table 8.

[0069] Table 8. pCDNA 3.1-tDimer2 plasmid digestion system

[0070] Reagent Volume (μL) pCDNA3.1-tDimer2 plasmid 4.0 Xba I 1.0 Eco R I 1.0 10 x Single Buffer 2.0 ddH2O 12.0 System 20.0

[0071] Add the reaction mixture to a 200 μL centrifuge tube and mix well. Incubate in a metal bath at 37°C for 1 hour for enzyme digestion, and then perform gel recovery according to the instructions of the gel recovery kit.

[0072] (6) Homologous recombination

[0073] The enzyme-digested plasmid vector pCDNA3.1-tDimer2, recovered from the gel, was homologously recombinated with the overlap PCR product EGFP-SNAP-25 at a molar ratio of 1:3. The system is shown in Table 9.

[0074] Table 9. pCDNA 3.1-EGFP-SNAP-25-tDimer2 homologous recombination reaction system

[0075] Reagent Volume (μL) pcDNA3.1-tDimer2 digested gel recovery product 5.0 Overlap PCR gel recovery product 0.5 2×M5 5.0 System 10.5

[0076] Add the reactants to a 200 μL centrifuge tube and mix well. Incubate in a metal bath at 50 °C for 1 hour.

[0077] (7) Heat shock transformation of Top 10 competent cells

[0078] ① Take one tube of Top 10 competent cells and thaw it on ice;

[0079] ② Add all the homologous recombination reaction products to 50 μL of acceptor cells, mix gently, and place on ice for 30 min;

[0080] ③ Heat shock: Place the centrifuge tubes in a metal bath at 42°C for 90 seconds;

[0081] ④ Quickly transfer the centrifuge tubes to ice and let them sit for 2 minutes;

[0082] ⑤ Resuscitation: Add 200 μL of LB liquid medium to the centrifuge tube and incubate gently with shaking at 37°C for 45 min to revive the bacteria;

[0083] ⑥ Spread the revived bacteria evenly on Amp-resistant LB solid culture plates;

[0084] ⑦ Incubate at 37℃ for 12-16 hours.

[0085] (8) Plasmid mini-extraction

[0086] ① Bacterial culture: After the colonies grow on the transformation plate, pick 3 single colonies and put them into 10 mL of sterilized LB liquid medium, add the corresponding volume of Amp antibiotic, and culture at 37℃, 180 rpm for 12-16 hours with shaking.

[0087] ② Plasmid extraction: Follow the instructions for the plasmid mini-extraction kit.

[0088] (9) PCR amplification of Lenti vector fragments

[0089] Using the Lenti-Cas9 plasmid as a template, the Lenti vector fragment was amplified. The amplification system is shown in Table 10, and the amplification program is shown in Table 11.

[0090] Table 10 Lenti fragment PCR amplification system

[0091]

[0092] Table 11 Lenti fragment PCR amplification program

[0093]

[0094] Perform gel extraction of PCR products according to the gel extraction kit instructions.

[0095] (10) Double digestion of pCDNA3.1-EGFP-SNAP-25-tDimer2 plasmid and Lenti vector fragment.

[0096] The enzyme digestion system is shown in Table 12.

[0097] Table 12 shows the double digestion of the CDNA 3.1-EGFP-SNAP-25-tDimer2 plasmid and Lenti vector fragment.

[0098]

[0099]

[0100] Gel recovery of the double enzyme digestion products was performed according to the gel recovery kit.

[0101] (11) Connection reaction

[0102] The Lenti plasmid vector recovered from the gel was ligated with the EGFP-SNAP-25-tDimer2 fragment at a molar ratio of 1:7, as shown in Table 13 below.

[0103] Table 13 Lenti-EGFP-SNAP-25-tDimer2 ligation reaction system

[0104] Reagent Volume (μL) Lenti vector PCR gel recovery product 2.0 EGFP-SNAP-25-tDimer2 fragment digested gel recovery product 2.0 10 x T4 ligase 1.0 10 x T4 ligase buffer 1.0 ddH2O 4.0 System 10.0

[0105] Add the reactants to a 200 μL centrifuge tube and mix well. Incubate in a metal bath at 16 °C for 12 hours.

[0106] (12) Thermal shock conversion

[0107] The steps are the same as in 1.3.2(7).

[0108] (13) Recombinant plasmid sequencing

[0109] ① Bacterial culture: After the colonies grow on the transformation plate, pick 3 single colonies and put them into 10 mL of sterilized LB liquid medium, add the corresponding volume of Amp antibiotic, and culture at 37℃, 180 rpm for 12-16 hours with shaking.

[0110] ② Plasmid extraction: Follow the instructions for the plasmid mini-extraction kit.

[0111] ③ Sequencing: The plasmid was sent to Tianjin Genewiz Co., Ltd. for sequencing. The sequencing results were compared with the theoretical sequence. If the comparison was consistent, it indicated that the clone was successfully constructed and could be used for subsequent experiments.

[0112] This embodiment constructs the Lenti-EGFP-SNAP-25-tDimer2 vector. The vector can also be any other commercially available cell expression vector, such as pLVX, pCMV, pSV2, pcDNA3.1, etc. The cloning strategy for recombining EGFP, tDimer2, and the linker peptide can employ any restriction endonuclease or a seamless ligation method. In other words, any restriction endonuclease or a seamless ligation method can be used between the sensor molecule and the vector, and between the various elements of EGFP, tDimer2, and the linker peptide, as will be understood by those skilled in the art.

[0113] Example 2: Construction of a Neuro-2a cell line transiently expressing EGFP-SNAP-25-tDimer2

[0114] 2.1 Biomaterials

[0115] Neuro-2a cells were purchased from BioWind.

[0116] 2.2 Reagents

[0117] DMEM high glucose medium (BI, catalog number 06-1055-57-1ACS); MEM (containing NEAA) (Pronos, catalog number PM150410); fetal bovine serum (VivaCell, catalog number C04001-500); liposome nucleic acid transfection reagent (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 40802ES03); MEM-α (BI, catalog number 01-042-1ACS); double antibody (Gibco, catalog number 17504-044).

[0118] 2.3 Implementation Steps

[0119] 2.3.1 Construction of Neuro-2a cell line transiently expressing EGFP-SNAP-25-tDimer2

[0120] (1) Transient expression of EGFP-SNAP-25-tDimer2 in Neuro-2a cells

[0121] ①Neuro-2a cell plating: One day before transfection, Neuro-2a cells were digested with trypsin and counted, at a concentration of 1×10⁻⁶. 5 Cells / well were seeded into 24-well plates and cultured at 37°C in a 5% CO2 cell culture incubator for 12 hours.

[0122] ② Liposome transfection: The ratio of plasmid (μg) to liposome transfection reagent (μL) is 1:1.5. Specifically, dilute 1 μg of Lenti-EGFP-SNAP-25-tDimer2 plasmid with 50 μL of MEM-α medium and mix well. Dilute 1.5 μL of liposome transfection reagent with 50 μL of MEM-α medium, incubate at room temperature for 5 min, then mix the diluted liposomes with the plasmid using a pipette and incubate at room temperature for 20 min to form a DNA-liposome complex.

[0123] ③ Protein Expression: Replace the old medium in the 24-well plate with medium without antibiotics. Add 100 μL of DNA-liposome complex to the 24-well plate and gently shake to mix. Incubate at 37℃ and 5% CO2 for 6 hours, then replace the medium with complete medium and incubate for another 18 hours. Green and red fluorescence and FRET phenomenon were observed using a Leica confocal microscope, indicating that the target protein EGFP-SNAP-25-tDimer2 was expressed in Neuro-2a cells.

[0124] The confocal image shows the BoNT / A conjugation result of Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2. Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 were transfected with liposomes. After co-incubation with BoNT / A, the EGFP channel fluorescence increased while the FRET channel fluorescence disappeared, indicating that BoNT / A exerted its activity. Figure 1 (As shown).

[0125] This invention uses the Neuro-2a cell line as the transiently transfected EGFP-SNAP-25-tDimer2 cell sensor. Chapman and Dong disclosed a rat adrenal pheochromocytoma cell line, PC12, in patent US2016356776(A1) in 2016 for BoNT / A cell viability assays. Similarly, we constructed a PC12 cell line transiently transfected with EGFP-SNAP-25-tDimer2. The results showed that although the sensitivity of PC12 cells to BoNT / A was indeed higher than that of Neuro-2a in Western blot analysis (…),… Figure 2(As shown in a and b). However, unexpectedly, after transient transfection of the EGFP-SNAP-25-tDimer2 cell sensor into cells and subsequent detection of BoNT / A activity, a comparison of the FRET / EGFP-Log[BoNT / A](nM) curves showed that the slope of the fitted curve obtained by Neuro-2a cells was more pronounced than that of PC12. This indicates that Neuro-2a has a wider response range to BoNT / A, is more sensitive to BoNT / A detection, and has a greater advantage. Figure 3 (As shown).

[0126] Example 3: Detection of BoNT / A ratio in Neuro-2a cell lines transiently expressing EGFP-SNAP-25-tDimer2

[0127] 3.1 Biomaterials

[0128] BoNT / A was prepared according to the method of Miyashita et al. (Miyashita SI, Zhang J, Zhang S, et al., Science translational medicine, 2021, 13(575): eaaz4197); Hengli, 100U, batch number 20211164; 96-well cell culture plate (Jet Biotech, catalog number TCP001096).

[0129] 3.2 Reagents

[0130] MEM (containing NEAA) (Pronos, catalog number PM150410); fetal bovine serum (VivaCell, catalog number C04001-500); penicillin antibody (Gibco, catalog number 17504-044).

[0131] 3.3 Implementation Steps

[0132] 3.3.1 BoNT / A Detection

[0133] (1) Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 were seeded at 1000 cells / well in 96-well plates, and BoNT / A was added in series of concentrations and incubated for 48 hours.

[0134] (2) Leica laser confocal microscopy imaging: Nine points evenly distributed in one well were selected, and the focus was automatically adjusted. Then, the whole well of the 96-well plate was automatically scanned and stitched to obtain whole well cell images of the EGFP channel (excitation, 488nm; emission, 507nm) and FRET channel (excitation, 488nm; emission, 579nm).

[0135] (3) The fluorescence intensity of EGFP and FRET channels in whole-well cells is automatically counted using confocal microscopy.

[0136] (4) Data Processing: Calculate FRET / EGFP, logarithmize the BoNT / A concentration, use GraphPad Prism to collect statistical data, create a FRET / EGFP-Log[BoNT / A](nM) curve, and plot a standard curve. Figure 4 Reaction step Reaction temperature (°C) Reaction time (S) Cycle number Pre-denaturation Denaturation Annealing Extension Final extension Reagent Volume (μL) pCDNA3.1-tDimer2 plasmid Xba I Eco R I 10 x Single Buffer System Reagent Volume (μL) pcDNA3.1-tDimer2 digested gel recovery product Overlap PCR gel recovery product System Reagent Volume (μL) Lenti vector PCR gel recovery product EGFP-SNAP-25-tDimer2 fragment digested gel recovery product 10 x T4 ligase 10 x T4 ligase buffer System Figure 1 Figure 2 Figure 3 Figure 4 Reaction step Reaction temperature (°C) Reaction time (S) Cycle number Pre-denaturation Denaturation Annealing Extension Final extension Reagent Volume (μ (As shown).

[0137] The results of this invention show that the detection limit for BoNT / A using the FRET-based cellular sensor EGFP-SNAP-25-tDimer2 for detecting the bioactivity of botulinum toxin type A is 100 pM, and the standard curve is y = -0.1392x + 0.9824. 2 =0.9991, exhibiting linearity between 3.125-50 nM, with recoveries of 86.3%-117.2%, showing a much higher response to BoNT / A than to BoNT / C, and exhibiting specificity to BoNT / A.

[0138] In this embodiment of the invention, a single-blind sample test was also performed on the diluted samples from other experimenters. The results showed that the actual 40U sample had a detection value of 41±4.97U.

[0139] The beneficial effects of this invention are that the FRET-based cell sensor for detecting the bioactivity or content of botulinum toxin type A does not require UV excitation, reducing the phototoxicity of UV to cells. The green fluorescent protein and red fluorescent protein used have minimal spectral overlap, minimizing crosstalk between the donor emission channel and the recipient emission channel, thus significantly simplifying the FRET quantification process. This cell sensor employs whole-well scanning for automated data processing during detection, achieving fully automated, rapid, high-resolution imaging and quantitative analysis of cells, avoiding operational errors and greatly reducing the time and effort required for manual analysis and statistical calculations.

[0140] This invention utilizes a FRET-based cell sensor with SNAP-25 as the linker between FRET pairs. Theoretically, in addition to detecting BoNT / A, it can also be used for the bioactivity detection of BoNT / E. Furthermore, by replacing the linker, such as with VAMP2 or Syntaxin, the bioactivity detection of other serotypes of BoNTs can be achieved, as will be understood by those skilled in the art.

[0141] The FRET-based BoNT / A bioactivity detection cell sensor provided by this invention is EGFP-SNAP-25-tDimer2. Theoretically, FRET will be generated as long as the donor fluorescent protein and the acceptor fluorescent protein are close enough. Therefore, tDimer2-SNAP-25-EGFP can also be used as a BoNT / A bioactivity detection cell sensor, which is understandable to those skilled in the art.

[0142] The FRET-based cell sensor for detecting the bioactivity or content of botulinum toxin type A provided by this invention can be co-transfected with the receptor of BoNT / A in cells to construct a stable cell line, making the detection more sensitive and convenient.

[0143] The receptor cell for the cell sensor provided by this invention is Neuro-2a. Inducing the differentiation of Neuro-2a cell lines using existing publicly available culture methods to express more botulinum toxin receptor molecules can significantly improve the sensitivity of these receptor cells. Furthermore, using a stable transfection method to screen for cell lines stably expressing the sensor molecule EGFP-SNAP-25-tDimer2 simplifies the detection process and shortens the detection cycle, which is understandable to those skilled in the art.

[0144] This invention uses the Neuro-2a cell line as the transiently transfected cell line for the EGFP-SNAP-25-tDimer2 cell sensor. Chapman and Dong disclosed a rat adrenal pheochromocytoma cell line, PC12, in patent US2016356776(A1) in 2016 for BoNT / A cell viability assays. Similarly, this invention constructs a PC12 cell line transiently transfected with EGFP-SNAP-25-tDimer2. Results show that, although the sensitivity of PC12 cells to BoNT / A is indeed higher than that of Neuro-2a in Western blot analysis... However, unexpectedly, when BoNT / A activity was detected after the EGFP-SNAP-25-tDimer2 cell sensor was transiently transfected into the cells, and the FRET / EGFP-Log[BoNT / A](nM) curves were compared, the results showed that the slope of the fitted curve obtained by Neuro-2a cells was more pronounced than that of PC12. This indicates that Neuro-2a has a wider response range to BoNT / A and is more sensitive and advantageous for the detection of BoNT / A.

[0145] The FRET-based detection method for type A botulinum toxin provided by this invention can be applied to evaluate the bioactivity of materials containing BoNT / A in the environment and pharmaceutical manufacturing process. At the same time, it can meet the in vitro screening requirements of botulinum toxin inhibitors and can also be used for the diagnosis of BoNT / A and BoNT / E botulinum toxin poisoning.

Claims

1. A FRET-based BoNT / A cell detection sensor, characterized in that, The cell sensor is composed of EGFP-SNAP-25-tDimer2, and its sequence is shown in SEQ ID No.

4.

2. The use of the cell sensor as described in claim 1 in the preparation of a detection reagent for BoNT / A, or in the preparation of a reagent for screening BoNT / A inhibitors.

3. The method for preparing the cell sensor as described in claim 1, characterized in that, The method includes the following steps: (1) Construct a recombinant plasmid expressing the cell sensor EGFP-SNAP-25-tDimer2; (2) Transfect Neuro-2a cells with the recombinant plasmid described in step (1) to obtain cell sensors.

4. A FRET-based BoNT / A detection kit, characterized in that, The kit contains the cell sensor as described in claim 1.

5. The method of using the detection kit as described in claim 4, characterized in that, The method includes: (1) Detection system: The cell sensor described in claim 1 is placed in the sample to be tested; (2) Results analysis: The FRET signal of the cell sensor before and after exposure to the sample was detected and compared. The decrease in FRET signal indicated the presence of BoNT / A in the sample. The method described is not for diagnostic purposes.

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