FRET (Fluorescence Resonance Energy Transfer)-based A-type and E-type botulinum toxin detection cell sensor and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an FRET (Fluorescence Resonance Energy Transfer)-based A-type and E-type botulinum toxin detection cell sensor and application thereof. The invention accidentally finds that EGFP / tDimer2 is used as a donor fluorophore and a receptor fluorophore, SNAP-25 is used as a connexon, and fusion expression of EGFP-SANP-25-tDimer2 is carried out; the method comprises the following steps: carrying out transient transfection on a Neuro-2a cell by using a plasmid for expressing EGFP-SANP-25-tDimer2, so as to construct a cell sensor; the cell sensor can be used for evaluating the biological activity of materials containing BoNT / A and BoNT / E in the environment and in the pharmaceutical production process. The in-vitro screening requirement of the botulinum toxin inhibitor can also be met, and the kit can also be used for diagnosing BoNT / A and BoNT / E botulinum toxin poisoning.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and provides a FRET-based cell sensor for detecting type A and type E botulinum toxins and applications thereof. The cell sensor can be used in the diagnosis of BoNT / A or BoNT / E botulinum toxin poisoning, or in the screening of BoNT / A or BoNT / E inhibitors, or in the determination of the content and potency of BoNT / A or BoNT / E products. Background Art

[0002] Botulinum neurotoxins (BoNTs) are the most toxic natural toxins known to humans. They are mainly produced by Gram-positive Clostridium botulinum in an anaerobic environment. The oral lethal dose for humans is 1 μg / kg, while the parenteral lethal dose for humans is 0.1-1 ng / kg. They are listed as one of the six Class A warfare agents by the Centers for Disease Control and Prevention (CDC) of the United States. BoNTs are divided into seven serotypes (AG) based on immunological differences. All BoNTs are composed of a heavy chain (HC) (~100 kDa) and a light chain (LC) (~50 kDa) connected by disulfide bonds. 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 acidic vesicles through receptor-mediated endocytosis. The acidic environment in the acidic vesicles causes HC to insert into the vesicle membrane and LC to unfold. HC assists LC in transferring to the cytoplasm of the cell, and then the disulfide bond reduction releases LC and refolds it 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. BoNTs serotypes each have specific SNARE protein substrates and cleavage sites. BoNT / A and E cleave SNAP-25 at different sites, while BoNT / B, D, G and F cleave synaptobrevin 2 (VAMP-2) at different sites, and BoNT / C cleaves SNAP-25 and syntaxin. The cleavage of SNARE proteins leads to the destruction of the exocytosis machinery, thereby blocking the release of neurotransmitters, resulting in the characteristic flaccid paralysis of botulinum toxin poisoning, and severe poisoning will lead to death due to respiratory failure.

[0003] Botulinum toxin infection causes botulism, which first manifests as blurred vision, oral dehydration, difficulty speaking and swallowing, followed by rapid onset of flaccid paralysis, and ultimately death in severe cases. The most common forms of poisoning are foodborne botulism, infant botulism, and wound botulism. In humans, botulism is mainly caused by toxin serotypes A, B, E, and F. According to the US CDC, there were 673 laboratory-confirmed cases of botulism in the United States from 2012 to 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, upper limb spasticity after stroke, urinary incontinence and hemifacial spasm, and depression; BoNTs are also used in medical cosmetology to treat frown lines and crow's feet, as well as to slim the face and legs. About 50% of BoNTs medical products are used for cosmetic medicine. So far, five BoNTs have been approved by my country's National Medical Products Administration (NMPA), all of which are botulinum toxin type A.

[0005] BoNTs are the most toxic substances known so far, among which BoNT / A is the most toxic among the 7 known serotypes and accounts for the largest proportion of human botulism. In addition, among the 7 serotypes of BoNTs, only BoNT / A and BoNT / B are used in medical and cosmetic applications, and BoNT / A is the most widely used. Therefore, the development of a bioactivity detection method for BoNT / A is of great significance for botulism diagnosis, inhibitor screening, and potency determination.

[0006] Mouse bioassay is the "gold standard" for BoNT / A detection, with a detection sensitivity of up to 20pg / mL. However, this method is time-consuming, costly, and faces ethical issues regarding animal protection. Cell-dependent ELISA is the second BoNT / A detection method approved by the FDA. This method uses the sample to be tested to treat SiMa / hiPSC, then lyses the cells, and uses the classic ELISA method to detect the cleavage of the substrate by BoNT / A. The activity of the tested toxin sample is proportional to the degree of cleavage of its substrate. This method simulates the entire process of BoNT / A exerting its effects, and has high sensitivity and short detection time. However, there are issues such as the ownership of SiMa cells and the time-consuming problem of inducing differentiation of hiPSC cells.

[0007] FRET-based BoNT / A detection is a very promising alternative method. Chapman and Dong disclosed in a patent in 2016 the use of CFP and YFP fluorescence pairs to construct a CFP-SNAP-25(1-206)-YFP sensor for BoNT / A cell activity detection. However, the disadvantages of the CFP / YFP pair are low dynamic range, phototoxicity induced by ultraviolet excitation, autofluorescence, and a large amount of spectral crosstalk, and the ultraviolet exciter required to excite CFP is expensive and most laser confocals are not equipped. EGFP / RFP fluorescence pairs, such as EGFP / DsRED, EGFP / mRuby, EGFP / mCherry, and EGFP / tDimer2, have greater spectral separation, and do not require single fluorescence controls of EGFP and RFP fluorescent proteins during detection, simplifying the FRET quantification step. The excitation light of EGFP is in the visible light range, so an expensive ultraviolet exciter is not required, which greatly reduces the detection cost and improves the applicability of the detection method.

[0008] U.S. Patent US20220390461A1 discloses a recombinant substrate of a green fluorescent protein / red fluorescent protein pair coupled with VAMP (27-94) and SNAP-25 (141-206) as a linker for detecting botulinum toxin in clinical samples and food. In cells, SNAP-25 is anchored to the cell membrane on the cytoplasmic side of the cell by palmitylation with its four Cys at positions 85, 88, 90 and 92, and after BoNT / A is taken up by the receptor cells, its LC active part is released and also transported and positioned on the inner side of the receptor cell membrane, thereby efficiently cutting SNAP-25. The VAMP (27-94) and SNAP-25 (141-206) linkers disclosed in U.S. Patent US20220390461A1 lack palmitylation anchoring sites and can only exist diffusely in the cytoplasm. Therefore, when detecting botulinum toxin type A, the sensitivity is low.

[0009] The present invention provides a cell sensor for detecting the activity or content of BoNT / A or BoNT / E in vitro, using SNAP-25 as a linker, fusion-expressing EGFP and tDimer2 at both ends, and transiently transfecting into Neuro-2a cells, which has the characteristics of being able to detect the entire process of BoNT / A or BoNT / E poisoning, low cost, and short detection time. The FRET-based cell sensor provided by the present invention can be used in the determination of the potency of BoNT / A or BoNT / E products, or in the diagnosis of BoNT / A or BoNT / E botulinum toxin poisoning, or in the screening of BoNT / A or BoNT / E inhibitors. Summary of the invention

[0010] Based on the above technical problems, the present invention unexpectedly found that an expression vector of EGFP-SNAP-25-tDimer2 fusion protein was constructed using EGFP / tDimer2 as the donor and acceptor fluorophores and SNAP-25 as the linker. After transiently transferring the expression vector into Neuro-2a cells, a FRET-based BoNT / A or BoNT / E detection cell sensor was obtained, which can realize the detection of BoNT / A or BoNT / E biological activity or content in living cells. Specifically, it includes the following contents:

[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 a sensor molecule; the cell sensor contains a linker peptide, a donor fluorophore and an acceptor fluorophore, wherein the linker peptide is a SNAP-25 protein; the donor fluorophore is EGFP; and the acceptor fluorophore is tDimer2.

[0012] Preferably, the cell is a Neuro-2a cell.

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

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

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

[0016] In a second aspect, the present invention provides use of the cell sensor described in the first aspect in preparing a detection reagent for BoNT / A or BoNT / E.

[0017] In a third aspect, the present invention provides use of the cell sensor described in the first aspect in preparing a reagent for screening BoNT / A or BoNT / E inhibitors.

[0018] In a fourth aspect, the present invention provides a method for preparing the cell sensor according to the first aspect, the method comprising the following steps:

[0019] (1) constructing 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 a cell sensor.

[0021] In a fifth aspect, the present invention provides a FRET-based BoNT / A or BoNT / E detection kit, wherein the kit contains the cell sensor described in the first aspect.

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

[0023] (1) Detection system: placing the cell sensor described in the first aspect in a sample to be tested;

[0024] (2) Result analysis: detecting and comparing the FRET signals of the cell sensor before and after being exposed to the sample to be tested, wherein a decrease in the FRET signal indicates the presence of BoNT / A or BoNT / E in the sample to be tested.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention uses SNAP-25 as a linker, fuses and expresses EGFP and tDimer2 fluorescent proteins at both ends, constructs an EGFP-SNAP-25-tDimer2 expression plasmid, and transiently transfects it 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) Detection of BoNT / A or BoNT / E cell bioactivity based on FRET cell sensors covers the entire BoNT / A activity process, including binding, internalization, translocation, and enzyme activity. Through laser confocal microscopy, whole-well automatic scanning imaging and data processing, it meets the requirements of high-throughput detection and can detect a large number of samples in a short time. It can be applied to the detection of the biological activity of recombinant BoNT / A and BoNT / A extracted by traditional methods during production. It can also meet the requirements of in vitro screening of botulinum toxin inhibitors and the diagnosis of botulinum toxin BoNT / A or BoNT / E poisoning.

[0028] (3) The FRET-based cell sensor used in the present invention uses SNAP-25 as the linker between the FRET pairs. In theory, in addition to being used for the detection of BoNT / A, it can also be used for the detection of the biological activity of BoNT / E. Furthermore, by replacing the linker, such as VAMP2 or Syntaxin, the biological activity detection of other serotypes of BoNTs can be achieved.

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

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

[0031] (6) It is well known in the art that PC12 cells are more sensitive to BoNT / A than Neuro-2a. However, the present invention unexpectedly discovered that after transient transfer of the EGFP-SNAP-25-tDimer2 recombinant plasmid to Neuro-2a, the cell sensor obtained has a wider response range to BoNT / A, is more sensitive to BoNT / A detection, has more advantages, and has unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Confocal results of Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 detecting BoNT / A; 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 Western blot detection of PC12 (a) and Neuro-2a (b) cells' sensitivity to BoNT / A. Lane 1 is the negative control, and lanes 2-6 are the results of Western blot analysis of proteins extracted after incubation of 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 3FRET / EGFP-Log[BoNT / A](nM) curve of BoNT / A activity detected in Neuro-2a cells and PC12 cells transiently expressing EGFP-SNAP-25-tDimer2; Neuro-2a and PC12 cells transiently expressing EGFP-SNAP-25-tDimer2 were incubated with BoNT / A in a 96-well plate at 37°C for 48 hours, and then the whole well was scanned with a laser confocal microscope to image the EGFP channel (excitation, 488nm; emission, 507nm), FRET channel (excitation, 488nm; emission, 579nm), and tDimer2 channel (excitation, 557nm, emission, 579nm). At the same time, automatic data processing was performed to obtain the fluorescence images and fluorescence intensities of the EGFP and FRET channels of the whole well cells, calculate FRET / EGFP, and take the logarithm of the BoNT / A concentration. GraphPad Prism statistical data was used to create a FRET / EGFP-Log[BoNT / A](nM) curve graph.

[0035] Figure 4 Standard curve of BoNT / A detected in Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2; Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 were incubated with BoNT / A in a 96-well plate at 37°C for 48 hours, and then the whole well was scanned and imaged using a laser confocal microscope using the EGFP channel (excitation, 488nm; emission, 507nm), FRET channel (excitation, 488nm; emission, 579nm), and tDimer2 channel (excitation, 557nm; emission, 579nm). At the same time, automatic data processing was performed to obtain the fluorescence images and fluorescence intensities of the EGFP and FRET channels of the whole well of cells, calculate FRET / EGFP, take the logarithm of the BoNT / A concentration, use GraphPad Prism to perform statistical data, make a FRET / EGFP-Log[BoNT / A](nM) curve, and draw a standard curve. DETAILED DESCRIPTION

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

[0037] Example 1 Lenti-EGFP-SNAP-25-tDimer2 plasmid construction

[0038] 1.1 Biomaterials

[0039] EGFP sequence (OK067273.1, fully synthesized by GeneWeizhi); SNAP-25 sequence (NM_001322903.2, fully synthesized by GeneWeizhi); pCDNA3.1-tDimer2 (Ubao Biotechnology); Top10 competent cells (Shanghai Sangon Biotechnology 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 recombination enzyme (Polymer, catalog number 2011A); SpeedyCut XbaI fast cutting enzyme, SpeedyCut BamHI fast cutting enzyme, SpeedyCut EcoRI fast cutting enzyme were purchased from Sangon Biotechnology Co., Ltd.; other reagents were of analytical grade.

[0042] 1.3 Implementation steps

[0043] 1.3.1 Primer design and synthesis

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

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

[0046]

[0047]

[0048] 1.3.2 Plasmid construction

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

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

[0051] Table 2 EGFP fragment PCR amplification system

[0052]

[0053] Table 3 EGFP fragment PCR amplification program

[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 steps in the gel recovery kit.

[0060] (3) Overlap PCR amplification of EGFP-SNAP-25 fragments

[0061] The EGFP and SNAP-25 fragments recovered from the gel were fused together by 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 steps Reaction temperature (℃) Reaction time(S) Number of cycles Pre-denaturation 98 180 1 transsexual 98 30 10 / 30 annealing 60 30 10 / 30 extend 72 60 10 / 30 Final extension 72 600 1

[0066] Without adding primers, the Overlap PCR amplification program was performed, with 10 cycles of denaturation, annealing and extension. After the program was completed, primers were added, and the number of denaturation, annealing and extension cycles was 30.

[0067] (4) Gel recovery of the EGFP-SNAP-25 fragment overlap PCR amplification product can be performed using the gel recovery kit.

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

[0069] Table 8 pCDNA3.1-tDimer2 plasmid restriction enzyme system

[0070] Reactants Volume (μL) pCDNA3.1-tDimer2 Plasmid 4.0 Xgf 1.0 EcoRI 1.0 10×Single Buffer 2.0 <![CDATA[ddH2O]]> 12.0 system 20.0

[0071] The reactants were added to a 200 μL centrifuge tube and mixed evenly. The mixture was incubated at 37°C in a metal bath for 1 hour for enzyme digestion. The gel recovery was performed according to the instructions of the gel recovery kit.

[0072] (6) Homologous recombination

[0073] The restriction plasmid vector pCDNA3.1-tDimer2 after gel recovery and the Overlap PCR product EGFP-SNAP-25 were homologously recombined at a molar ratio of 1:3. The system is shown in Table 9:

[0074] Table 9 pCDNA3.1-EGFP-SNAP-25-tDimer2 homologous recombination reaction system

[0075] Reactants Volume (μL) pcDNA3.1-tDimer2 digestion and gel recovery products 5.0 Overlap PCR gel recovery product 0.5 2×M5 5.0 system 10.5

[0076] The reactants were added to a 200 μL centrifuge tube and mixed well. The mixture was placed in a metal bath at 50°C and reacted for 1 hr.

[0077] (7) Heat shock transformation of Top10 competent cells

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

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

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

[0081] ④ Quickly transfer the centrifuge tube to ice and place it for 2 minutes;

[0082] ⑤Resuscitation: Add 200 μL LB liquid culture medium to the centrifuge tube and incubate at 37°C with gentle shaking for 45 minutes to allow the bacteria to recover;

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

[0084] ⑦ Culture in 37℃ incubator for 12-16 hours.

[0085] (8) Plasmid extraction

[0086] ① Bacterial liquid culture: When clones grow on the transformation plate, pick 3 single clones and place them in 10 mL of sterilized LB liquid culture medium, add the corresponding volume of Amp antibiotic, and culture at 37°C, 180 rpm, with shaking for 12-16 hours;

[0087] ②Extract plasmid: follow the instructions of the plasmid extraction kit.

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

[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] PCR product gel recovery was performed according to the gel recovery kit steps.

[0095] (10) Double restriction 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 Double restriction enzyme digestion of CDNA3.1-EGFP-SNAP-25-tDimer2 plasmid and Lenti vector fragment

[0098]

[0099]

[0100] The double enzyme digestion product was recovered by gel recovery kit.

[0101] (11) Ligation 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. The system is shown in Table 13 below.

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

[0104] Reactants Volume (μL) Lenti vector PCR post-gel recovery product 2.0 The EGFP-SNAP-25-tDimer2 fragment was digested and the product was recovered from the gel 2.0 10×T4 ligase 1.0 10×T4 ligase buffer 1.0 <![CDATA[ddH2O]]> 4.0 system 10.0

[0105] The reactants were added to a 200 μL centrifuge tube and mixed well. The metal bath was kept at 16°C and reacted for 12 hours.

[0106] (12) Thermal shock conversion

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

[0108] (13) Recombinant plasmid sequencing

[0109] ① Bacterial liquid culture: When clones grow on the transformation plate, pick 3 single clones and place them in 10 mL of sterilized LB liquid culture medium, add the corresponding volume of Amp antibiotic, and culture at 37°C, 180 rpm, with shaking for 12-16 hours;

[0110] ②Extract plasmid: follow the instructions of the plasmid extraction kit.

[0111] ③Sequencing: The plasmid was sent to Tianjin GeneWeizhi Co., Ltd. for sequencing. The sequencing results were compared with the theoretical sequence. The alignment was consistent, indicating that the clone was successfully constructed and can be used for subsequent experiments.

[0112] In this embodiment, a Lenti-EGFP-SNAP-25-tDimer2 vector is constructed, wherein 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 segment can use any restriction endonuclease or a seamless connection. In other words, between the sensor molecule and the vector, between EGFP, tDimer2 and the linker peptide segment, any restriction endonuclease can be used, or a seamless connection can be used, which can be understood by those skilled in the art.

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

[0114] 2.1 Biomaterials

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

[0116] 2.2 Reagents

[0117] DMEM high-glucose medium (BI, catalog number 06-1055-57-1ACS); MEM (containing NEAA) (Punosai, catalog number PM150410); fetal bovine serum (VivaCell, catalog number C04001-500); liposome nucleic acid transfection reagent (Shanghai Yishen 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, trypsinize Neuro-2a cells and count them, according to 1×10 5 cells / well were inoculated into 24-well plates and cultured in a 37°C, 5% CO2 cell culture incubator for 12 hr.

[0122] ② Liposome transfection: Plasmid (μg): liposome transfection reagent (μL) is 1:1.5, that is, 1μg Lenti-EGFP-SNAP-25-tDimer2 plasmid is diluted with 50μL MEM-α medium and mixed. 1.5μL liposome transfection reagent is diluted with 50μL MEM-α medium, and allowed to stand at room temperature for 5min. Then, the diluted liposomes and plasmids are mixed with a pipette and incubated at room temperature for 20min to form a DNA-liposome complex.

[0123] ③Protein expression: The old culture medium in the 24-well plate was replaced with a culture medium without double antibody, and 100 μL of DNA-liposome complex was added to the 24-well plate and gently shaken to mix. After 6 hours of culture in a 37°C, 5% CO2 incubator, the culture medium was replaced with a complete culture medium and cultured 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] Confocal results of Neuro-2a cells transiently expressing EGFP-SNAP-25-tDimer2 detecting BoNT / A; 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 increased and the fluorescence of the FRET channel disappeared, indicating that BoNT / A exerted its activity ( Figure 1 shown).

[0125] In the present invention, Neuro-2a cell line was used as the transient cell for transient transduction of 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 activity detection. Similarly, we constructed a PC12 cell line transiently transfected with EGFP-SNAP-25-tDimer2, and the results showed that although PC12 cells were indeed more sensitive to BoNT / A than Neuro-2a in Western blot detection ( Figure 2However, unexpectedly, after the EGFP-SNAP-25-tDimer2 cell sensor was transiently transferred into the cells and then the BoNT / A activity was detected, the FRET / EGFP-Log[BoNT / A](nM) curves were compared. The results showed that the slope of the fitting curve obtained by Neuro-2a cells was more obvious than that of PC12, indicating that Neuro-2a has a wider response range to BoNT / A, is more sensitive to BoNT / A detection, and has more advantages ( Figure 3 shown).

[0126] Example 3 Detection of BoNT / A in Neuro-2a Cell Line 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 (Jie Te Bio, catalog number TCP001096).

[0129] 3.2 Reagents

[0130] MEM (containing NEAA) (Pnocell, catalog number PM150410); fetal bovine serum (VivaCell, catalog number C04001-500); double 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 plated in 96-well plates at 1000 cells / well, and a series of concentrations of BoNT / A were added and incubated for 48 hr.

[0134] (2) Leica laser confocal microscopy imaging: 9 evenly distributed points were selected in a well, and the focus was automatically adjusted. Then, the entire well of the 96-well plate was automatically scanned and imaged, and stitched to obtain images of the entire well of cells in the EGFP channel (excitation, 488nm; emission, 507nm) and FRET channel (excitation, 488nm; emission, 579nm).

[0135] (3) Confocal microscopy automatically counts the fluorescence intensity of the entire well of cells in the EGFP and FRET channels.

[0136] (4) Data processing: Calculate FRET / EGFP, take the logarithm of BoNT / A concentration, use GraphPad Prism to calculate the statistics, make a FRET / EGFP-Log[BoNT / A](nM) curve, and draw a standard curve graph ( Figure 4 shown).

[0137] The results of the present invention show that the detection limit of BoNT / A by the FRET-based cell sensor EGFP-SNAP-25-tDimer2 is 100 pM, and the standard curve is y=-0.1392x+0.9824, R 2 =0.9991, linear between 3.125-50nM, recovery rate of 86.3%-117.2%, much higher response to BoNT / A than BoNT / C, and specific for BoNT / A.

[0138] In the embodiment of the present invention, single-blind sample testing was also performed on the diluted ion concentration of other experimenters, and the results showed that the actual 40U sample had a detection value of 41±4.97U.

[0139] The beneficial effect of the present invention is that the FRET-based cell sensor for detecting the biological activity or content of botulinum toxin type A does not require UV excitation, which reduces the phototoxicity of UV to cells, and the green fluorescent protein and red fluorescent protein used have less spectral overlap, which minimizes the crosstalk of the donor emission channel to the acceptor emission channel, thereby significantly simplifying the FRET quantitative process. This cell sensor uses whole-well scanning for automatic data processing during the detection process, realizes fully automatic, rapid high-resolution imaging and quantitative analysis of cells, avoids operational errors, and greatly reduces the time and effort required for manual analysis and statistical calculations.

[0140] The present invention uses a FRET-based cell sensor with SNAP-25 as a linker between FRET pairs. In theory, in addition to being used for the detection of BoNT / A, it can also be used for the detection of the biological activity of BoNT / E. Furthermore, by replacing the linker, such as VAMP2 or Syntaxin, the biological activity detection of other serotypes of BoNTs can be achieved, which can be understood by those skilled in the art.

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

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

[0143] The receptor cell of the cell sensor provided by the present invention is Neuro-2a. The Neuro-2a cell line is induced to differentiate by the existing public culture method so that they express more botulinum toxin receptor molecules, which can also significantly improve the sensitivity of these receptor cells. In addition, the cell line stably expressing the sensor molecule EGFP-SNAP-25-tDimer2 can be screened by the stable transfection method, which can further simplify the detection operation and shorten the detection cycle, which can be understood by those skilled in the art.

[0144] The Neuro-2a cell line is used as a transient cell for transiently transfecting the EGFP-SNAP-25-tDimer2 cell sensor in the present invention. Chapman and Dong disclosed a rat adrenal pheochromocytoma cell line PC12 in patent US2016356776 (A1) in 2016 for BoNT / A cell activity detection. Similarly, the present invention 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 detection. However, unexpectedly, when the EGFP-SNAP-25-tDimer2 cell sensor was transiently transferred into the cells and the BoNT / A activity was detected, the FRET / EGFP-Log[BoNT / A](nM) curves were compared. The results showed that the slope of the fitting curve obtained by Neuro-2a cells was more obvious than that of PC12, indicating that Neuro-2a has a wider response range to BoNT / A, is more sensitive to BoNT / A detection, and has more advantages.

[0145] The FRET-based botulinum toxin type A detection method provided by the present invention can be used to evaluate the biological activity of materials containing BoNT / A in the environment and in the pharmaceutical production process. At the same time, it can meet the in vitro screening needs 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 or BoNT / E detection cell sensor, characterized in that: The cell sensor is obtained by transfecting cells with a plasmid expressing a molecular sensor; the molecular sensor contains a linker peptide, a donor fluorophore and an acceptor fluorophore, wherein the linker peptide is SNAP-25 protein; the donor fluorophore is EGFP; and the acceptor fluorophore is tDimer2.

2. The cell sensor according to claim 1, characterized in that The cells are Neuro-2a cells.

3. The cell sensor according to claim 2, characterized in that The amino acid sequence of the EGFP is shown as SEQ ID No.1, the amino acid sequence of the tDimer2 is shown as SEQ ID No.3, and the sequence of the linker peptide is shown as SEQ ID No.

2.

4. The cell sensor according to claim 3, characterized in that The molecular sensor consists of EGFP-SNAP-25-tDimer2, and the sequence is shown in SEQ ID No.

4.

5. Use of the cell sensor according to any one of claims 1 to 4 in preparing a detection reagent for BoNT / A or BoNT / E, or in preparing a reagent for screening a BoNT / A or BoNT / E inhibitor.

6. The method for preparing a cell sensor according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: (1) constructing 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 a cell sensor.

7. A FRET-based BoNT / A or BoNT / E detection kit, characterized in that: The kit contains the cell sensor according to any one of claims 1 to 4.

8. The method for using the detection kit according to claim 7, characterized in that: The method comprises: (1) Detection system: placing the cell sensor according to any one of claims 1 to 4 in a sample to be detected; (2) Result analysis: detecting and comparing the FRET signals of the cell sensor before and after being exposed to the sample to be tested, wherein a decrease in the FRET signal indicates the presence of BoNT / A or BoNT / E in the sample to be tested.

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