FRET-based botulinum toxin type A and E detection molecular 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 botulinum toxin A and E detection molecular sensor and application thereof. The invention accidentally finds that the BoNT / A or BoNT / E biological activity detection molecular sensor is prepared by taking EGFP / DsRED as donor and receptor fluorophores and SNAP-25 (141-206aa) as a connexon through an after-ripening process, and the detection molecular sensor has the characteristics of high signal-to-noise ratio, rapidness, simplicity, convenience, sensitivity, good accuracy, high sensitivity, good repeatability and high stability. The biological activity or content detection on BoNT / A or BoNT / E at a molecular level can be realized; the kit can be applied to detection of the biological activity of the light chain in the preparation process of BoNT / A or BoNT / E extracted by a recombination or traditional method, can also be used for in-vitro screening of botulinum toxin inhibitors, and can also be used for poisoning diagnosis of BoNT / A or BoNT / E.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and provides a FRET-based botulinum toxin type A and type E molecular sensor for detecting botulinum toxin and its application. The molecular 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 toxins (BoNTs) are neurotoxic proteins produced by Clostridium botulinum in an anaerobic environment. The action of BoNTs on neuronal cells includes four steps: BoNTs recognize and bind to neuronal cells. The binding of toxins to nerve cells involves a dual receptor mechanism of ganglioside compounds such as GT1b and protein receptors. BoNTs bind to the presynaptic membrane through dual receptor recognition on the surface of target cells through its heavy chain receptor binding domain HC; BoNTs further form an acidic vesicle that encapsulates the toxin through endocytosis of the cell, carrying the toxin into the cell; the acidic environment in the acidic vesicle changes the conformation of the toxin, and the reduction of the interchain disulfide bond causes the heavy chain to separate from the light chain, and the heavy chain translocation domain H N The phospholipid bilayer embedded in the vesicle membrane mediates the entry of the light chain into the neuronal cytoplasm; the light chain of the toxin specifically cuts the SNARE protein, blocking the fusion of the transmitter vesicles with the plasma membrane, thereby preventing the release of acetylcholine into the synaptic cleft, resulting in the inhibition of muscle neuron activation, affecting the motor neurons of the peripheral nervous system, and causing flaccid paralysis. Among them, BoNT / A, C and E cut SNAP-25, BoNT / B, D, F and G cut VAMP, and BoNT / C also cuts syntaxin.

[0003] BoNTs can enter the human or vertebrate circulation and cause botulism through different pathways, including ingestion of food contaminated with Clostridium botulinum, infection with Clostridium botulinum through wounds, and colonization of the intestine by Clostridium botulinum, causing infant botulism or adult intestinal botulism. If not treated in time, it will lead to respiratory failure and death. The lethal dose of botulinum toxin in humans is about 1.3-2.1 ng / kg when administered by intravenous or intramuscular injection, and 10-13 ng / kg when administered by inhalation. Although botulinum toxin is highly toxic, it can be used in medical cosmetology to achieve effects such as wrinkle removal when injected locally in low doses; it can also be used for long-term relief of many neurological diseases, including but not limited to cervical dystonia, strabismus, blepharospasm, chronic migraine, hyperhidrosis, urinary incontinence, and multiple sclerosis. In addition, due to the high potency of BoNTs and the fact that only nanogram doses of intravenous injection can be lethal, BoNT / A is the most toxic of the seven known serotypes and accounts for the largest proportion of human botulism. In addition, among the seven 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 bioactivity detection methods for BoNT / A is of great significance for botulism diagnosis, inhibitor screening, and potency determination.

[0004] At present, the "gold standard" for BoNT / A detection is the mouse lethality test, which has high sensitivity, but is cumbersome to operate, has a long test cycle, poor reproducibility between batches, and relatively high capital costs. It also does not meet the current principles of animal experiment replacement, reduction, and optimization. Therefore, it is urgent to establish a rapid, sensitive, and accurate method for detecting the biological activity of BoNT / A that can replace MBA.

[0005] The FRET-based BoNT / A bioactivity detection method is a very promising alternative method. Chapman and Dong disclosed in a patent in 2016 the use of SNAP-25 as a linker to connect CFP to YFP as a FRET-based biosensor to detect BoNT / A. However, the spectral characteristics of the CFP and YFP fluorescence pair have defects when used in the FRET-based BoNT / A bioactivity detection method, such as low dynamic range, ultraviolet excitation-induced phototoxicity, autofluorescence, and a large amount of spectral crosstalk. 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 (141-206aa) as a linker for detecting botulinum toxin in clinical samples and food. The linker between the green fluorescent donor and the red fluorescent acceptor provided by the invention is too long and has low FRET efficiency. Therefore, after the substrate sensor provided by the invention is cleaved by the toxin, it can only use the electrophoresis method to detect the cleavage product to semi-quantitate the activity of botulinum toxin, but cannot use high-throughput detection of FRET signal changes to quantify the activity of botulinum toxin. In addition, the invention states that the fusion linker provided can be cleaved by all 7 types of botulinum toxin, which is not applicable in scenarios where specific identification of the type of botulinum toxin to be tested is required.

[0006] The green fluorescent protein / red fluorescent protein pair has high brightness and emission efficiency, but because DsRed is a tetramer and tends to form oligomers, the protein fused with it will form aggregates with it, which is not conducive to recognition and cutting. It is generally necessary to mutate it when used. Therefore, people in this field generally choose EGFP / mRuby, EGFP / mCherry, EGFP / tDimmer2 and other fluorescent pairs to prepare BoNT / A sensors. The present invention unexpectedly found that the BoNT / A or BoNT / E biological activity detection molecular sensor prepared by using EGFP / DsRED as donor and acceptor fluorophores, SNAP-25 (141-206aa) as a linker, and after the ripening process has the characteristics of high signal-to-noise ratio, and can realize the detection of BoNT / A or BoNT / E biological activity or content at the molecular level, and has the characteristics of rapidity, simplicity, sensitivity and good accuracy. The FRET-based molecular sensor provided by the present invention 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 potency determination during the preparation process of BoNT / A or BoNT / E. Summary of the invention

[0007] Based on the above technical problems, the present invention unexpectedly found that the molecular sensor for detecting the biological activity of BoNT / A or BoNT / E prepared by using EGFP / DsRED as the donor and acceptor fluorophores, SNAP-25 (141-206aa) as the linker, and after a post-ripening process has the characteristics of high signal-to-noise ratio, and can realize the detection of the biological activity or content of BoNT / A or BoNT / E at the molecular level. Specifically, it includes the following contents:

[0008] In a first aspect, the present invention provides a FRET-based BoNT / A or BoNT / E detection molecular sensor, wherein the molecular sensor comprises 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 DsRED.

[0009] Preferably, the linker peptide is 141-206aa of SNAP-25.

[0010] Preferably, the amino acid sequence of the EGFP is shown as SEQ ID No.1, the amino acid sequence of the DsRED is shown as SEQ ID No.3, and the sequence of the linker peptide is shown as SEQ ID No.2.

[0011] Preferably, the molecular sensor composition is EGFP-SNAP-25(141-206aa)-DsRED.

[0012] Preferably, the sequence of the EGFP-SNAP-25(141-206aa)-DsRED is shown as SEQ ID No.4.

[0013] Preferably, the EGFP-SNAP-25(141-206aa)-DsRED is fused with a His tag.

[0014] Preferably, the His tag is located at the N-terminus of EGFP-SNAP-25(141-206aa)-DsRED.

[0015] Preferably, the length of the His tag is 6.

[0016] In a second aspect, the present invention provides use of the molecular 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 molecular 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 molecular sensor according to the first aspect, the method comprising the following steps:

[0019] (1) constructing a recombinant plasmid expressing the detection molecular sensor EGFP-SNAP-25 (141-206aa)-DsRED;

[0020] (2) Induce expression and purify to obtain EGFP-SNAP-25 (141-206aa)-DsRED fusion protein;

[0021] (3) The EGFP-SNAP-25 (141-206aa) -DsRED fusion protein was placed at 4°C for at least 72 hours, then quickly frozen in liquid nitrogen using 5%-20% glycerol as a protective agent, and stored in a -80°C refrigerator.

[0022] Preferably, the EGFP-SNAP-25(141-206aa)-DsRED is fused with a His tag to facilitate purification.

[0023] Preferably, the His tag is located at the N-terminus of EGFP-SNAP-25(141-206aa)-DsRED.

[0024] Preferably, the length of the His tag is 6.

[0025] Preferably, during the purification process, the initial binding buffer composition is 20 mM PB pH 7.4, 150 mM NaCl, 100 mM imidazole.

[0026] Preferably, the liquid nitrogen quick-freezing buffer is 50 mM HEPES-NaOH, 10 mM NaCl, pH 7.1, containing 5-20% glycerol.

[0027] Preferably, the glycerol content is 10%.

[0028] Preferably, the temperature is kept at 4°C for 96 hours.

[0029] In a fifth aspect, the present invention provides a FRET-based BoNT / A or BoNT / E detection kit, wherein the kit contains the molecular sensor described in the first aspect.

[0030] In a sixth aspect, the present invention provides a method for using the detection kit described in the fifth aspect, the method comprising:

[0031] (1) Detection system: placing the molecular sensor described in the first aspect in a sample to be tested;

[0032] (2) Result analysis: detecting and comparing the FRET signals of the molecular 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.

[0033] Preferably, the detection system further comprises providing DTT and Zn to the sample to be detected. 2+ .

[0034] The beneficial effects of the present invention are:

[0035] (1) The present invention uses SNAP-25 (141-206aa) as a linker, and fusion-expresses EGFP and DsRED fluorescent proteins at both ends to construct a pET28a (+) -EGFP-SNAP-25 (141-206aa) -DsRED plasmid, and purifies the EGFP-SNAP-25 (141-206aa) -DsRED fusion protein; the EGFP-SNAP-25 (141-206aa) -DsRED fusion protein is used as a fluorescent probe molecular sensor to realize FRET-based BoNT / A biological activity detection.

[0036] (2) The detection of BoNT / A bioactivity based on FRET molecular sensors meets the requirements of high-throughput detection and can detect a large number of samples in a short time. It has high detection sensitivity and good accuracy. It can be applied to the detection of the bioactivity of the light chain of recombinant BoNT / A and BoNT / A extracted by traditional methods during production, and can also meet the in vitro screening requirements of botulinum toxin inhibitors.

[0037] (3) The FRET-based molecular sensor used in the present invention uses SNAP-25 (141-206aa) 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 SNAP-25 (93-202), VAMP2 or Syntaxin, the biological activity detection of other serotypes of BoNTs can be achieved.

[0038] (4) Compared with EGFP / mRuby, EGFP / mCherry, and EGFP / tDimmer2, the EGFP / DsRED fluorescence pair used in the FRET-based BoNT / A bioactivity detection molecular sensor of the present invention has a high signal-to-noise ratio; in actual detection, the CFP and YFP single fluorescent protein controls can be omitted, thereby greatly simplifying the FRET bioactivity quantitative detection method.

[0039] (5) It is well known to those skilled in the art that DsRed is a tetramer and tends to form oligomers, which causes the protein fused with it to form aggregates together with it, which is not conducive to recognition and cleavage. When used, other red fluorescent protein monomers obtained by gene mutation are generally selected. However, the present invention unexpectedly discovered that the BoNT / A bioactivity detection molecular sensor prepared by using EGFP / DsRED as donor and acceptor fluorophores, SNAP-25 (141-206aa) as a linker, and after a post-ripening process has the characteristics of a high signal-to-noise ratio, and can realize the detection of BoNT / A bioactivity or content at the molecular level. It has unexpected technical effects and overcomes technical bias. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Molecular sensor EGFP-SNAP-25 (141-206aa) -DsRED protein purification result diagram; wherein, M is Protein Ladder, lane 1 is the pre-column sample of the supernatant of the lysis solution, lane 2 is the chromatography penetration sample, lane 3 is the Wash sample, and lane 4 is the elution peak Elution sample.

[0041] Figure 2 Figure 3. SDS-PAGE staining results of the cleavage effect of BoNT / A-LC on the molecular sensor. After IPTG-induced expression of pET28a(+)-BoNT / A-LC in E. coli BL21(DE3), the cells were collected and lysed and purified by Ni 2 + His6-BoNT / A-LC was purified by affinity chromatography; His6-BoNT / A-LC was mixed with 500nM EGFP-SNAP25(141-206aa)-DsRED at 37°C and reacted in a 100μl reaction system for 15 minutes; Lane 1 is Protein Ladder, Lane 2 is negative control, and Lanes 3-9 are samples after the reaction of EGFP-SNAP25(141-206aa)-DsRED with 4nM His6-BoNT / A-LC diluted 2 times in series.

[0042] Figure 3BoNT / A in vitro bioactivity detection standard curve based on FRET molecular sensor; In a 96-well full black plate, GFP-SNAP-25 (141-206aa)-DsRED protein was co-incubated with BoNT / A at 37°C for 4 hours, and then excited at 488nm using a multifunctional microplate reader, and the emission fluorescence intensity at 510nm (EGFP) and 585nm (DsRED) was collected and labeled as Em510 and Em585, respectively, and Em585 / Em510 was calculated. The concentration of BoNT / A was taken as a logarithm, and statistical data was used in GraphPad Prism to prepare the Em585 / Em510-Log[BoNT / A](pM) curve and draw the standard curve. DETAILED DESCRIPTION

[0043] 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.

[0044] Example 1 Construction of pET28a(+)-EGFP-SNAP-25(141-206aa)-DsRED plasmid

[0045] 1.1 Biomaterials

[0046] EGFP sequence (OK067273.1, fully synthesized by GeneWeizhi); SNAP-25 sequence (NM_001322903.2, fully synthesized by GeneWeizhi); pET-28a(+)-DsRED plasmid (Fenghui Biotechnology, catalog number ZT721); Top10 competent cells (Shanghai Sangon Biotechnology Co., Ltd., catalog number B528412).

[0047] 1.2 Reagents

[0048] 2×Hieff 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 BamHⅠ fast cutting enzyme was purchased from Sangon Biotechnology Co., Ltd.; other reagents were of analytical grade.

[0049] 1.3 Implementation steps

[0050] 1.3.1 Primer design and synthesis

[0051] According to the EGFP sequence, SNAP-25 sequence and pET-28a(+)-DsRED vector sequence, two pairs of appropriate upstream and downstream primers were designed and synthesized as shown in Table 1.

[0052] Table 1 Primer sequences for construction of pET28a(+)-EGFP-SNAP-25(141-206aa)-DsRED plasmid

[0053]

[0054]

[0055] 1.3.2 Plasmid construction

[0056] (1) PCR amplification of EGFP fragment and SNAP-25 (141-206aa) fragment

[0057] The amplification systems are shown in Tables 2 and 4, respectively, and the amplification procedures are shown in Tables 3 and 5, respectively.

[0058] Table 2 EGFP fragment PCR amplification system

[0059]

[0060] Table 3 EGFP fragment PCR amplification program

[0061]

[0062] Table 4 SNAP-25 (141-206aa) fragment PCR amplification system

[0063]

[0064] Table 5 SNAP-25 (141-206aa) fragment PCR amplification program

[0065]

[0066]

[0067] (2) Gel recovery of EGFP and SNAP-25 (141-206aa) PCR amplification products: Follow the steps of the gel recovery kit.

[0068] (3) Overlap PCR amplification of EGFP-SNAP-25 (141-206aa) fragment

[0069] The EGFP and SNAP-25 (141-206aa) fragments recovered from the gel were fused together by Overlap PCR to obtain the EGFP-SNAP-25 (141-206aa) fragment. The amplification system and amplification program are shown in Tables 6 and 7, respectively.

[0070] Table 6 Overlap PCR amplification system of EGFP-SNAP-25 (141-206aa) fragment

[0071]

[0072] Table 7 Overlap PCR amplification procedure of EGFP-SNAP-25 (141-206aa) fragment

[0073] Reaction steps Reaction temperature (℃) Reaction time(S) Number of cycles Pre-denaturation 98 240 1 transsexual 98 10 10 / 30 annealing 60 15 10 / 30 extend 72 50 10 / 30 Final extension 72 600 1

[0074] 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.

[0075] (4) Gel recovery of the EGFP-SNAP-25 (141-206aa) fragment overlap PCR amplification product can be performed using the gel recovery kit.

[0076] (5) pET28a(+)-DsRED plasmid was digested with BamHI. The digestion system is shown in Table 8.

[0077] Table 8 pET28a(+)-DsRED plasmid restriction enzyme system

[0078] Reactants Volume (μL) pET28a(+)-DsRED Plasmid 4.0 B H 2.0 10 × SpeedyOne Cutter 4.0 <![CDATA[ddH2O]]> 12.0 system 20.0

[0079] Add the reactants to a 200 μL centrifuge tube and mix well. Incubate in a metal bath at 37°C for 1 hour.

[0080] (6) The pET28a(+)-DsRED plasmid digestion product was recovered by gel recovery according to the gel recovery kit.

[0081] (7) Homologous recombination ligation of pET28a(+)-DsRED and EGFP-SNAP-25 (141-206aa) fragments

[0082] The pET28a(+)-DsRED plasmid vector recovered from the gel was homologously recombined with the EGFP-SNAP-25 (141-206aa) fragment at a molar ratio of 1:3. The system is shown in 9:

[0083] Table 9 pET28a(+)-EGFP-SNAP-25(141-206aa)-DsRED homologous recombination reaction system

[0084] Reactants Volume (μL) Gel recovery after pET28a(+)-DsRED digestion 5.0 Overlap PCR gel recovery product 0.5 2×M5 5.0 system 10.5

[0085] 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.

[0086] (8) Heat shock transformation of Top10 competent cells

[0087] ①Take 1 tube of Top 10 competent cells and place it on ice to thaw;

[0088] ② Add all the homologous recombination reaction products to 50 μL of receptor cells, mix gently, and place on ice for 30 minutes;

[0089] ③ Heat shock: Place the centrifuge tube in a metal bath at 42°C for 90 seconds;

[0090] ④ Quickly transfer the centrifuge tube to ice and place it for 2 minutes;

[0091] 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;

[0092] ⑥ Spread the revived bacteria evenly on the Amp-resistant LB solid culture plate;

[0093] ⑦ Culture in 37℃ incubator for 12-16 hours.

[0094] (9) Recombinant plasmid sequencing

[0095] 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;

[0096] ②Extract plasmid: follow the instructions of the plasmid extraction kit.

[0097] 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.

[0098] In this embodiment, a pET28a(+)-EGFP-SNAP-25(141-206aa)-DsRED vector is constructed, wherein the vector can also be any other commercially available E. coli expression vector pET series such as pET29, pET31b(+), pET32, pET42, pTrx3c, etc. The cloning strategy for recombining EGFP and DsRED and the linker peptide segment can use any restriction endonuclease or a seamless connection. In other words, between the molecular sensor and the vector, between the molecular sensor EGFP, DsRED 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.

[0099] Example 2 EGFP-SNAP-25 (141-206aa) -DsRED protein induced expression and purification

[0100] 2.1 Biomaterials

[0101] BL21(DE3) competent cells were purchased from Sangon Biotechnology Co., Ltd.

[0102] 2.2 Reagents

[0103] IPTG (Sangon Biotechnology Co., Ltd., Catalog No. A600168-0025); Ni Sepharose TM 6FastFlow (GE Healthcare, catalog number 17-5318-01); other reagents were of analytical grade.

[0104] 2.3 Implementation steps

[0105] 2.3.1 EGFP-SNAP-25(141-206aa)-DsRED protein induced expression

[0106] (1) Bacteria culture:

[0107] ① Transformation: Transform the correctly sequenced pET28a(+)-EGFP-SNAP-25(141-206aa)-DsRED recombinant plasmid into BL21(DE3) competent cells.

[0108] ② Shake the strain: After clones grow on the transformation plate, pick a single clone and place it in 20mL LB liquid culture medium. Add 20μL of 100mg / mL Kan antibiotics. Incubate at 37℃, 180rpm, and shake for 12-16hr. When the LB liquid becomes turbid, it can be stored as a strain and placed at 4℃ for later use.

[0109] (2) Induced expression:

[0110] 500mL LB liquid culture medium was inoculated with 10mL of bacterial strain at a volume ratio of 2%, 500μL of 100mg / mL Kan antibiotic was added, and cultured at 37℃, 200rpm for 3hr. 200μL of bacterial solution was taken as the uninduced sample. At this time, the OD value of the bacterial solution was about 0.8. 200μL of 1M IPTG was added to make the final concentration of 0.4mmol / L. After continuing to culture at 16℃, 200rpm for 20hr, 200μL of bacterial solution was taken as the induced sample. The other bacterial solution was centrifuged at 8000rpm for 3min to collect the bacteria, calculate the bacterial weight, mark the batch number, and store at -20℃.

[0111] 2.3.2 EGFP-SNAP-25(141-206aa)-DsRED protein purification

[0112] (1) Bacteria destruction:

[0113] For the induced bacteria stored at -20℃, add Binding buffer at a ratio of 1:30 between wet weight of bacteria (g) and Binding buffer (mL). Place the resuspended bacteria in a high-pressure homogenizer at 850 Bar for 3 minutes to break the bacteria. After the breaking of the bacteria, the bacterial liquid becomes clear. Centrifuge at 10000 rpm for 10 minutes and collect the supernatant, which is marked as S.

[0114] (2)Ni 2+ Column affinity chromatography

[0115] ① Column loading: Clean the chromatography column and fix it vertically on the iron frame with a fixing clamp. After tightening the lower cover of the chromatography column, add the filler mixed with distilled water into the chromatography column, let it stand and wait for the filler to settle to the bottom of the chromatography column, install the column height adjuster and tighten it. The column diameter is 1.6 cm and the column height is 5 cm.

[0116] ②Chromatographic conditions

[0117] Binding buffer: 20mM PB pH7.4, 150mM NaCl, 100mM imidazole;

[0118] Elution buffer: 20mM PB pH7.4, 150mM NaCl, 150mM imidazole; 20mM PB pH7.4, 150mM NaCl, 300mM imidazole; 20mM PB pH7.4, 150mM NaCl, 500mM imidazole.

[0119] ③Chromatography process:

[0120] a. Connect the assembled chromatography column to the peristaltic pump and detector, and adjust the peristaltic pump to a flow rate of 1.5 mL / min;

[0121] b. Cleaning: Pass 10CV of distilled water through the column;

[0122] c. Balance: 10CV Binding buffer is passed through the column;

[0123] d. Sample loading: The sample loading volume is generally 10-20CV, and the flow-through sample is collected by LC;

[0124] e. Washing: After loading, pass 10CV Binding buffer through the column and collect the washing sample W;

[0125] f. Elution: Elution with elution buffer containing different concentrations of imidazole, and collect sample E according to the change of detector value;

[0126] g. Wash the column with 10 CV of distilled water; disassemble the column and store the filler in 20% ethanol in a 4°C refrigerator.

[0127] The collected samples were detected by SDS-PAGE electrophoresis to observe the purification effect. The eluted EGFP-SNAP-25(141-206aa)-DsRED protein was dialyzed into 50 mM HEPES-NaOH, 10 mM NaCl, pH 7.1.

[0128] In the process of purifying EGFP-SNAP-25(141-206aa)-DsRED molecular sensor by nickel ion affinity chromatography, we found that the target protein was more impure when 300mM NaCl was used as recommended in the filler manual. Reducing the salt concentration did not affect the yield and a purer target protein was obtained. At the same time, the yield of the target protein was not affected when the imidazole concentration in the binding buffer was increased to 100mM, and the sample purity was higher than that of the sample loaded with low concentration of imidazole (e.g. Figure 1 shown).

[0129] In this embodiment, the His6 tag is fused to the N-terminus of the molecular sensor. Theoretically, the His6 tag can also be fused to the C-terminus of the molecular sensor, or the His6 tag can be fused to both the N-terminus and the C-terminus of the molecular sensor. In addition, His6 can be superimposed on two His6 or three His6, which is understandable to those skilled in the art.

[0130] Since DsRED matures slowly, it initially exhibits green fluorescence properties and takes several days to reach full red fluorescence. Therefore, it is necessary to wait until the DsRED protein is fully mature before use to ensure the occurrence and stability of FRET. This step requires that DsRED be placed in a 4°C refrigerator for no less than 96 hours to allow it to mature, then quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0131] After freezing the EGFP-SNAP-25 (141-206aa) -DsRED molecular sensor, it was found that the frozen sample was unstable and the color of the fluorescent protein disappeared. Although it recovered after thawing, the detection value of BoNT / A bioactivity was significantly different from that of the freshly prepared sample. The present invention uses 5%-20% glycerol as a protective agent, and flash freezing in liquid nitrogen after packaging, which is conducive to the stability of the target protein, among which 10% glycerol has the best effect.

[0132] The present invention uses different concentrations of BoNT / A to cut the EGFP-SNAP-25 (141-206aa) -DsRED molecular sensor, and then uses 12% SDS-PAGE electrophoresis to detect that BoNT / A can completely cut the EGFP-SNAP-25 (141-206aa) -DsRED molecular sensor (such as Figure 2 shown).

[0133] Example 3 BoNT / A detection based on EGFP-SNAP-25 (141-206aa)-DsRED protein

[0134] 3.1 Biomaterials

[0135] BoNT / A, 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 full black board (Biosharp, product number BS-MP-96B).

[0136] 3.2 Reagents

[0137] DTT (Beijing Solebow Technology Co., Ltd., catalog number D8220); the other reagents were of analytical grade.

[0138] 3.3 Implementation steps

[0139] 3.3.1 Solution preparation

[0140] (1) 10× digestion buffer: 500mM HEPES-NaOH, 50mM NaCl, 1% Tween-20, 100μM ZnCl2, 50mM DTT, pH 7.1.

[0141] (2) Supplement buffer: 50 mM HEPES-NaOH, 0.1% Tween-20, pH 7.1.

[0142] 3.3.2 BoNT / A detection based on EGFP-SNAP-25(141-206aa)-DsRED protein

[0143] (1) BoNT / A reduction: 2 mM DTT and 10 μM ZnCl2 were added to the supplement buffer, and BoNT / A was serially diluted using the supplement buffer. The solution was incubated in a metal bath at 37°C for 1 hr. DTT reduces the disulfide bond between BoNT / A-HC and BoNT / A-LC, thereby releasing BoNT / A-LC to exert its zinc-dependent metalloenzyme activity.

[0144] (2) According to the detection system described in Table 10, the EGFP-SNAP-25-DsRED protein, 5× enzyme digestion buffer, and supplement buffer were mixed in advance, added to a 96-well black plate, and then the reduced BoNT / A was added and incubated in a 37°C constant temperature incubator for 4 hours.

[0145] Table 10 EGFP-SNAP-25 (141-206aa)-DsRed protein and serial dilution BoNT / A action detection system

[0146]

[0147]

[0148] (3) Use a multifunctional microplate reader to excite at 488 nm, measure the emission values ​​of EGFP and FRET at emission wavelengths of 510 nm and 585 nm, respectively, and calculate Em585 / Em510. Take the logarithmic value of the concentration of BoNT / A, and use GraphPad Prism to calculate the statistics, make the Em585 / Em510-Log[BoNT / A](pM) curve, and draw the standard curve (such as Figure 3 shown).

[0149] The results showed that the detection limit of the FRET-based botulinum toxin type A bioactivity detection molecular sensor EGFP-SNAP-25 (141-206aa) -DsRED for BoNT / A was 4 U, and the standard curve was y = -0.1255x + 0.4314, R 2 =0.9931, linear between 7.8U-125U, recovery rate of 96.8%-122.7%, precision within 20%, response to BoNT / A is much higher than BoNT / C, and it is specific for BoNT / A.

[0150] 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 sample of 36U had a detection value of 36.4±6.4U.

[0151] The results of the present invention also show that the molecular sensor is dialyzed into 50mM HEPES-NaOH pH 7.1, 10mM NaCl, and then flash-frozen in liquid nitrogen and stored at -80°C after packaging, which can ensure the stability of the fluorescent protein (CV% <10%). The stability of the substrate protein EGFP-SNAP-25 (141-206aa) -DsRED in the FRET in vitro assay is crucial to the reproducibility of the detection and the credibility of the results, which can be understood by those skilled in the art.

[0152] The beneficial effect of the present invention is that the EGFP / DsRED fluorescence pair used in the FRET-based botulinum toxin type A biological activity detection molecular sensor has excellent spectral separation properties, which minimizes the crosstalk of the donor emission channel on the acceptor emission channel. In actual detection, the CFP and YFP single fluorescent protein controls can be omitted, thereby significantly simplifying the FRET quantitative detection process to meet the requirements of high-throughput detection, and can detect a large number of samples in a short time with high detection sensitivity and good repeatability.

[0153] The fluorescence pair used in the embodiment of the present invention is EGFP / DsRED. The EGFP and DsRed fluorescent proteins in the fluorescence pair have high brightness and emission efficiency. The fluorescence pair of EGFP / DsRED is more suitable for short-time, high-brightness experiments than other green fluorescent protein / red fluorescent protein pairs such as EGFP / mRuby, EGFP / mCherry, EGFP / tDimmer2, etc., and has a high signal-to-noise ratio when used in the FRET-based BoNT / A biological activity detection molecular sensor of the present invention.

[0154] The present invention selects DsRED as the acceptor fluorescent protein for FRET, which breaks through the traditional cognition of DsRed by those skilled in the art, namely, the well-known defect of DsRed: it is a tetramer, tends to form oligomers, and causes the protein fused thereto to form aggregates with it. Therefore, the substrate molecule aggregates are not conducive to being recognized and cut by the BoNT / A metal ion protease. The FRET-based BoNT / A bioactivity detection molecular sensor provided by the present invention uses the EGFP / DsRED fluorescence pair, and unexpectedly, BoNT / A completely cuts the molecular sensor.

[0155] The FRET-based BoNT / A bioactivity detection molecular sensor provided by the present invention is EGFP-SNAP-25(141-206aa)-DsRED. Theoretically, as long as the distance between the donor fluorescent protein and the acceptor fluorescent protein is short enough, FRET will occur. Therefore, DsRED-SNAP-25(141-206aa)-EGFP can also be used as a BoNT / A bioactivity detection molecular sensor, which can be understood by technicians in the same field.

[0156] The present invention uses a FRET-based molecular 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 SNAP-25 (93-202), 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.

[0157] The FRET-based botulinum toxin detection method provided by the present invention can be applied to the detection of the biological activity of the light chain during the production of recombinant BoNT / A and BoNT / A extracted by traditional methods, can also meet the in vitro screening needs of botulinum toxin inhibitors, and can also be used for the diagnosis of BoNT / A botulinum toxin poisoning.

Claims

1. A FRET-based BoNT / A or BoNT / E detection molecular sensor, characterized in that: 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 DsRED.

2. The molecular sensor according to claim 1, characterized in that The linker peptide is 141-206aa of SNAP-25.

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

2.

4. The molecular sensor according to claim 3, characterized in that The molecular sensor consists of EGFP-SNAP-25 (141-206aa)-DsRED, and the sequence is shown in SEQ ID No.

4.

5. The molecular sensor according to claim 4, characterized in that The EGFP-SNAP-25(141-206aa)-DsRED was fused with a His tag.

6. Use of the molecular sensor according to any one of claims 1 to 5 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.

7. The method for preparing a molecular sensor according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) constructing a recombinant plasmid expressing the molecular sensor EGFP-SNAP-25 (141-206aa)-DsRED; (2) Induce expression and purify to obtain EGFP-SNAP-25 (141-206aa)-DsRED fusion protein; (3) The EGFP-SNAP-25 (141-206aa) -DsRED fusion protein was placed at 4°C for at least 72 hours, then quickly frozen in liquid nitrogen using 5%-20% glycerol as a protective agent, and stored in a -80°C refrigerator.

8. A FRET-based BoNT / A or BoNT / E detection kit, characterized in that: The kit contains the molecular sensor according to any one of claims 1 to 5.

9. The method for using the detection kit according to claim 8, characterized in that: The method comprises: (1) Detection system: placing the molecular sensor described in any one of claims 1 to 5 in a sample to be tested; (2) Result analysis: detecting and comparing the FRET signals of the molecular 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.

10. The method of use according to claim 9, characterized in that: The detection system also includes providing DTT and Zn 2+ .

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