DlucCage biosensor for detecting fumonisin B1 as well as preparation method and application of dlucCage biosensor

Abstract

The invention relates to a biosensor for detecting fumonisin B1 as well as a preparation method and application of the biosensor. The biosensor comprises dlucCage protein and lucKey protein, the amino acid sequence of the dlucCage protein is as shown in SEQ ID NO: 1, or the dlucCage protein is a polypeptide with one, two or three amino acids mutated into serine on the sequence SEQ ID NO: 1; the amino acid sequence of the lucKey protein is as shown in SEQ ID NO: 2, or the lucKey protein is a polypeptide with one, two or three amino acids mutated on the sequence SEQ ID NO: 2. The biosensor creatively realizes a good synergistic effect of an antigen mimic epitope and a dlucCage protein allosteric function, the connecting peptide designed at the head and the tail of a molecule in an extending manner can be stably connected with a target binding motif with a larger size, the problem of space limitation of a bolt structural domain in a traditional lucCage protein on target binding motif grafting is effectively broken through, and the biosensor has a good application prospect. The method has the advantages of high sensitivity, strong specificity, rapid signal reading and the like, and has a good application prospect in the field of food safety detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the detection of harmful substances, specifically to a dlucCage biosensor for detecting fumonisin B1, its preparation method, and its application. Background Technology

[0002] Fumonisins (FBs) are a class of mycotoxins mainly produced by Fusarium moniliformes, primarily contaminating grain crops such as corn. Fumonisins are significantly toxic to animal and human health, causing pulmonary edema in pigs, leukomalacia in horses, diarrhea in poultry, liver and kidney damage in various animals, and are associated with esophageal cancer and gastrointestinal diseases in humans. Fumonisin B1 (FB1) is the most prevalent and toxic analogue, accounting for over 60% of fumonisin contamination. FB1 exhibits neurotoxicity, teratogenicity, genotoxicity, carcinogenicity, hepatotoxicity, immunosuppression, and reproductive toxicity. Therefore, establishing a fumonisin B1 detection technology applicable to the entire food supply chain and capable of real-time analysis is of great significance for ensuring food safety.

[0003] Traditional methods for detecting fumonisin B1 include rapid screening and quantitative methods. Rapid screening methods, including the most widely used enzyme-linked immunosorbent assay (ELISA), suffer from cross-reactivity, potentially reacting with other structural analogs (such as FB2 and FB3), and slightly lower specificity. They are also susceptible to matrix interference, requiring precise sample pretreatment. Immunochromatographic strip methods are prone to false positives or false negatives. Quantitative methods primarily include high-performance liquid chromatography (HPLC) and the currently recognized most authoritative confirmatory method, liquid chromatography-mass spectrometry (LC-MS / MS). LC-MS / MS offers ultra-high sensitivity and simultaneous detection of multiple toxins, but the equipment is very expensive, with high operating and maintenance costs; operation is complex, requiring highly skilled technicians; and sample pretreatment requirements are extremely high.

[0004] Allosteric proteins are a class of proteins whose biological activity is regulated by the non-covalent binding of specific molecules (called effectors or regulators), resulting in changes in the protein's three-dimensional conformation. This regulation typically occurs at a different site on the protein than its active site, i.e., the allosteric site. Based on the results of effector binding, they can be classified as allosteric activation (positive co-efficacy), where effector binding enhances protein activity, and allosteric inhibition (negative co-efficacy), where effector binding reduces protein activity. Utilizing allosteric proteins as biosensing elements combines the specificity of detection targets (antigens / antibodies) with the conformational switching properties of allosteric proteins. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a dlucCage biosensor for detecting fumonisin B1 that is convenient to use, highly sensitive, and has good specificity, and its application.

[0006] A first aspect of the present invention is to provide a dlucCage biosensor for detecting fumonisin B1, comprising dlucCage protein and lucKey protein, wherein the amino acid sequence of the dlucCage protein is as shown in SEQ ID NO:1, or is a polypeptide having one, two, or three amino acids mutated in the sequence SEQ ID NO:1.

[0007] The amino acid sequence of the lucKey protein is shown in SEQ ID NO:2, or it is a polypeptide with one, two, or three amino acid mutations in the sequence SEQ ID NO:2.

[0008] In some implementations, the amino acid is mutated to serine.

[0009] In some embodiments, the dlucCage protein contains a polypeptide with mutated amino acids comprising at least one of the following: amino acid mutated to serine at position 366, amino acid mutated to serine at position 380, and amino acid mutated to serine at position 387, counting from the N-terminus.

[0010] In some embodiments, the lucKey protein contains a polypeptide with mutated amino acids comprising at least one of the following: amino acid mutated to serine at position 220, amino acid mutated to serine at position 230, or amino acid mutated to serine at position 238, starting from the N-terminus.

[0011] In some preferred embodiments, the amino acid sequence of the dlucCage protein is as shown in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8.

[0012] In some preferred embodiments, the amino acid sequence of the dlucCage protein is shown in SEQ ID NO:6; or the nucleotide sequence of the gene encoding the dlucCage protein is shown in SEQ ID NO:7.

[0013] In some preferred embodiments, the amino acid sequence of the lucKey protein is as shown in SEQ ID NO:9; or the nucleotide sequence of the gene encoding the lucKey protein is as shown in SEQ ID NO:10.

[0014] A second aspect of the present invention is to provide a recombinant expression vector or genetically engineered bacterium for expressing the dlucCage protein in a biosensor for detecting fumonisin B1, wherein the recombinant expression vector or genetically engineered bacterium is inserted with the coding gene for the dlucCage protein in any of the above-mentioned biosensors; and / or a recombinant expression vector or genetically engineered bacterium for expressing the lucKey protein, wherein the recombinant expression vector or genetically engineered bacterium is inserted with the coding gene for the lucKey protein in any of the above-mentioned biosensors.

[0015] A third aspect of the present invention provides a method for preparing the biosensor, the method comprising the following steps:

[0016] S1. Construct recombinant expression vectors for dlucCage and lucKey proteins, respectively;

[0017] S2. The two recombinant expression vectors were transformed into different genetically engineered bacteria to obtain two recombinant engineered bacteria expressing two proteins;

[0018] S3. Induce the recombinant engineered bacteria to express dlucCage protein, and purify the dlucCage protein;

[0019] S4. Induce the recombinant engineered bacteria to express lucKey protein, collect inclusion bodies, denature, renature and purify the inclusion bodies to obtain biologically active lucKey protein.

[0020] In some embodiments, step S4, denaturing and refolding the inclusion bodies includes: repeatedly washing the inclusion bodies 3-5 times with washing buffer until the supernatant is clear; dissolving the inclusion bodies with denaturing lysis buffer and collecting the supernatant by centrifugation; purifying the supernatant and then refolding it by staged gradient dialysis; the staged gradient dialysis refolding includes dialysis in refolding buffer I for 12-16 hours, followed by dialysis in refolding buffer II for 12-16 hours; preferably, refolding buffer I contains: pH 8.5, 20 mM Tris-HCl, 1 mM EDTA, 50 mM urea, 2.2 mM reduced glutathione, and 5% glycerol by volume; and / or refolding buffer II contains: pH 8.5, 20 mM Tris-HCl, 1 mM EDTA, and 50 mM urea.

[0021] A third aspect of the present invention provides a method for detecting fumonisin B1, using any of the above-described dlucCage biosensors, comprising the following steps:

[0022] S1. Add incubation buffer to the reaction wells;

[0023] S2. Add the dlucCage protein and fumonisin B1 antibody from the above biosensor and mix well;

[0024] S3. Add the fumonisin B1 antibody, mix well, and the molar ratio of the dlucCage protein to the lucKey protein is 1:(1-3);

[0025] S4. Add the sample to be tested, mix well, and incubate;

[0026] S5. Add furimazine substrate and measure the luminescence intensity.

[0027] In some implementations, in step S4, the incubation is carried out at 36°C-38°C for 50-70 minutes.

[0028] In some embodiments, the molar ratio of the dlucCage protein to the lucKey protein is 1:(1-2), more preferably 1:1.

[0029] In some embodiments, the incubation buffer comprises: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA, pH 7.4.

[0030] In some embodiments, the gene encoding the dlucCage protein is inserted between the NdeⅠ and XhoⅠ restriction sites of the pET-21b(+) vector to construct a recombinant expression vector expressing the dlucCage protein.

[0031] In some embodiments, the gene encoding the lucKey protein is inserted between the NdeⅠ and XhoⅠ restriction sites of the pET-21b(+) vector to construct a recombinant expression vector expressing the lucKey protein.

[0032] In some embodiments, the recombinant expression vector may be an Escherichia coli (E. coli) expression vector, including the pET series, such as pET-21b(+).

[0033] In some embodiments, the genetically engineered bacteria is Escherichia coli, such as the Lemo21(DE3) strain.

[0034] The dlucCageFB1 protein-based biosensor described in this invention has the advantage of significantly broadening target applicability. This biosensor can stably connect to larger target-binding motifs by using a linker designed to extend the ends of the molecule, effectively overcoming the spatial limitation of the gate plug domain in traditional lucCage proteins on the grafting of target-binding motifs.

[0035] The dlucCage biosensor of this invention comprises an optimized dlucCage (dlucCageFB1_S) and the lucKey protein. The N-terminus of the enveloping domain and the C-terminus of the latching domain in dlucCage are each linked by a specific linker peptide to a mimic epitope polypeptide of fumonisin B1 antibody. The latching domain also integrates a luciferase SmBit fragment. This invention utilizes the bivalent binding characteristic of the fumonisin B1 antibody to regulate sensor conformational changes: when the target analyte fumonisin B1 is present, it competitively blocks the binding of the antibody to the mimic epitope, releasing the latching domain and allowing the lucKey protein to trigger a complementary luciferase reaction through competitive binding, generating a signal output. This sensor innovatively achieves the synergistic effect of the antigen mimic epitope and the allosteric function of the dlucCage protein, possessing advantages such as simple preparation, low cost, high sensitivity, strong specificity, and rapid signal readout, showing promising application prospects in the field of food safety detection. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the detection principle of the biosensor of the present invention.

[0037] Figure 2 This is an SDS-PAGE gel image of dlucCage protein (A) and lucKey protein (B) in the biosensor of the present invention.

[0038] Figure 3 The image shows the sensor's response to different concentrations of fumonisin B1 antibody when the dlucCage:lucKey concentration ratio is 50 nM:50 nM.

[0039] Figure 4A A schematic diagram of a biosensor for dlucCage proteins with different linker lengths.

[0040] Figure 4B The image shows the detection response of dlucCage with different linker lengths.

[0041] Figure 4C The graph shows the response of dlucCageFB1_S:lucKey protein at different ratios.

[0042] Figure 5 The graph shows the response of mutants of dlucCageFB1_S and lucKey proteins with different affinities to fumonisin B1 antibody.

[0043] Figure 6 This is a standard curve established based on the inhibitory effect of fumonisin B1 standard when dlucCage:lucKey is 50 nM:50 nM.

[0044] Figure 7 This is a specific detection diagram of the biosensor described in Embodiment 6 of the present invention. Detailed Implementation

[0045] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0046] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or under conditions recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0047] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0048] See Figure 1 Some embodiments of the present invention relate to a dlucCage biosensor for detecting fumonisin B1, comprising dlucCage protein and lucKey protein, wherein the amino acid sequence of the dlucCage protein is as shown in SEQ ID NO:1, or is a polypeptide having one, two, or three amino acid mutations in the sequence SEQ ID NO:1, and the amino acid sequence of the lucKey protein is as shown in SEQ ID NO:2, or is a polypeptide having one, two, or three amino acid mutations in the sequence SEQ ID NO:2.

[0049] The dlucCage protein includes a shroud domain and a latching domain. The shroud domain contains a shroud domain body and a fumonisin B1 polypeptide mimic epitope. The shroud domain body and the fumonisin B1 polypeptide mimic epitope are linked by a specific linker. The fumonisin B1 polypeptide mimic epitope is located at the N-terminus of the shroud domain and the C-terminus of the latching domain. The latching domain also integrates a luciferase SmBit fragment. The lucKey protein contains a polypeptide that strongly binds to the shroud domain of the dlucCage protein and a luciferase LgBit fragment. This invention utilizes the bivalent binding characteristic of the fumonisin B1 antibody to regulate the conformational change of the biosensor: when the target fumonisin B1 is present, it competitively blocks the binding of the antibody to the mimic epitope, releasing the latching domain and allowing the lucKey protein to trigger a luciferase complementary reaction through competitive binding, generating a signal output.

[0050] In some embodiments of the present invention, a method for preparing the dlucCage protein in the biosensor is also involved, comprising:

[0051] (a) The nucleotide sequence shown in SEQ ID NO:11 was inserted between the NdeⅠ and XhoⅠ restriction sites of the pET-21b(+) vector to construct a recombinant expression vector;

[0052] (b) The recombinant expression vector obtained in step (a) was transformed into Escherichia coli Lemo21(DE3) strain to obtain recombinant engineered bacteria;

[0053] (c) Inducing the recombinant engineered bacteria to express the dlucCage protein;

[0054] (d) Purify to obtain the dlucCage protein.

[0055] In some further embodiments, steps (c) and (d) include: inoculating positive recombinant engineered bacteria into LB liquid medium containing ampicillin and culturing at 37°C to the logarithmic growth phase; adding IPTG to a final concentration of 0.4 mM and inducing culture at 18°C ​​for 16 hours; collecting bacterial cells by centrifugation and resuspending them in lysis buffer; disrupting cells by sonication and collecting the supernatant by centrifugation; loading the supernatant onto a equilibrated nickel affinity chromatography column and performing washing and elution operations sequentially; collecting and concentrating the eluent and storing the purified protein at -80°C.

[0056] In some embodiments, the lysis buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, 1 mM PMSF, and 0.02 mg / ml DNase.

[0057] In some embodiments, the washing buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 30 mM imidazole.

[0058] In some embodiments, the elution buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 300 mM imidazole.

[0059] Some embodiments of the present invention also relate to a method for preparing the lucKey protein in a biosensor, including:

[0060] (a) The nucleotide sequence shown in SEQ ID NO:12 was inserted between the NdeⅠ and XhoⅠ restriction sites of the pET-21b(+) vector to construct a recombinant expression vector;

[0061] (b) The recombinant expression vector obtained in step (a) was transformed into Escherichia coli Lemo21(DE3) strain to obtain recombinant engineered bacteria;

[0062] (c) Induce the recombinant engineered bacteria to express the lucKey protein and collect inclusion bodies;

[0063] (d) The inclusion bodies are denatured, renatured and purified to obtain active lucKey protein.

[0064] In some of these embodiments, steps (c) and (d) specifically include:

[0065] Positive recombinant engineered bacteria were inoculated into LB liquid medium containing ampicillin and cultured at 37°C to the logarithmic growth phase. IPTG was added to a final concentration of 0.4 mM, and the culture was induced at 18°C ​​for 16 hours. The bacterial cells were collected by centrifugation and resuspended in lysis buffer. The cells were sonicated and the precipitate was collected by centrifugation as inclusion bodies. The inclusion bodies were washed 3–5 times with washing buffer until the supernatant was clear. The inclusion bodies were dissolved in denaturing lysis buffer, and the supernatant was collected by centrifugation. The supernatant was purified using a nickel affinity chromatography column, undergoing equilibration, loading, washing, and elution. The eluent containing lucKey protein was collected and refolded by staged gradient dialysis. The refolded protein was concentrated and stored at -80°C.

[0066] In some embodiments, the lysis buffer comprises: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, 1 mM PMSF and 0.02 mg / ml DNase.

[0067] In some further embodiments, the washing buffer comprises: 20 mM Tris-HCl (pH 8.0), 0.5% (v / v) Triton X-100, 1 mM EDTA and 0.5 M NaCl.

[0068] In some embodiments, the denaturing lysis buffer comprises: 8M urea, 20mM Tris-HCl (pH 8.0), 2mM imidazole and 3mM DTT.

[0069] In some further embodiments, the equilibration buffer comprises: 8M urea, 20mM Tris-HCl (pH 8.0), 2mM imidazole and 1mM 2-mercaptoethanol.

[0070] In some further embodiments, the elution buffer comprises: 8M urea, 20mM Tris-HCl (pH 8.0), 500mM imidazole and 1mM 2-mercaptoethanol.

[0071] In some embodiments, the staged gradient dialysis includes:

[0072] Dialyze in refolding buffer I for 12–16 hours, wherein the refolding buffer I contains: 20 mM Tris-HCl (pH 8.5), 1 mM EDTA, 50 mM urea, 2.2 mM reduced glutathione and 5% (v / v) glycerol;

[0073] Dialyze in refolding buffer II for 12–16 hours, wherein the refolding buffer II contains 20 mM Tris-HCl (pH 8.5), 1 mM EDTA and 50 mM urea.

[0074] The fumonisin B1 monoclonal antibody described in the examples can be obtained by the following preparation method:

[0075] 1. Animal immunization

[0076] Preparation of fumonisin B1-BSA: 1.1 mg BSA and 0.15 mg FB1 (FB1 standard, catalog number: Sigma, F1147-1MG, specification: 1 mg) were dissolved in 0.45 mL of 0.01 mol / L PBS (pH 7.4). 0.45 mL of 0.5% glutaraldehyde solution was added dropwise, and the mixture was stirred at room temperature for 2 h. 0.1 mL of glycine solution (1 mol / L, pH 7.0) was added, and stirring was continued for 10 min. The mixture was dialyzed against PBS at 4 °C. Three 6-8 week old female Balb / c mice were selected. Fumonisin B1-BSA was used as the immunogen, and an equal volume of Freund's adjuvant was mixed. The mixture was vortexed until the immunogen was completely emulsified, and the mice were immunized by multiple subcutaneous injections in the abdomen. For the initial immunization, Freund's complete adjuvant was used, with an immunogen dose of 120 μg / mouse. Subsequent immunizations used Freund's incomplete adjuvant, with the immunogen dose decreasing each time, immunized every 3 weeks for a total of 3 immunizations. One week after the second immunization, tail blood was collected from mice, and the serum antibody titer and inhibition rate in the tail blood were determined by indirect ELISA. Three days before fusion, the mice with the highest titer were directly immunized with the immunogen at a dose of 40 μg / mouse.

[0077] 2. Preparation and screening of hybridoma cells

[0078] Selected immunized mice were euthanized by cervical dislocation. The spleens were removed and ground in a clean bench. Mouse myeloma cells SP2 / 0 and spleen cells were mixed at a 1:10 ratio, and preheated 50% PEG was added for cell fusion. The mixture was then added to 96-well cell culture plates containing feeder cells. Cell status was observed under a microscope after fusion. A semi-quantitative medium change was performed on day 5 post-fusion, and culture continued until day 7, after which the entire medium was replaced.

[0079] Approximately 7–10 days after cell fusion, the cell supernatant from the cell culture plate was aspirated, and the amount of secreted antibodies in the supernatant was determined using an indirect ELISA method. Positive cells were screened in the wells, and subcloning was performed using a limiting dilution method. Fumonisin B1-OVA was coated in the wells; PBS was used as the blank control; culture medium was used as the negative control; and serum from the ocular blood of immunized mice was used as the positive control. When the selected monoclonal cells showed a 100% positivity rate in a 96-well cell culture plate, they were considered positive monoclonal cells and were cryopreserved and expanded for further culture as appropriate.

[0080] 3. Preparation and purification of monoclonal antibody ascites fluid

[0081] Eight-week-old Balb / c mice were pre-stimulated with liquid paraffin one week in advance to promote the secretion and accumulation of nutrients in the peritoneal cavity. The resulting hybridoma cell line was expanded to the required number, centrifuged, carefully washed, resuspended in sterile 75% physiological saline, and injected into the mouse abdomen via intraperitoneal injection. After approximately one week, when the mouse abdomen showed significant distension, ascites fluid was collected, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected as the monoclonal antibody ascites fluid.

[0082] The collected ascites fluid was purified using a Protein G affinity chromatography column. Before loading, the ascites fluid was filtered through a microporous membrane and used as the loading solution. The Protein G column was equilibrated with binding buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.4). The filtered ascites fluid was then loaded onto the chromatography column. After equilibration, elution was performed using elution buffer (0.1M citric acid, pH 2.5–3.0), and the antibody-rich eluent was collected. The elution buffer is acidic, and the pH should be adjusted to neutral immediately using a neutralization buffer (1 M Tris-HCl, pH 9.0) to obtain fumonisin B1 monoclonal antibody (for the specific sequence, see: Cai C, Xia Y, Guo Y, et al. Biosynthetic small molecule antigens mimics medicated lateral flow immunoassay for cotoxin fumonis in B1 using nanobody fusion proteins [J]. Journal of Hazardous Materials, 2025, 487: 137194).

[0083] In the following sequence: Bold: Linker; Italic: FB1 analog epitope; Underline: SmBit dlucCageFB1_S amino acid sequence: SEQ ID NO:1

[0084] dlucCageFB1_S nucleotide sequence: SEQ ID NO:2

[0085]

[0086] Linker peptide SEQ ID NO:3 in the amino acid sequence of dlucCageFB1_M

[0087] Linking peptide in the amino acid sequence of GGSAEAAAKEAAAKEAGGSGGSGGS dlucCageFB1_L: SEQ ID NO:4

[0088] GGSGGSAEAAAKEAAAKEAAAKEAGGSGGSGGS dlucCageFB1_S_L1: Amino acid sequence: SEQ ID NO:5

[0089] dlucCageFB1_S_L2 amino acid sequence: SEQ ID NO:6

[0090] dlucCageFB1_S_L2 nucleotide sequence: SEQ ID NO:7

[0091]

[0092] dlucCageFB1_S_L3 amino acid sequence: SEQ ID NO:8

[0093] MGSHHHHHHGSGSENLYFQGNNAAMYSEMATDGGGGSGGGGSSKEAAKKLQDLNIELAR

[0094] KLLEASTKLQRLNIRLAEALLEAIARLQELNLELVYLAVELTDPKRIRDEIKEVKDKSKEIIRR

[0095] AEKEIDDAAKESKKILEEARKAIRDAAEESRKILEEGSGSGSDALDELQKLNLELAKLLLKA

[0096] IAETQDLNLRAAKAFLEAAAKLQELNIRAVELLVKLTDPATIRRALEHAKRRSKEIIDEAERA

[0097] IRAAKRESERIIEEARRLIEKAKEESERIIREGSGSGDPDIKKLQDLNIELARELLRAHAQLQR [[ID=​​​​​​​​​​​​​​​​​GGSGGGSGGSDEARKAIARVKRESKRIVEDAERLIREAAAASEKISREAERLIREAAAASEKISRE*

[0104] lucKey nucleotide sequence: SEQ ID NO:10

[0105] 5’-atgggtagccatcatcatcatcaccacggtagcggtagtgaaaatctgtattttcagggcagcggcatggtgtttaccctggaagattttgtgg gtgactgggaacagaccgcagcatataatctggatcaggtgctggaacagggtggcgttagtagcctgctgcagaatctggccgttagtgtgaccccgattcagcgtattgtgcgcagcggcgaaaatgccctgaaaattgatattcatatcatcccgtatgagggtctgagcgcagatcagatggcccagattgaagaagtttttaaagtggtttacccggtggatgatcatcattttaaagttattctgccgtatggtacactggttattgatggcgttaccccgaatatgctgaattattttggccgcccgtatgaaggcattgccgtttttgatggtaaaaagattaccgtgaccggtacactgtggaatggtaataagattattgatgaacgcctgattaccccggatggtagcatgctgtttcgcgttaccattaatagtggcggcagcggcggcggtggtagtggtggtggtagcggcggtagtgatgaagcacgcaaagccattgcacgtgtgaaacgcgaaagtaaacgcattgttgaagatgccgaacgcctgatccgcgaagcagcagccgccagtgaaaaaattagccgcgaagccgaacgcttaattcgtgaagcagcagcggcaagcgaaaaaattagtcgtgaataa-3’ lucKey_k1 amino acid sequence: SEQ ID NO:11

[0106] MGSHHHHHHGSGSENLYFQGSGMVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLA

[0107] VSVTPIQRIVRSGENALKIDIHIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVID

[0108] GVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSGGSGG

[0109] GGSGGGSGGSDEARKAIARVKRESKRIVEDAERLSREAAAASEKISREAERLIREAAAASEKISRE*

[0110] lucKey_k2 amino acid sequence: SEQ ID NO:12

[0111] ​​​​​​​​​​​​​​​​​​​​​GVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSGGSGG

[0119] GGSGGGSGGSDEARKAIARVKRESKRIVEDAERLSREAAAASEKSSREAERLSREAAAASE

[0120] KISRE*

[0121] '

[0122] The present invention will be further described in detail below with reference to specific embodiments.

[0123] Example 1: Prokaryotic expression and purification of dlucCage protein

[0124] The prokaryotic expression and purification of dlucCage protein includes the following steps:

[0125] The genes encoding the target proteins (dlucCageFB1_S / dlucCageFB1_M / dlucCageFB1_L / dlucCageFB1_S_L1 / dlucCageFB1_S_L2 / dlucCageFB1_S_L3) were cloned into the NdeⅠ and XhoⅠ restriction sites of the pET-21b(+) vector to construct recombinant expression plasmids. The recombinant plasmids were transformed into *E. coli* Lemo21(DE3) competent cells, plated on LB agar plates containing ampicillin, and positive clones were screened.

[0126] Single colonies were picked and inoculated into 5 ml of LB broth containing ampicillin, and cultured at 37°C with shaking for 8 hours. The culture was then transferred at a 1:50 ratio to 100 ml of LB broth containing ampicillin, and cultured at 37°C with shaking for 4 hours. IPTG was added to a final concentration of 0.4 mM, and the cells were induced at 18°C ​​for 16 hours. The cells were collected by centrifugation at 4000 g for 15 minutes at 4°C. The cells were resuspended in 30 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole, 1 mM MPMSF, 0.02 mg / ml DNase). Cells were lysed using an ultrasonic homogenizer under ice bath conditions (5 seconds on, 5 seconds off, total 20 minutes). The lysate was centrifuged at 12000 rpm for 20 minutes at 4°C, and the supernatant was collected.

[0127] The supernatant was loaded onto a 1 mL HisTrap nickel affinity chromatography column pre-equilibrated with wash buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole). The column was washed twice with 10 column volumes (CV) of wash buffer. The target protein was eluted with 8 CV of elution buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 300 mM imidazole). The eluent was verified by SDS-PAGE gel electrophoresis (results are shown in the figure). Figure 2 As shown in Figure A), the eluent with the correct bands was concentrated using an Ultra-15 centrifugal filter, aliquoted, and stored at -80°C.

[0128] Example 2: Preparation of lucKey protein

[0129] The preparation method of lucKey protein includes the following steps:

[0130] The genes encoding the target proteins (lucKey / lucKey_k1 / lucKey_k2 / lucKey_k3) were cloned into the NdeⅠ and XhoⅠ restriction sites of the pET-21b(+) vector to construct recombinant expression plasmids. The recombinant plasmids were then transformed into *E. coli*.

[0131] Lemo21(DE3) competent cells were plated on LB agar plates containing ampicillin, and positive clones were selected.

[0132] Single colonies were picked and inoculated into 5 ml of LB broth containing ampicillin, and cultured at 37°C with shaking for 8 hours. The culture was then transferred at a 1:50 ratio to 100 ml of LB broth containing ampicillin, and cultured at 37°C with shaking for 4 hours. IPTG was added to a final concentration of 0.4 mM, and the cells were induced at 18°C ​​for 16 hours. The cells were collected by centrifugation at 4000g for 15 minutes at 4°C. The cells were resuspended in 30 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole, 1 mM MPMSF, 0.02 mg / ml DNase). The cells were then disrupted using an ultrasonic disruptor under ice bath conditions (5 seconds on, 5 seconds off, total duration 20 minutes). After disruption, the solution was centrifuged at 12,000 rpm for 20 minutes at 4°C. The precipitate was collected and washed 3–5 times with washing buffer (20 mM Tris-HCl (pH 8.0), 0.5% (v / v) Triton X-100, 1 mM EDTA, and 0.5 M NaCl) until the supernatant was clear. The inclusion bodies were dissolved with denaturing lysis buffer (8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole, and 3 mM DTT), and the supernatant was collected by centrifugation. The supernatant was purified by nickel affinity chromatography, equilibrated with equilibration buffer (8 M urea, 20 mM Tris-HCl (pH 8.0), 2 mM imidazole, and 1 mM 2-mercaptoethanol), then loaded, washed, and eluted with elution buffer (8 M urea, 20 mM Tris-HCl (pH 8.0), 500 mM imidazole, and 1 mM 2-mercaptoethanol). The eluent containing lucKey protein was collected and then loaded into a container. The dialysate was placed in a dialysis bag and dialyzed in refolding buffer I (20 mM Tris-HCl (pH 8.5), 1 mM EDTA, 50 mM urea, 2.2 mM reduced glutathione, and 5% (v / v) glycerol) for 12–16 hours; then the dialysis bag was placed in refolding buffer II (20 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 50 mM urea) and dialyzed for 12–16 hours. The dialysate was then verified by SDS-PAGE gel electrophoresis (results are shown in Figure 1). Figure 2 As shown in Figure B), the eluent with the correct bands was concentrated using an Ultra-15 centrifugal filter, aliquoted, and stored at -80°C.

[0133] Example 3: Sensor Response Characteristics Test to Fumonisin B1 Antibody

[0134] Dilute dlucCageFB1_S and lucKey proteins to 500 nM. In each well of a white, opaque 96-well plate, pre-fill with 20 μL of incubation buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA, pH 7.4). Then, add 10 μL of dlucCage protein and 10 μL of serially diluted fumonisin B1 antibody solution, mixing thoroughly. Next, add 10 μL of lucKey protein to each well and incubate at 37°C for 60 min. After incubation, add 50 μL of 15 μM furimazine substrate and measure the luminescence intensity. Results are as follows: Figure 3 As shown, the sensor exhibits a concentration-dependent gradient response to fumonisin B1 antibody. Based on this, the sensor architecture was subsequently modified and optimized.

[0135] Example 4: Optimization of the linker length for the dlucCage sensor

[0136] To investigate the effect of linker length on the mimic epitope-antibody binding effect, we designed three linkers of different lengths to link the fumonisin B1 mimic epitope peptide, namely: short... (named dlucCageFB1_S), medium (named dlucCageFB1_M) and long (Named dlucCageFB1_L), meaning that except for the linker peptide sequence, it is identical to the corresponding component of dlucCageFB1_S. For example... Figure 4A As shown. The response of the three linker length designs to the fumonisin B1 antibody was then characterized, with the specific implementation steps being the same as in Example 3. Experimental results showed that only the short linker length design responded to the antibody; the other two did not. Figure 4B As shown. Therefore, the design with a short linker length (dlucCageFB1_S) was chosen as the target for subsequent experimental optimization.

[0137] The ratio of dlucCage sensor elements was also optimized, with the ratio of dlucCageFB1_S:lucKey protein elements set to 50nM:25nM, 50nM:50nM, 50nM:100nM, and 50nM:150nM. Experiments were then conducted following the steps in Example 3. The results showed that the best response signal to the target antibody was observed when the molar ratio of dlucCageFB1_S:lucKey protein elements in the sensor was 1:(1-3), and the highest signal gain was provided when the molar ratio was 1:1. Figure 4C ).

[0138] Example 5: Construction and Screening of Mutants with Reduced Affinity Gradients between dlucCageFB1 and lucKey Proteins To finely regulate the binding affinity between lucKey and dlucCageFB1_S proteins, we performed site-directed mutagenesis on specific hydrophobic amino acid residues involved in the interaction within their key peptide domains, replacing them with more hydrophobic serine residues (Serine, Ser, S). Specifically: In dlucCageFB1_S, starting from the N-terminus, alanine (Alanine, Ala, A), isoleucine (Isoleucine, Ile, I), and leucine (Leucine, Leu, L) at position 366 were mutated to serine (Serine, Ser, S), and the mutant at position 366 (Alanine, Ala, A) was named dlucCageFB1_S_L1; the mutant at position 366 (Alanine, Ala, A) was mutated to serine (Serine, Ser, S). The mutant with Alanine (Ala,A), isoleucine (Ile,I) at position 380 mutated to serine (Ser,S) was named dlucCageFB1_S_L2; the mutant with alanine (Ala,A) at position 366, isoleucine (Ile,I) at position 380, and leucine (Leu,L) at position 387 mutated to serine (Ser,S) was named dlucCageFB1_S_L3. In lucKey, starting from the N-terminus, isoleucine (Ile,I) at positions 220, 230, and 238 is mutated to serine (Ser,S). The mutant with isoleucine (Ile,I) at position 220 is named lucKey_k1; the mutant with isoleucine (Ile,I) at positions 220 and 230 is named lucKey_k2; and the mutant with isoleucine (Ile,I) at positions 220, 230, and 238 is named lucKey_k3. Through cumulative mutations, the dlucCageFB1_S_L1 / L2 / L3 and lucKey_k1 / k2 / k3 series with decreasing affinity were constructed.

[0139] Subsequently, following the experimental steps of Example 3, luminescent response detection was performed on each mutant combination in the presence of the target antibody. The results showed (e.g.) Figure 5 (as shown), Figure 5 In the image, from left to right, the first heatmap shows the background luminescence intensity of different mutant combinations of dlucCageFB1_S and lucKey (each grid represents a combination); the second heatmap shows the luminescence intensity of each combination after the addition of fumonisin B1 antibody; the third heatmap shows the signal enhancement of each combination after the addition of fumonisin B1 antibody, calculated as the luminescence intensity of the +FB1 antibody group / the luminescence intensity of the background group. The combination with the most significant increase in luminescence intensity is marked in red in the image.

[0140] The combined mutant of dlucCageFB1_S_L2 and lucKey showed the most significant change in luminescence intensity and the highest response to the target antibody. Therefore, the dlucCageFB1_S_L2 protein (SEQ ID NO:6) and lucKey protein (SEQ ID NO:9) were ultimately selected as components of the biosensor for the subsequent competitive detection of fumonisin B1.

[0141] Example 6

[0142] (1) Detection of fumonisin B1 samples using the dlucCage biosensor:

[0143] Dilute dlucCageFB1_S_L2 and lucKey proteins to 500 nM. Add 10 μl of incubation buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA, pH 7.4) to each well of a white opaque 96-well plate, then add 10 μl of the diluted dlucCageFB1_S_L2 protein. Dilute fumonisin B1 antibody to 300 nM and add 10 μl / well to each 96-well plate; then add serially diluted fumonisin B1 standard solution (10 μl / well). Finally, add lucKey protein (10 μl / well) and incubate at 37°C for 60 minutes. Add 50 μl of 15 μM furimazine substrate and measure the luminescence intensity. Plot a standard curve with the logarithm of the fumonisin B1 standard concentration on the x-axis and the relative luminescence intensity on the y-axis (e.g., ...). Figure 6 (As shown). The results show that when the sensor dlucCageFB1_S_L2:lucKey is used at a concentration of 50 nM:50 nM, the half-maximum inhibitory concentration (IC50) of fumonisin B1 is [value missing]. 50 The limit of detection (LOD) was 32.24 ng / ml, the limit of detection (LOD) was 7.027 ng / ml, and the linear range was 11.09-52.70 ng / ml.

[0144] (2) Specific detection of the sensor:

[0145] To evaluate the specificity of the biosensor of this invention, the interference of (dlucCageFB1_S_L2 protein and lucKey protein) on common mycotoxins such as ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin B1 (AFB1) at a concentration of 1 μg / ml was tested. Following the steps described above, various interfering toxin solutions (replacing fumonisin B1 standard) were added to the sample to be tested, and the luminescence intensity was detected and recorded.

[0146] The results are as follows Figure 7 As shown, this demonstrates that the sensor of the present invention has high specificity for fumonisin B1, and there is no obvious cross-reactivity with other toxins at higher concentrations.

[0147] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A dlucCage biosensor for detecting fumonisin B1, characterized in that, The biosensor includes dlucCage protein and lucKey protein; wherein... The amino acid sequence of the dlucCage protein is shown in SEQ ID NO:1, or it is a polypeptide with one, two, or three amino acids mutated to serine in the sequence SEQ ID NO:

1. The amino acid sequence of the lucKey protein is shown in SEQ ID NO:2, or it is a polypeptide with one, two, or three amino acid mutations in the sequence SEQ ID NO:

2.

2. The dlucCage biosensor according to claim 1, characterized in that, In the dlucCage protein, the mutated amino acid peptides include those starting from the N-terminus, with amino acid mutated to serine at positions 366, 380, and 387.

3. The dlucCage biosensor according to claim 1, characterized in that, In the lucKey protein, the mutated amino acid peptides include those starting from the N-terminus, with amino acid mutated to serine at positions 220, 230, and 238.

4. The dlucCage biosensor according to claim 2, characterized in that, The amino acid sequence of the dlucCage protein is shown in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8; Preferably, it is SEQ ID NO:6, or the nucleotide sequence of the gene encoding the dlucCage protein as shown in SEQ ID NO:

7.

5. The dlucCage biosensor according to claim 3 or 4, characterized in that, The amino acid sequence of the lucKey protein is as shown in SEQ ID NO:9; or the nucleotide sequence of the gene encoding the lucKey protein is as shown in SEQ ID NO:

10.

6. A recombinant expression vector or genetically engineered bacterium for expressing the dlucCage protein, characterized in that, The recombinant expression vector or genetically engineered bacteria is inserted with the encoding gene of the dlucCage protein in any of the biosensors of claims 1-5; and / or a recombinant expression vector or genetically engineered bacteria for expressing the lucKey protein, wherein the recombinant expression vector or genetically engineered bacteria is inserted with the encoding gene of the lucKey protein in any of the biosensors of claims 1-5.

7. The method for preparing the dlucCage biosensor according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: S1. Construct recombinant expression vectors for dlucCage and lucKey proteins, respectively; S2. The two recombinant expression vectors were transformed into different genetically engineered bacteria to obtain two recombinant engineered bacteria expressing two proteins; S3. Induce the recombinant engineered bacteria to express dlucCage protein, and purify the dlucCage protein; S4. Induce the recombinant engineered bacteria to express lucKey protein, collect inclusion bodies, denature, renature and purify the inclusion bodies to obtain biologically active lucKey protein.

8. The preparation method according to claim 7, characterized in that, In step S4, the denaturation and refolding of the inclusion bodies includes: repeatedly washing the inclusion bodies 3-5 times with washing buffer until the supernatant is clear; dissolving the inclusion bodies with denaturing lysis buffer and collecting the supernatant by centrifugation; purifying the supernatant and then refolding it by staged gradient dialysis; the staged gradient dialysis refolding includes dialysis in refolding buffer I for 12-16 hours, followed by dialysis in refolding buffer II for 12-16 hours; preferably, refolding buffer I contains: pH 8.5, 20mM Tris-HCl, 1mM EDTA, 50mM urea, 2.2mM reduced glutathione, and 5% glycerol by volume; and / or refolding buffer II contains: pH 8.5, 20mM Tris-HCl, 1mM EDTA, and 50mM urea.

9. A method for detecting fumonisin B1, characterized in that, Includes the following steps: S1. Add incubation buffer to the reaction wells; S2. Add the dlucCage protein and fumonisin B1 antibody from any of the biosensors described in claims 1-5, and mix well; S3. Add the lucKey protein to any of the biosensors described in claims 1-5, mix well, and the molar ratio of the dlucCage protein to the lucKey protein is 1:(1-3); S4. Add the sample to be tested, mix well, and incubate; S5. Add furimazine substrate and measure the luminescence intensity.

10. The method for detecting fumonisin B1 according to claim 9, characterized in that, In step S4, incubate at 36℃-38℃ for 50-70 minutes; and / or The molar ratio of the dlucCage protein to the lucKey protein is 1:1; and / or The incubation buffer contains: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 1 mg / ml BSA, pH 7.4.

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