Use of a luciferase fusion nanobody in the detection of vomitoxin

Abstract

The application belongs to the technical field of biological genes and particularly relates to application of a luciferase fusion nanobody in detection of vomitoxin. The application constructs a novel nanobody-luciferase fusion protein, which is composed of a vomitoxin nanobody, a flexible connecting peptide and a luciferase reporter protein in sequence. The connecting peptide serves as a flexible "hinge" to spatially separate the nanobody and the luciferase two domains, so as to ensure that they can be independently and correctly folded and do not interfere with the biological activity of each other. The nanobody-luciferase fusion protein can be specifically combined with vomitoxin and is used for rapid and high-sensitivity detection of vomitoxin.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a luciferase-fused nanobody in the detection of vomitoxin. Background Technology

[0002] Vomitoxin, also known as deoxynivalenol, is mainly produced by fungi such as *Fusarium graminearum* and is one of the most common and serious mycotoxins contaminating grains such as wheat, corn, and barley, as well as their products. Vomitoxin exhibits strong cytotoxicity, immunotoxicity, and emetic effects, posing a serious threat to human and animal health. Therefore, establishing rapid, sensitive, and reliable methods for detecting vomitoxin is crucial for ensuring food security and safety.

[0003] Currently, detection methods for vomitoxin are mainly divided into two categories: large-scale instrumental analysis methods and rapid immunoassay methods. Large-scale instrumental analysis methods mainly include high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Their advantages include high accuracy, high sensitivity, and the ability to simultaneously detect multiple toxins. Disadvantages include expensive equipment, complex operation, and difficulty in on-site, real-time detection; they are typically performed only in central laboratories. The other category, rapid immunoassay methods, mainly includes enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic strips. Their advantages include relatively simple operation, fast detection speed, low cost, and certain specificity and sensitivity, making them suitable for preliminary screening of batches of samples. Their disadvantages include limited sensitivity and quantification capabilities; the sensitivity of traditional ELISA methods is usually limited to the μg / kg level, insufficient for trace detection needs, and lower quantitative accuracy compared to instrumental methods. Traditional enzyme-labeled antibodies have large molecular weights, complex coupling processes, and difficulty in ensuring batch-to-batch stability. Antibodies have poor tolerance to high temperatures and extreme pH conditions, and are easily inactivated during transportation, storage, or in complex sample matrices, affecting the reliability of the detection.

[0004] To overcome these shortcomings, researchers have explored a variety of novel detection strategies. Among them, nanobodies and bioluminescent reporter systems have shown unique advantages.

[0005] Nanobodies are variable region fragments derived from heavy chain antibodies of camelids. They are characterized by their small molecular weight, high stability, ease of large-scale, low-cost expression in prokaryotic systems such as *E. coli*, and ease of genetic engineering modification. Bioluminescent reporter systems, exemplified by nanoluciferase, exhibit low background, high signal-to-noise ratio, and sensitivity far exceeding traditional colorimetric methods in the chemiluminescent signals generated by their catalytic substrates. In particular, the NanoLuc-based complementary system restores activity when the fragmented LgBiT and SmBiT subunits are brought close together, providing an ideal platform for homogeneous detection.

[0006] Despite the progress made by nanobodies and luciferase technology, some reference patents have reported cases of fusing nanobodies with luciferase for the detection of SARS-CoV-2 virus (202211649210) or African swine fever virus (CN202410557290.3).

[0007] However, there is still a gap in the innovative integration of these technologies and their application to the direct detection of small molecule toxins, especially vomitoxins.

[0008] Therefore, there is an urgent need to develop a novel fusion protein for the bioluminescent detection of vomitoxin to overcome the shortcomings of existing detection technologies. Summary of the Invention

[0009] The purpose of this invention is to combine the high affinity and high specificity of nanobodies for vomitoxin with the high catalytic efficiency and high signal output of luciferase to establish an unprecedented bioluminescent detection method for vomitoxin with ultra-high sensitivity and ease of operation, so as to overcome many shortcomings of existing detection technologies.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a nanobody-luciferase fusion protein, wherein the nanobody-luciferase fusion protein is composed of a vomitoxin nanobody, a flexible linker peptide, and a luciferase reporter protein connected in sequence. The amino acid sequence of the vomiting toxin nanobody is shown in SEQ ID NO.1; the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.2; and the amino acid sequence of the luciferase reporter protein is shown in SEQ ID NO.3.

[0011] Preferably, the linker peptide is a flexible "hinge" that spatially separates the nanobody and the luciferase domains.

[0012] Preferably, the nanobody-luciferase fusion protein can specifically bind to vomitoxin.

[0013] The present invention also provides a nucleic acid molecule encoding the above-mentioned nanobody-luciferase fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0014] The present invention also provides a kit for detecting vomitoxin, the kit comprising the above-mentioned nanobody-luciferase fusion protein.

[0015] The present invention also provides a recombinant plasmid comprising the above-described nucleic acid molecules.

[0016] The present invention also provides an expression vector containing the above-mentioned nucleic acid molecule, wherein the expression vector is pET-22b.

[0017] The present invention also provides a host cell containing the above-mentioned expression vector, wherein the host cell is an Escherichia coli BL21(DE3) cell.

[0018] The present invention also provides the use of the above-mentioned nanobody-luciferase fusion protein, or the above-mentioned recombinant plasmid, or the above-mentioned expression vector in the preparation of reagents or kits for binding vomitoxin.

[0019] The beneficial effects of this invention are: 1. High sensitivity: The high affinity of the fusion protein to the nanobody and the high catalytic efficiency of luciferase significantly improve the detection sensitivity.

[0020] 2. Rapid detection: Short reaction time, detection can be completed within 30 minutes.

[0021] 3. Easy to operate: No complicated pretreatment is required, suitable for rapid on-site testing.

[0022] 4. Low cost: Fusion proteins can be prepared in large quantities through prokaryotic expression systems, which is low cost.

[0023] 5. High throughput: Applicable to various detection platforms such as microplate, enabling batch sample detection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the fusion protein structure; Figure 2 Image showing the SDS-PAGE detection results; Figure 3 This is a schematic diagram of the principle of the vomitoxin-luciferase assay. Figure 4 This is a standard curve for the immunofluorescence detection of vomitoxin. Detailed Implementation

[0026] This invention provides a novel nanobody-luciferase fusion protein for construction and innovatively applies it to the rapid and highly sensitive detection of vomitoxin.

[0027] The fusion protein of this invention is composed of three key functional elements connected in series in a specific order: vomiting toxin nanobody → flexible linker peptide → luciferase reporter protein.

[0028] First, the vomitoxin nanobody (VHH) was developed by immunizing alpacas with a vomitoxin-carrier protein conjugate (e.g., DON-BSA) to construct a phage-display nanobody library. Then, using vomitoxin as the target molecule, a multi-round "adsorption-elution-amplification" panning process was performed to ultimately obtain a high-affinity, high-specificity anti-vomitoxin nanobody clone. The variable region amino acid sequence of the nanobody is shown in SEQ ID NO.1. Its function is to specifically recognize and bind to free vomitoxin molecules in the sample.

[0029] VHH: MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQ (SEQ ID NO. 1).

[0030] Secondly, the flexible linker of this invention employs a flexible amino acid sequence rich in glycine (Gly) and serine (Ser), such as "(GGGGS)n", where n is typically 2-4. In a preferred embodiment of this invention, the linker sequence used is (GGGGS)3 (as shown in SEQ ID NO.2). Linker: GGGGSGGGGSGGGGS (SEQ ID NO.2).

[0031] Its function is as follows: the linker peptide acts as a flexible "hinge" to spatially separate the two structural domains of the nanobody and the luciferase, ensuring that they can fold independently and correctly without interfering with each other's biological activity.

[0032] Finally, the luciferase reporter protein of this invention is preferably a nano-luciferase. It has advantages such as small molecular weight (approximately 19 kDa), high luminescence intensity, and good stability, which are beneficial for the expression and function of the fusion protein. The amino acid sequence of the nano-luciferase (Nluc) is shown in SEQ ID NO.3.

[0033] Nluc: MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (SEQ ID NO. 3).

[0034] Its function is to generate a high-intensity, stable chemiluminescent signal when it reacts with a substrate (such as furan luciferin) for quantitative detection.

[0035] This invention also provides an encoding amino acid sequence, an expression vector, and a host cell.

[0036] The amino acid sequence encoded by the present invention is based on the amino acid sequence of the above-mentioned fusion protein (VHH-Linker-NanoLuc), using E. coli preferred codons, and is artificially synthesized to encode the fusion protein, the amino acid sequence of which is shown in SEQ ID NO.4.

[0037] VHH-Linker-NanoLuc:MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQGGGGSGG GGSGGGGSVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (SEQ IDNO.4).

[0038] The expression vector of this invention is obtained by cloning the amino acid sequence shown in SEQ ID NO.4, after codon optimization in E. coli, into the multiple cloning site of a prokaryotic expression vector (such as the pET series). The pET-22b vector is preferred, as it introduces a 6×His tag at the N-terminus or C-terminus of the fusion protein for easier subsequent purification.

[0039] The host cell described in this invention involves transforming the constructed recombinant plasmid into the expression host. The preferred strain is *Escherichia coli* BL21 (DE3), which contains the T7 RNA polymerase gene and is suitable for efficient protein expression driven by the T7 promoter.

[0040] This invention is based on the principle of competitive immunoassay, combined with bioluminescent signals for detection. Detailed steps are as follows: (1) Add the test sample (or standard) and a fixed concentration of fusion protein to a microplate coated with vomitoxin-bovine serum albumin (DON-BSA).

[0041] (2) Free vomitoxin in the sample competitively binds to the nanobody sites on the fusion protein with DON-BSA immobilized on the microplate.

[0042] (3) After incubation and washing, unbound fusion proteins are washed away.

[0043] (4) Add the substrate solution of nano-luciferase.

[0044] (5) Detection of chemiluminescence signal value (RLU). The higher the concentration of vomitoxin in the sample, the less fusion protein binds to the plate, and the lower the luminescence signal produced. By plotting a standard curve, vomitoxin in unknown samples can be accurately quantified.

[0045] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0047] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0048] Example 1: Screening of anti-vomiting toxin nanobodies A nanobody with high affinity for vomitoxin was obtained through screening using immunized alpacas and phage display technology. The amino acid sequence of its variable region (VHH) is shown in SEQ ID NO.1. This sequence was confirmed by gene sequencing.

[0049] Example 2 Construction of fusion protein expression vector 2.1 Gene Synthesis Based on the designed amino acid sequence of the fusion protein (SEQ ID NO.1+SEQ ID NO.2+SEQ ID NO.3), the schematic diagram of the fusion protein structure is shown below. Figure 1 As shown, a professional biotechnology company was commissioned to perform whole-genome synthesis to obtain the DNA fragment shown in SEQ ID NO.4, and then introduced DNA fragments at both ends of the fragment. Bam HI and Not I restriction site.

[0050] 2.2 Enzyme digestion and ligation The synthesized gene and pET22b(+) vector plasmid were respectively used... Bam HI and Not I was subjected to double enzyme digestion. The target fragment and linearized vector were recovered by agarose gel electrophoresis.

[0051] 2.3 Connection Conversion The recovered gene fragment was ligated to the vector fragment using T4 DNA ligase to construct the recombinant plasmid pET22b-VHH-Nluc. The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing kanamycin (50 μg / mL), and incubated overnight at 37°C.

[0052] 2.4 Identification of positive clones Single colonies were picked for amplification by shaking, and plasmids were extracted for double enzyme digestion verification and DNA sequencing to confirm that the sequences were completely correct.

[0053] Example 3 Expression and purification of fusion protein 3.1 Induced Expression The verified recombinant plasmid was transformed into the expression host *Escherichia coli* BL21(DE3). Single colonies were picked and inoculated into LB broth containing kanamycin and cultured at 37°C with shaking at 220 rpm until OD500 was reached. 600 The concentration was approximately 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and expression was induced at 25°C for 16 hours.

[0054] 3.2 Protein purification Collect bacterial cells by centrifugation, resuspend in lysis buffer, and sonicate. Centrifuge to collect the supernatant, and load the supernatant onto a pre-equilibrated Ni-NTA agarose affinity chromatography column. Wash with wash buffer containing 20 mM imidazole to remove non-specifically bound proteins. Elute the target fusion protein with elution buffer containing 300 mM imidazole. Dialyze the eluted protein into PBS buffer (pH 7.4) to remove imidazole.

[0055] 3.3 Protein Identification The purified product was analyzed by SDS-PAGE, and a single, clear band was visible at a molecular weight of approximately 40 kDa. Figure 2 As shown, the results are consistent with theoretical calculations. Protein concentration was determined by the BCA method, and the protein was aliquoted and stored at -80°C for later use.

[0056] Example 4: Vomitoxin Detection Method and Performance Evaluation Based on Fusion Protein 4.1 The testing process is as follows: Figure 3 As shown, the specific testing steps are as follows: (1) Coating: Coat a white opaque 96-well plate with DON-BSA conjugate (1 μg / mL, diluted with carbonate buffer), 100 μL / well, overnight at 4°C.

[0057] (2) Blocking: Discard the coating solution, add 300 μL of PBS solution containing 3% BSA to each well, and block at 37°C for 2 hours.

[0058] (3) Sample addition and competition: Mix the vomitoxin standard (concentration gradient of 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 ng / mL) or the extract of the sample to be tested with an equal volume of fusion protein solution of a certain working concentration, add it to the washed microplate, 100 μL / well, and incubate at 37°C in the dark for 30 minutes.

[0059] (4) Washing: Wash the microplate 5 times with PBST buffer and pat dry.

[0060] (5) Signal detection: Add 100 μL of freshly prepared nano-luciferase substrate solution to each well immediately, and use a chemiluminescence detector to read the luminescence value (RLU) of each well.

[0061] 4.2 Performance Evaluation Results (1) Standard curve and sensitivity: A standard curve was plotted with the logarithm of the concentration of the vomitoxin standard as the abscissa and the fluorescence inhibition rate (B / B0%) as the ordinate. The results are as follows: Figure 4 As shown.

[0062] The calculated half-maximal inhibitory concentration (IC50) of this method is 0.47 ng / mL, and the linear detection range is 0.10–1.87 ng / mL.

[0063] (2) Specificity: Cross-reactivity experiments were conducted using structurally similar fungal toxins (such as T-2 toxin, zearalenone, and fumonisin B1). The results showed that the cross-reactivity rates were all below 30%, indicating that the fusion protein of the present invention has extremely high specificity for vomitoxin.

[0064] (3) Accuracy and precision: Spiking recovery experiments with high, medium and low concentrations were conducted on blank wheat samples. The average recovery rate was between 92% and 106%, and the intra-batch and inter-batch coefficients of variation were both less than 10%, indicating that the method is accurate and reliable.

[0065] Example 5 Assembly of the detection kit The fusion protein and other necessary components of the present invention are assembled into a kit, the contents of which include: (1) One white 96-well microplate coated with DON-BSA (removable).

[0066] (2) Lyophilized or liquid luciferase fusion protein (1 bottle).

[0067] (3) Vomitoxin series standard solutions (0, 0.05, 0.1, 0.5, 2.5, 10 ng / mL, 1 bottle each).

[0068] (4) Concentrated detergent (20×, 1 bottle).

[0069] (5) Nano-luciferase substrate (lyophilized powder or concentrate, 1 bottle / set).

[0070] (6) Sample extraction solution / diluent (1 bottle).

[0071] (7) Instruction manual (1 copy).

[0072] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nanobody-luciferase fusion protein, characterized in that, The nanobody-luciferase fusion protein is composed of a vomiting toxin nanobody, a flexible linker peptide, and a luciferase reporter protein connected in sequence. The amino acid sequence of the vomiting toxin nanobody is shown in SEQ ID NO.1; the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.2; and the amino acid sequence of the luciferase reporter protein is shown in SEQ ID NO.

3.

2. The nanobody-luciferase fusion protein according to claim 1, characterized in that, The linker peptide is a flexible "hinge" that spatially separates the nanobody and luciferase domains.

3. The nanobody-luciferase fusion protein according to claim 1, characterized in that, The nanobody-luciferase fusion protein can specifically bind to vomitoxin.

4. A nucleic acid molecule encoding the nanobody-luciferase fusion protein of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

5. A kit for detecting vomitoxin, characterized in that, The kit includes the nanobody-luciferase fusion protein of claim 1.

6. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleic acid molecule of claim 4.

7. An expression vector containing the nucleic acid molecule of claim 4, characterized in that, The expression vector is pET-22b.

8. A host cell containing the expression vector of claim 7, characterized in that, The host cell is Escherichia coli BL21(DE3) cell.

9. The use of the nanobody-luciferase fusion protein of claim 1, the recombinant plasmid of claim 6, or the expression vector of claim 7 in the preparation of reagents or kits for binding vomitoxin.

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