A Nanobody against Staphylococcal Enterotoxin B and Its Application

Abstract

The present invention provides a nanobody against staphylococcal enterotoxin B and its application, belonging to the technical field of nanobody technology. The nanobody against staphylococcal enterotoxin B of the present invention includes H9-VHH and H9-D7-VHH. The nanobody against staphylococcal enterotoxin B (SEB) provided by the present invention carries a Trx tag. A fusion expression plasmid of the SEB nanobody is constructed using the coding gene of the nanobody, realizing the soluble expression of the nanobody in Escherichia coli. Based on this nanobody, the present invention also establishes a time-resolved fluorescence immunochromatographic detection system for SEB. This system is sensitive, fast, accurate, with strong stability and high specificity, and has broad application prospects in the detection of SEB.

Description

Technical Field

[0001] The present invention relates to the technical field of nanobody, and particularly relates to a staphylococcal enterotoxin B nanobody and its application. Background Art

[0002] Staphylococcal enterotoxin B (SEB) is a major virulence factor of Staphylococcus aureus. Among them, the median lethal dose (LD 50 ) of SEB is about 20 ng / kg. Due to its characteristics of being easy to prepare and highly toxic, it is considered a potential biological warfare agent and bioterrorism agent. At the same time, SEB also poses a great threat to human health and food safety. However, in the past few decades, there has been no approved SEB detection reagent clinically, and enzyme-linked immunosorbent assay (ELISA) is commonly used for auxiliary diagnosis. The ELISA method usually takes 4 - 5 hours to complete, which is difficult to meet the need for rapid detection. Moreover, most of the current commercial SEB ELISA detection kits use polyclonal antibodies, with poor specificity. Therefore, it is urgent to prepare high-specificity antibodies against SEB and develop rapid detection methods with strong specificity and high sensitivity.

[0003] Nanobodies are a class of heavy-chain antibodies that are naturally lacking light chains, isolated from camel and shark sera. The molecular weight of nanobodies is only one-tenth of that of traditional complete antibodies (about 15 kDa), and they can act on antigens alone, with complete antigen recognition ability. Compared with traditional antibodies, nanobodies have the characteristics of small size, good stability, strong specificity, high affinity, heat stability, and easy modification. Moreover, they can be stably produced with high quality through in vitro recombinant expression, effectively avoiding the batch-to-batch differences of traditional antibodies, and are particularly suitable for large-scale preparation and standardized production.

[0004] Time resolved fluorescence immunochromatography assay (TRFICA) is a new analytical method based on immunochromatography technology and fluorescence analysis. This technology uses lanthanide rare earth ions as markers, which well avoids the autofluorescence of biological materials and the easy quenching of fluorescent dyes in fluorescence immunoassay. In addition, before signal acquisition, the short-lived fluorescence can be effectively eliminated by setting a delay time, making it more suitable for the detection of SEB in complex matrices. Summary of the Invention

[0005] The object of the present invention is to provide a nanobody against staphylococcal enterotoxin B (SEB). By constructing a fusion expression plasmid of the SEB nanobody, soluble expression of the nanobody in Escherichia coli is achieved. Then, based on this nanobody, a time-resolved fluorescence immunochromatographic detection system for SEB is expected to be established, making the detection system sensitive, rapid, accurate, highly stable and highly specific.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a nanobody against staphylococcal enterotoxin B, comprising H9-VHH and H9-D7-VHH;

[0008] The amino acid sequence of framework region FR1 of the H9-VHH is as shown in SEQ ID NO.1, the amino acid sequence of FR2 is as shown in SEQ ID NO.2, the amino acid sequence of FR3 is as shown in SEQ ID NO.3, and the amino acid sequence of FR4 is as shown in SEQ ID NO.4; the amino acid sequence of complementary determining region CDR1 is as shown in SEQ ID NO.5, the amino acid sequence of CDR2 is as shown in SEQ ID NO.6, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.7;

[0009] The amino acid sequence of framework region FR1 of the H9-D7-VHH is as shown in SEQ ID NO.8, the amino acid sequence of FR2 is as shown in SEQ ID NO.9, the amino acid sequence of FR3 is as shown in SEQ ID NO.10, and the amino acid sequence of FR4 is as shown in SEQ ID NO.11; the amino acid sequence of complementary determining region CDR1 is as shown in SEQ ID NO.12, the amino acid sequence of CDR2 is as shown in SEQ ID NO.13, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.14.

[0010] Preferably, the amino acid sequence of the H9-VHH is as shown in SEQ ID NO.15; the amino acid sequence of the H9-D7-VHH is as shown in SEQ ID NO.16.

[0011] The present invention also provides a gene encoding the above-mentioned nanobody against staphylococcal enterotoxin B. The nucleotide sequence encoding the H9-VHH is as shown in SEQ ID NO.17; the nucleotide sequence encoding the H9-D7-VHH is as shown in SEQ ID NO.18.

[0012] The present invention also provides a recombinant plasmid, which contains the above-mentioned gene.

[0013] The present invention also provides a recombinant microorganism, into which the above recombinant plasmid is transfected into Escherichia coli to obtain the recombinant microorganism.

[0014] The present invention also provides the use of the above recombinant plasmid or the above recombinant microorganism in the preparation of a nanobody against staphylococcal enterotoxin B.

[0015] The present invention also provides the use of the above staphylococcal enterotoxin B nanobody in the preparation of a reagent, test strip or kit for detecting staphylococcal enterotoxin B.

[0016] The present invention also provides a test strip for detecting staphylococcal enterotoxin B, which comprises a PVC bottom plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane and a blotting paper are sequentially lapped from left to right;

[0017] The conjugate pad contains microsphere-labeled H9-VHH and chicken IgY; on the nitrocellulose membrane, a detection line and a quality control line are coated in the chromatography direction; the detection line is coated with H9-D7-VHH, and the quality control line is coated with goat anti-chicken IgY.

[0018] Preferably, the microsphere is a europium ion fluorescent microsphere, and the antibody labeling mass concentration of the final H9-VHH is 0.04 - 0.06 mg / mL.

[0019] Preferably, the coating concentration of the detection line is 0.3 - 0.5 mg / mL, and the coating amount is 0.8 - 1.2 μL / cm; the coating concentration of the quality control line is 0.4 - 0.6 mg / mL, and the coating amount is 0.8 - 1.2 μL / cm.

[0020] The present invention provides a staphylococcal enterotoxin B nanobody and its application. The present invention has the following beneficial effects:

[0021] (1) The SEB nanobodies (H9-VHH, H9-D7-VHH) designed and synthesized by the present invention carry Trx tags and can be stably expressed in Escherichia coli. After purifying and detecting the expressed products, it is found that the binding constant of H9-VHH to SEB is 2.52E+05, the dissociation constant is 1.41E-04, the affinity constant is 5.60E-10, the highest signal value is 0.09, and the correlation coefficient is 0.965, indicating a relatively high affinity. The binding constant of H9-D7-VHH to SEB is 3.86E+04, the dissociation constant is 1.42E-04, the affinity constant is 3.68E-09, the highest signal value is 0.1419, and the correlation coefficient is 0.9848, indicating a relatively high affinity.

[0022] (2) Establish a time-resolved fluorescence immunochromatographic detection system based on the SEB nanobody of the present invention. Using H9-VHH as the labeled antibody and H9-D7-VHH as the coated antibody, it can quickly and accurately detect SEB. The detection sensitivity can reach 0.041 ng / mL, and there is no cross-reaction with other types of Staphylococcus aureus enterotoxins (SEA, SEC, SED, SEE), botulinum toxin type A (BoNT / A), and Clostridium perfringens epsilon toxin (ETX). The detection system established by the present invention has good application prospects in the detection of SEB in food samples. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the amino acid sequence structure of the H9-VHH antibody.

[0024] Figure 2 It is a schematic diagram of the amino acid sequence structure of the H9-D7-VHH antibody.

[0025] Figure 3 It is an SDS-PAGE electrophoresis analysis diagram of the products induced and expressed by the recombinant bacteria. Among them, M is the marker, and lanes 1-2 are the precipitate and supernatant after induction of the H9-VHH antibody respectively, and lanes 3-4 are the precipitate and supernatant after induction of the H9-D7-VHH antibody respectively.

[0026] Figure 4 It is an SDS-PAGE electrophoresis analysis diagram of the purified H9-VHH antibody and H9-D7-VHH antibody.

[0027] Figure 5 It is the pasting position and detection principle diagram of the time-resolved immunochromatographic test strip for detecting SEB.

[0028] Figure 6 It is the test result diagram of the detection limit of the time-resolved immunochromatographic test strip for detecting SEB.

[0029] Figure 7 It is the test result diagram of the specificity of the time-resolved immunochromatographic test strip for detecting SEB.

[0030] Figure 8 It is the test result diagram of the stability of the time-resolved immunochromatographic test strip for detecting SEB.

[0031] Figure 9 It is the test result diagram of the detection limit of the enzyme-linked immunosorbent assay for detecting SEB. Detailed Embodiments

[0032] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0033] Example 1

[0034] 1. Design the amino acid sequences of SEB nanobodies H9-VHH and H9-D7-VHH

[0035] The H9-VHH antibody starts from the start codon, followed by the start codon M, amino acid G (to prevent frameshift mutations in the target sequence), Trx protein tag (positions 3-111 of the full-length sequence), (G4S)3 linker peptide (positions 112-126 of the full-length sequence), VHH amino acid sequence (positions 127-271 of the full-length sequence, also known as H9), 6×His tag (positions 272-277 of the full-length sequence), and a stop codon. Its schematic diagram is as shown in Figure 1 shown ( Figure 1 where * represents the stop codon).

[0036] Among them, the amino acid sequence of framework region FR1 is: EVQLQASGGGLVQAGGSLRLTCAVS (SEQ ID NO.1);

[0037] The amino acid sequence of framework region FR2 is: WFRQAPGKEREFVA (SEQ ID NO.2);

[0038] The amino acid sequence of framework region FR3 is: RFIISRDNAKNTVYLQMNNLEPEDTAVYSCAR (SEQ ID NO.3);

[0039] The amino acid sequence of framework region FR4 is: WGQETQVTVSSEPKTPKPQPAASGAEFAAAVD (SEQ ID NO.4);

[0040] The amino acid sequence of complementary determining region CDR1 is: GPTFGSYALG (SEQ ID NO.5);

[0041] The amino acid sequence of complementary determining region CDR2 is: AVSWSGGDTYADSVKG (SEQ ID NO.6);

[0042] The amino acid sequence of complementary determining region CDR3 is: MQDRYYISKDAKDYGY (SEQ ID NO.7).

[0043] The H9-D7-VHH antibody starts from the start codon, followed by the start amino acid M, Trx protein tag (positions 1-111 of the full-length sequence), (G4S)3 linker peptide (positions 112-126 of the full-length sequence), VHH amino acid sequence (positions 127-263 of the full-length sequence, also known as H9-D7), 6×His tag (positions 264-269 of the full-length sequence), and a stop codon. Its schematic diagram is as shown inFigure 2 as shown Figure 2 where * indicates a stop codon).

[0044] Among them, the amino acid sequence of framework region FR1 is: DVQLQASGGGLVQVGGSLRLSCAAS (SEQ ID NO.8);

[0045] The amino acid sequence of framework region FR2 is: WHRQAPGKSRELVA (SEQ ID NO.9);

[0046] The amino acid sequence of framework region FR3 is: RFTISRDNTKNTVYLQMNSLEPEDTAVYYCNV (SEQ ID NO.10);

[0047] The amino acid sequence of framework region FR4 is: WGQGTQVAVSSEPKTPKPQPAASGAEFAAAVD (SEQ ID NO.11);

[0048] The amino acid sequence of complementary determining region CDR1 is: GSTFRIGYMS (SEQ ID NO.12);

[0049] The amino acid sequence of complementary determining region CDR2 is: RISSGGTTDYLDSVKD (SEQ ID NO.13);

[0050] The amino acid sequence of complementary determining region CDR3 is: VDYRANEY (SEQ ID NO.14).

[0051] The full-length amino acid sequence of H9-VHH antibody is:

[0052] MGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGGGSGGGGSGGGGSEVQLQASGGGLVQAGGSLRLTCAVSGPTFGSYALGWFRQAPGKEREFVAAVSWSGGDTYADSVKGRFIISRDNAKNTVYLQMNNLEPEDTAVYSCARMQDRYYISKDAKDYGYWGQETQVTVSSEPKTPKPQPAASGAEFAAAVDHHHHHH* (SEQ ID NO.15).

[0053] The full-length amino acid sequence of H9-D7-VHH antibody is:

[0054] MGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGGGSGGGGSGGGGSDVQLQASGGGLVQVGGSLRLSCAASGSTFRIGYMSWHRQAPGKSRELVARISSGGTTDYLDSVKDRFTISRDNTKNTVYLQMNSLEPEDTAVYYCNVVDYRANEYWGQGTQVAVSSEPKTPKPQPAASGAEFAAAVDHHHHHH*(SEQ ID NO.16).

[0055] 2. Design and synthesize the coding genes of SEB nanobodies H9-VHH and H9-D7-VHH

[0056] According to the amino acid sequences of H9-VHH and H9-D7-VHH designed in step 1, design the coding gene sequences of recombinant nanobodies, add restriction enzyme sites and stop codons to the coding gene sequences, and then optimize the coding gene sequences according to the codon preference of Escherichia coli to obtain the coding gene sequences of the recombinant nanobodies shown in Table 1.

[0057] Table 1. Coding gene nucleotide sequences of H9-VHH and H9-D7-VHH

[0058]

[0059]

[0060]

[0061] 3. Construct recombinant plasmids and recombinant bacteria

[0062] The coding genes of H9-VHH and H9-D7-VHH in Table 1 were respectively cloned and constructed onto the pET-28a(+) vector (constructed by GenScript Biotech Corporation, Nanjing, China) through NcoⅠ and XhoⅠ restriction enzyme sites to obtain recombinant plasmids. The recombinant plasmids were transformed into E.coli T7-K12 (transformed by Bomed Gene Technology Co., Ltd., Beijing, China) competent cells, and then spread on LB solid medium containing kanamycin resistance for culture. Single colonies were picked and sent for sequencing verification. The correctly sequenced H9-VHH recombinant bacteria and H9-D7-VHH recombinant bacteria were preserved.

[0063] 4. Induced expression of SEB nanobodies

[0064] (1) Inoculate the monoclonal recombinant bacteria with correct sequencing alignment into 5 mL of LB liquid medium containing kanamycin, and activate overnight at 30 °C and 180 r / min.

[0065] (2) Transfer the bacterial strain from (1) to a 5 mL test tube of LB medium containing kanamycin at an inoculation amount of 1%, and culture with shaking at 180 r / min until the absorbance value (A 600 ) reaches between 0.6 and 0.8.

[0066] (3) Add 5 μL of IPTG with a concentration of 1 M to the test tube to make its final concentration 1 mM, and induce at low temperature of 16 °C for about 16 h.

[0067] (4) After induction, the bacterial cells are collected by centrifugation at 8000 g for 20 min, the supernatant is removed, and the cells are resuspended with 2 mL of PBS and sonicated. The set parameters are sonication for 3 s, pause for 2 s, power of 75%, and sonication for 3 - 5 min.

[0068] (5) Centrifuge the sonicated bacterial liquid at 12000 r / min for 10 min, and detect the protein expression after induction of the precipitate and supernatant by SDS-PAGE.

[0069] The SDS-PAGE results of the precipitate and supernatant after induction are as Figure 3 shown. The theoretical molecular weights of H9-VHH and H9-D7-VHH are approximately 29.5 kDa and 28.6 kDa, respectively. Figure 3 It shows that obvious bands are observed at around 30 kDa for both H9-VHH and H9-D7-VHH, indicating that both nanobodies are soluble expressed. With other conditions unchanged, transfer the bacterial strain to a 500 mL test tube of LB medium containing kanamycin according to the steps of (2) for scale-up culture and mass induction expression.

[0070] 5. Purification of SEB nanobody

[0071] (1) Centrifuge the 500 mL bacterial liquid after induction expression at 8000 g for 20 min, discard the medium, resuspend the precipitate with 200 mL of PBS, mix well and centrifuge at 8000 g for 20 min, repeat twice. Resuspend the precipitate with 80 mL of affinity chromatography working solution (hereinafter referred to as Solution A: 2.197 g of Na2HPO4, 0.53 g of NaH2PO4, 29.22 g of NaCl, 1.36 g of imidazole, 1 L of water), set the sonication working program parameters as sonication for 3 s, pause for 2 s, power of 75%, sonicate for 1 h until the bacterial liquid becomes clear and transparent, place it in a centrifuge at 10000 r / min, and centrifuge for 20 min. Filter the supernatant with a 0.45 μm filter for standby.

[0072] (2) Use the AKTA protein purification system and HisTrapTM HP column (Cytiva, USA). Set the program sequence as follows: Wash the instrument and pipelines with deionized water - Install the HisTrapTM HP column - Rinse the column with deionized water - Flush buffer A through pump A to equilibrate the column - Load the bacterial solution processed in step (1) through pump A - Flush buffer A until equilibrium - Set gradient elution (100% B, 60 min) - Collect samples after peak elution - Wash the column - Dismantle and store the column. Collect the purified samples for SDS-PAGE electrophoresis analysis and storage.

[0073] (3) Use the AKTA protein purification system and HiPrep SepHacryl TM S-100HR column (Cytiva, USA). Set the program sequence as follows: Wash the instrument and pipelines with deionized water - Install the HiPrep SepHacryl TM S-100HR column - Rinse the column with deionized water - Flush buffer A through pump A to equilibrate the column - Load the antibody collected in step (2) through the sample loop - Select the Inject program - Rinse with 15 mL of PBS and then close the Inject program - Collect samples after peak elution - Wash the column - Dismantle and store the column. Perform SDS-PAGE electrophoresis analysis on the purified samples.

[0074] The electrophoresis analysis results of step (3) are as Figure 4 shown, Figure 4 indicating that after purification by gel filtration chromatography, the lanes are basically clean without impurity bands and the purity is relatively high.

[0075] 6. Activity analysis of SEB nanobody

[0076] Use bio-layer interferometry (BLI) to analyze the antigen-binding activity of the nanobody. The molecular interaction analysis system used is Octet-RED96e (ForteBio, USA), and the selected sensor is Anti-Penta-HIS HISK sensor. The specific steps are as follows:

[0077] (1) Hydrate the sensor in pure water for 10 min.

[0078] (2) Use SEB as the immobilized substance at a concentration of 1 μg / mL. H9-VHH and H9-D7-VHH are used as analytes, and the concentrations are set at gradients of 800, 400, 200, 100, 50, 25, 12.5, 0 nM respectively.

[0079] (3) Add the 96-well plates in the following order. Add PBS to the first column as the baseline, add the immobilized substance (SEB) to the second column, add PBS to the third column for baseline 2 and the dissociation step, and add the serially diluted analyte (nanobody) to the fourth column for the binding step. Set the time and rotation speed for the analysis steps. The baseline washing time is 60 s, the immobilization binding time is 180 s, the baseline 2 washing time is 180 s, the binding time is 300 s, the dissociation time is 600 s, and the kinetic rotation speed is selected as 1000 r / min.

[0080] (4) Open the Octet analysis software, sort according to the analyte concentration, set the analyte with a concentration of 0 nM as the control group, and obtain the results.

[0081] The parameters of the affinity analysis of H9-VHH, H9-D7-VHH and SEB are shown in Table 2. The highest concentration of H9-VHH detected is 800 nM, the binding constant is 2.52E+05, the dissociation constant is 1.41E-04, the affinity constant is 5.60E-10, the highest signal value is 0.09, the correlation coefficient is 0.965, and the affinity is relatively high. The highest concentration of H9-D7-VHH is 800 nM, the binding constant is 3.86E+04, the dissociation constant is 1.42E-04, the affinity constant is 3.68E-09, the highest signal value is 0.1419, and the correlation coefficient is 0.9848, and the affinity is relatively high.

[0082] Table 2 Parameters of the affinity analysis of H9-VHH, H9-D7-VHH and SEB

[0083] antibody Conc(nM) ka(1 / Ms) kd(1 / s) H9-VHH 800 2.52E+05 1.41E-04 H9-D7-VHH 800 3.86E+04 1.42E-04

[0084] Example 2

[0085] This example provides a method for preparing a time-resolved immunochromatographic test strip for detecting SEB using SEB nanobody. The process is as follows:

[0086] Main reagents: microspheres, coating diluent, labeling diluent, and sample diluent are all purchased from Chengdu Weirui Biotechnology Co., Ltd.

[0087] 1. Preparation of the test strip

[0088] Add MES buffer (2 mL, pH 7.2) to 150 μL of europium ion fluorescent microspheres (1 mg), mix well for 15 min, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the mixture at a ratio of 1:2 to activate the carboxyl groups on the microspheres, and vortex for 20 min. After centrifugation at 15,000 r / min for 15 min, remove the supernatant, suspend the precipitate with 2 mL of boric acid buffer, then add 0.1 mg of H9-VHH, add 1% BSA blocking solution, place it in a 3D rotator at room temperature, rotate overnight and then disperse by ultrasonic treatment. The next morning, centrifuge and remove the supernatant again, and resuspend the precipitate in 2 mL of boric acid marker diluent containing 1% BSA to obtain the fluorescent probe, which is stored in the dark at 4 °C for standby.

[0089] Dilute H9-D7-VHH to 0.4 mg / mL and goat anti-chicken IgY to 0.5 mg / mL with coating diluent, and spray them on the NC membrane at a rate of 1 μL / cm using a dot membrane spraying instrument as the T line and C line respectively, and place them in a 37 °C forced air drying oven overnight.

[0090] Dilute the prepared fluorescent probe 3-fold with the marker diluent added with plant pigment, and add the microspheres conjugated with chicken IgY (added with pigment, the labeling method is the same as that of H9-VHH labeling), mix, and then spray it on the conjugate pad at a rate of 2 μL / cm, and place it in a 37 °C forced air drying oven in the dark for drying for more than 4 h.

[0091] Paste the prepared sample pad, conjugate pad, NC membrane coated with the C line and T line, and absorbent pad on the PVC bottom plate in sequence. The upper and lower ends of the conjugate pad are cross-linked with the sample pad and the NC membrane by 1-2 mm respectively, cut it into strips with a width of 4.12 mm, put it into the cartridge and press it tightly, and seal it and place it at 4 °C. Place it in a foil bag, add desiccant and seal it for storage; the pasting position and detection principle are as Figure 5 shown.

[0092] 2. Detection limit test

[0093] Gradient dilute the SEB standard product with sample diluent (0, 0.048, 0.097, 0.195, 0.391, 0.781, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 125, 250, 500 ng / mL), and detect each concentration 3 times. Take the SEB concentration value as the abscissa and the sample titer ratio (T / C value) as the ordinate to draw the detection curve of this method, as Figure 6 shown. Figure 6 It shows that the detection limit is 0.041 ng / mL and the visual detection limit is 1.56 ng / mL.

[0094] 3. Specificity test

[0095] The established TRFCIA method was used to detect other types of Staphylococcus aureus enterotoxins (SEA, SEC, SED, SEE), botulinum toxin type A (BoNT / A), and Clostridium perfringens epsilon toxin (ETX). The concentration of each toxin was set at 1000 ng / mL, and each concentration was repeated 3 times. The specificity of the method was verified under optimized conditions. The detection results of each toxin are as Figure 7 shown. Figure 7 It shows that the set interfering biotoxins have little or no effect on the detection of SEB. Therefore, the test strip prepared by the present invention exhibits high specificity for SEB.

[0096] 4. Stability

[0097] The heat stability acceleration experiment was used to determine the stability of the immunochromatographic test strip. The prepared test strips were sealed and placed in an electric heating blast drying oven at 37 °C, and the fluorescence intensity of different concentrations of SEB was measured every 10 days for a total of 30 days. The accelerated stability of the method was evaluated by the change in fluorescence intensity at different times. The results show that in the 30-day accelerated test, the T / C of 50 ng / mL and 1 ng / mL SEB was controlled within 10% of the initial detection value ( Figure 8 ). According to the Arrhenius formula, storing stably at 37 °C for at least 30 days is equivalent to storing at 4 °C for one year. Therefore, the test strip prepared by the present invention can be stored stably at 37 °C for at least 30 days and can be stored at 4 °C for one year.

[0098] 5. Accuracy

[0099] The accuracy of the immunochromatographic test strip was evaluated by the recovery experiment. Three different concentrations of positive spiked samples (45, 15, 5 ng / mL) were prepared with SEB protein standard in skim milk. The within-batch difference was calculated by repeating the measurement 6 times for each spiked concentration in the same batch, and the between-batch difference was calculated by repeating each spiked sample 3 times for different batches. The average sample recovery rates at different concentrations and the within-batch and between-batch differences of the test strip were obtained to evaluate the accuracy and precision of the method. Before testing, the skim milk was diluted 5 times as the diluent. The results are shown in Table 3.

[0100] Table 3 results show that the within-batch recovery rate of the test strip in skim milk samples was 94.81% - 118.4%, the between-batch recovery rate was 97.77% - 113.69%, the within-batch coefficient of variation < 11.73%, and the between-batch coefficient of variation < 12.28%. This indicates that the established TRFICA of the present invention has good accuracy and practicability in the detection of SEB in food samples.

[0101] Table 3 Results of accuracy and precision in skim milk samples

[0102]

[0103] Example 3

[0104] In this example, an enzyme-linked immunosorbent assay for detecting SEB was established using SEB nanobody. The process is as follows:

[0105] (1) Immobilize the capture antibody: Dilute H9-VHH with 1× coating buffer (Chengdu Weirui Biotechnology Co., Ltd., Chengdu, China), add 100 μL to each well of the enzyme-linked immunosorbent assay (ELISA) plate, and place it at 4 °C overnight. The next day, discard the liquid, and use a plate washer to add 350 μL of PBST to wash the ELISA plate 3 times.

[0106] (2) Blocking: Add 350 μL of 5% BSA and incubate at 37 °C for blocking, and then use PBST to wash 3 times again.

[0107] (3) Incubate the toxin: Dilute SEB with 5% BSA to 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19, 0.097, 0.048 ng / mL, add 100 μL to each well, repeat 3 wells for each concentration, place it in a shaking incubator at 37 °C at 1200 r / min, and shake for 20 min. After incubation, wash 3 times.

[0108] (4) Incubate the detection antibody: Dilute the biotinylated H9-D7-VHH detection antibody with 5% BSA, add 100 μL to each well, place it in a shaking incubator at 37 °C at 1200 r / min, and shake for 20 min. After incubation, wash 3 times.

[0109] (5) Incubate the chromogenic antibody: Dilute SA-HPR with 5% BSA, add 100 μL to each well, place it in a shaking incubator at 37 °C at 1200 r / min, and shake for 20 min. After incubation, wash 3 times.

[0110] (6) Chromogenic reaction: Add TMB, 100 μL to each well, let it stand in the dark for 20 min, and then add 50 μL of sulfuric acid termination solution to terminate the reaction. Read the value at A 450 using an enzyme-linked immunosorbent assay reader.

[0111] The detection results are as Figure 9 shown. The horizontal axis is the logarithm of the SEB concentration, the vertical axis is the value of A 450 , the cut-off value is 0.371, the dotted line is the cut-off value, Y = 0.1372 + 2.143 / (1 + 10^((0.611 - X)×1.025)), R 2 is greater than 0.99. Among the set SEB concentration gradients, the A at 0.78 ng / mL450 The value is still greater than 2.1 times that of the negative. Therefore, the lower limit of detection of the established ELISA method is 0.78 ng / mL.

[0112] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A nanobody against staphylococcal enterotoxin B, characterized in that, The staphylococcal enterotoxin B nanobody is H9-VHH; The amino acid sequence of framework region FR1 of the H9-VHH is shown in SEQ ID NO.1, the amino acid sequence of FR2 is shown in SEQ ID NO.2, the amino acid sequence of FR3 is shown in SEQ ID NO.3, and the amino acid sequence of FR4 is shown in SEQ ID NO.4; the amino acid sequence of complementary determining region CDR1 is shown in SEQ ID NO.5, the amino acid sequence of CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

7.

2. The nanobody against staphylococcal enterotoxin B according to claim 1, characterized in that, The amino acid sequence of the H9-VHH is shown in SEQ ID NO.

15.

3. The gene encoding the staphylococcal enterotoxin B nanobody according to claim 2, characterized in that, The nucleotide sequence encoding the H9-VHH is shown in SEQ ID NO.

17.

4. A recombinant plasmid, characterized in that, The recombinant plasmid contains the gene described in claim 3.

5. A recombinant microorganism, characterized in that, The recombinant plasmid described in claim 4 is transfected into Escherichia coli to obtain the recombinant microorganism.

6. Use of the recombinant plasmid described in claim 4 or the recombinant microorganism described in claim 5 in the preparation of staphylococcal enterotoxin B nanobody.

7. Use of the staphylococcal enterotoxin B nanobody described in claim 1 or 2 in the preparation of a reagent, test strip or kit for detecting staphylococcal enterotoxin B.

8. A test strip for detecting staphylococcal enterotoxin B, characterized in that, The test strip includes a PVC bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and a blotting paper are sequentially lapped on the PVC bottom plate from left to right; The conjugate pad contains the microsphere-labeled H9-VHH as described in claim 1 and chicken IgY; the nitrocellulose membrane is coated with a test line and a quality control line in the chromatography direction; the test line is coated with the staphylococcal enterotoxin B nanobody H9-D7-VHH, and the quality control line is coated with goat anti-chicken IgY; The amino acid sequence of framework region FR1 of the H9-D7-VHH is shown in SEQ ID NO.8, the amino acid sequence of FR2 is shown in SEQ ID NO.9, the amino acid sequence of FR3 is shown in SEQ ID NO.10, and the amino acid sequence of FR4 is shown in SEQ ID NO.11; the amino acid sequence of complementary determining region CDR1 is shown in SEQ ID NO.12, the amino acid sequence of CDR2 is shown in SEQ ID NO.13, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

14.

9. The test strip according to claim 8, characterized in that, The microsphere is an europium ion fluorescent microsphere, and the labeling concentration of the H9-VHH is 0.04-0.06 mg / mL.

10. The test strip according to claim 8, characterized in that, The coating concentration of the test line is 0.3-0.5 mg / mL, and the coating amount is 0.8-1.2 μL / cm; the coating concentration of the quality control line is 0.4-0.6 mg / mL, and the coating amount is 0.8-1.2 μL / cm.

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