Staphylococcus enterotoxin B nano antibody and application thereof

Abstract

The invention provides a staphylococcal enterotoxin B nano antibody and application thereof, and belongs to the technical field of nano antibodies. The staphylococcal enterotoxin B nano antibody disclosed by the invention comprises H9-VHH and H9-D7-VHH, and the staphylococcal enterotoxin B nano antibody disclosed by the invention comprises H9-VHH. The staphylococcal enterotoxin B (SEB) nano-antibody provided by the invention is provided with a Trx label, and a fusion expression plasmid of the SEB nano-antibody is constructed by utilizing a coding gene of the nano-antibody, so that the soluble expression of the nano-antibody in escherichia coli is realized. Based on the nano antibody, the invention also establishes a time-resolved fluorescence immunochromatography detection system aiming at SEB, and the system is sensitive, rapid and accurate in detection, strong in stability and high in specificity, and has a wide application prospect in the aspect of SEB detection.

Description

Technical Field

[0001] The present invention relates to the technical field of nano antibodies, and in particular to a staphylococcal enterotoxin B nano antibody and an application thereof. Background Art

[0002] Staphylococcal enterotoxin B (SEB) is the main virulence factor of Staphylococcus aureus. The median lethal dose (LD50) of SEB is 50 ) is about 20ng / kg, and because of its easy preparation and high toxicity, it is considered a potential biological warfare agent and bioterrorism agent. At the same time, SEB also poses a huge threat to human health and food safety. However, in the past few decades, there has been no approved SEB detection reagent in clinical practice, and enzyme-linked immunosorbent assay (ELISA) is commonly used for auxiliary diagnosis. The ELISA method usually takes 4-5h to complete, which is difficult to meet the needs of rapid detection, and most of the current commercial SEB ELISA detection kits use polyclonal antibodies with poor specificity. Therefore, it is urgent to prepare highly specific antibodies against SEB and develop rapid detection methods with strong specificity and high sensitivity.

[0003] Nanobodies are a type of heavy chain antibodies that are naturally missing light chains and are isolated from camel and shark sera. The molecular weight of nanobodies is only one-tenth of that of traditional complete antibodies (about 15kDa), and they can act on antigens alone and have complete antigen recognition capabilities. Compared with traditional antibodies, nanobodies have the characteristics of small size, good stability, strong specificity, high affinity, thermal stability, and easy transformation. They can also achieve high-quality and stable production through in vitro recombinant expression, effectively avoiding the batch-to-batch differences of traditional antibodies, and are particularly suitable for mass preparation and standardized production.

[0004] Time resolved fluorescence immunochromatography (TRFICA) is a new analytical method based on immunochromatography and fluorescence analysis. This technology uses lanthanide rare earth ions as markers, which effectively avoids the problems of spontaneous fluorescence of biological materials and easy quenching of fluorescent dyes in fluorescence immunoassay. In addition, before signal acquisition, 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 purpose of the present invention is to provide a Staphylococcal enterotoxin B (SEB) nanobody, and to achieve soluble expression of the nanobody in Escherichia coli by constructing a fusion expression plasmid of the SEB nanobody. It is then expected to establish a time-resolved fluorescence immunochromatography detection system for SEB based on the nanobody, so that the detection system has the characteristics of sensitivity, rapidity, accuracy, strong stability and high specificity.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0008] The amino acid sequence of the 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 the 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;

[0009] The amino acid sequence of the 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 the 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.

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

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

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

[0013] The present invention also provides a recombinant microorganism, which is obtained by transducing the recombinant plasmid into Escherichia coli.

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

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

[0016] The present invention also provides a test strip for detecting staphylococcal enterotoxin B, the test strip comprising a PVC bottom plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing paper are overlapped in sequence from left to right;

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

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

[0019] Preferably, 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.

[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 nanoantibodies (H9-VHH, H9-D7-VHH) designed and synthesized by the present invention carry Trx tags and can be stably expressed in Escherichia coli. The expressed products were purified and tested to find that the binding constant of H9-VHH to SEB was 2.52E+05, the dissociation constant was 1.41E-04, the affinity constant was 5.60E-10, the highest signal value was 0.09, and the correlation coefficient was 0.965, indicating a high affinity. The binding constant of H9-D7-VHH to SEB was 3.86E+04, the dissociation constant was 1.42E-04, the affinity constant was 3.68E-09, the highest signal value was 0.1419, the correlation coefficient was 0.9848, and the affinity was high.

[0022] (2) Based on the SEB nanobody of the present invention, a time-resolved fluorescent immunochromatographic detection system is established, with H9-VHH as the labeled antibody and H9-D7-VHH as the coated antibody, which can quickly and accurately detect SEB, with a detection sensitivity of up to 0.041 ng / mL, and 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 SEB detection of food samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the amino acid sequence structure of the H9-VHH antibody.

[0024] Figure 2 Schematic diagram of the amino acid sequence structure of the H9-D7-VHH antibody.

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

[0026] Figure 4 Figure 2 is the SDS-PAGE electrophoresis analysis of the purified H9-VHH antibody and H9-D7-VHH antibody.

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

[0028] Figure 6 This is a graph showing the detection limit test results of a time-resolved immunochromatographic test strip for detecting SEB.

[0029] Figure 7 This is a graph showing the specificity test results of a time-resolved immunochromatographic test strip for detecting SEB.

[0030] Figure 8 This is a graph showing the stability test results of a time-resolved immunochromatographic test strip for detecting SEB.

[0031] Fig. 9 This is a graph showing the detection limit test results of the enzyme-linked immunosorbent assay for detecting SEB. DETAILED DESCRIPTION

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

[0033] Example 1

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

[0035] The H9-VHH antibody starts with the start codon, followed by the start codon M, amino acid G (to prevent frameshift mutation in the target sequence), Trx protein tag (positions 3-111 of the full-length sequence), (G4S)3 connecting 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 stop codon. The schematic diagram of its structure is shown in the figure. Figure 1 As shown ( Figure 1 * indicates stop codon).

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

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

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

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

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

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

[0042] The amino acid sequence of the complementarity 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, the Trx protein tag (positions 1-111 of the full-length sequence), the (G4S)3 connecting peptide (positions 112-126 of the full-length sequence), the VHH amino acid sequence (positions 127-263 of the full-length sequence, also known as H9-D7), the 6×His tag (positions 264-269 of the full-length sequence) and the stop codon. The schematic diagram of its structure is shown in FIG. Figure 2 As shown ( Figure 2 * indicates stop codon).

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

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

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

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

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

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

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

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

[0052] MGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGGGSGGGGSGGGGSEVQLQASGGGLVQAG GSLRLTCAVSGPTFGSYALGWFRQAPGKEREFVAAVSWSGGDTYADSVKGRFIISRDNAKNTVYLQMNNLEPEDTAVYSCARMQDRYYISKDAKDYGYWGQETQVTVSSEPKTPKPQPAASGAEFAAAVDHHHHHH*(SEQ ID NO.15).

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

[0054] MGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGGGSGGGGSGGGGSDVQLQASGGGL VQVGGSLRLSCAASGSTFRIGYMSWHRQAPGKSRELVARISSGGTTDYLDSVKDRFTISRDNTKNTVYLQMNSLEPEDTAVYYCNVVDYRANEYWGQGTQVAVSSEPKTPKPQPAASGAEFAAAVDHHHHHH*(SEQ ID NO.16).

[0055] 2. Design and synthesis of genes encoding SEB nanoantibodies H9-VHH and H9-D7-VHH

[0056] According to the amino acid sequences of H9-VHH and H9-D7-VHH designed in step 1, the coding gene sequence of the recombinant nanobody was designed, restriction sites and stop codons were added to the coding gene sequence, and then the coding gene sequence was optimized according to the codon preference of Escherichia coli to obtain the coding gene sequence of the recombinant nanobody shown in Table 1.

[0057] Table 1 Nucleotide sequences of genes encoding H9-VHH and H9-D7-VHH

[0058]

[0059]

[0060]

[0061] 3. Construction of recombinant plasmids and recombinant bacteria

[0062] The coding genes of H9-VHH and H9-D7-VHH in Table 1 were cloned into pET-28a(+) vector (submitted to GenScript Biotech, Nanjing, China) through NcoⅠ and XhoⅠ restriction sites, respectively, to obtain recombinant plasmids. The recombinant plasmids were transformed into E. coli by thermosensitive method. After T7-K12 (transformed by Bomed Gene Technology Co., Ltd., Beijing, China) became competent, it was plated on LB solid medium containing kanamycin resistance and cultured, and a single clone was picked for sequencing verification. The H9-VHH recombinant bacteria and H9-D7-VHH recombinant bacteria with correct sequencing were preserved.

[0063] 4. Inducible expression of SEB nanobody

[0064] (1) The monoclonal recombinant bacteria with correct sequencing alignment were inoculated into 5 mL of LB liquid culture medium containing kanamycin and activated at 30°C and 180 r / min overnight.

[0065] (2) Transfer the bacteria from (1) to a 5 mL LB medium tube containing kanamycin at a 1% inoculum volume and culture at 180 rpm until the absorbance value (A 600 ) is between 0.6 and 0.8.

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

[0067] (4) After induction, the cells were collected by centrifugation at 8000 g for 20 min, the supernatant was removed, and the cells were resuspended in 2 mL of PBS and ultrasonically disrupted. The parameters were set as ultrasonication for 3 s, pause for 2 s, power of 75%, and disruption for 3 to 5 min.

[0068] (5) The bacterial solution after ultrasound was centrifuged at 12000 r / min for 10 min, and the precipitate and supernatant were used to detect the induced protein expression by SDS-PAGE.

[0069] The SDS-PAGE results of the precipitate and supernatant after induction are as follows Figure 3 The theoretical molecular weights of H9-VHH and H9-D7-VHH are approximately 29.5 kDa and 28.6 kDa, respectively. Figure 3 The results showed that H9-VHH and H9-D7-VHH both had obvious bands at around 30 kDa, indicating that both nanobodies were expressed in a soluble form. The other conditions remained unchanged, and the strain was transferred to a 500 mL LB medium tube containing kanamycin according to step (2) for expansion culture and large-scale induction of expression.

[0070] 5. Purification of SEB Nanobodies

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

[0072] (2) Using the AKTA protein purification system and HisTrapTM HP column (Cytiva, USA), the program sequence was set as follows: washing the instrument and pipeline with deionized water - installing the HisTrapTM HP column - flushing the column with deionized water - flushing the A solution through the A pump to balance the column - loading the sample (the bacterial solution treated in step (1)) through the A pump - flushing the A solution to equilibrium - setting the gradient elution (100% B, 60 min) - loading the sample after the peak - flushing the column - disassembling and storing the column. Collect the purified samples for SDS-PAGE electrophoresis analysis and store them.

[0073] (3) Using the AKTA protein purification system and HiPrep SepHacryl TM The S-100HR column (Cytiva, USA) was set up in the following order: deionized water to clean the instrument and pipes - install HiPrep SepHacryl TM S-100HR column - rinse the column with deionized water - flush the A solution through the A pump to balance the column - load the sample (the antibody collected in step (2)) through the sample loop - select the Inject program - rinse with 15mL of PBS and then turn off the Inject program - load the sample after the peak - rinse the column - disassemble and store the column. Perform SDS-PAGE electrophoresis analysis on the purified sample.

[0074] The results of electrophoresis analysis in step (3) are as follows Figure 4 As shown, Figure 4 It shows that after purification by gel filtration chromatography, the lane is basically clean and free of impurities, and the purity is relatively high.

[0075] 6. Activity analysis of SEB nanobody

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

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

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

[0079] (3) The 96-well plate was added in the following order: PBS was added to the first column as the baseline, the immobilized substance (SEB) was added to the second column, PBS was added to the third column for the baseline 2 and dissociation step, and the gradient diluted analyte (nanoantibody) was added to the fourth column as the binding step. The time and speed of the analysis step were set, the baseline cleaning time was 60 s, the immobilized binding time was 180 s, the baseline 2 cleaning time was 180 s, the binding time was 300 s, the dissociation time was 600 s, and the kinetic speed was selected as 1000 r / min.

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

[0081] The parameters of affinity analysis of H9-VHH, H9-D7-VHH and SEB are shown in Table 2. The highest concentration of H9-VHH detected is 800nM, 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 high. The highest concentration of H9-D7-VHH is 800nM, 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, the correlation coefficient is 0.9848, and the affinity is high.

[0082] Table 2 Parameters of 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 nanobodies. The process is as follows:

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

[0087] 1. Preparation of test strips

[0088] MES buffer (2 mL, pH 7.2) was added to 150 μL europium ion fluorescent microspheres (1 mg) and mixed for 15 min, then carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the mixture at a ratio of 1:2 to activate the carboxyl groups on the microspheres, and vortexed for 20 min, centrifuged at 15000 r / min for 15 min, and the supernatant was removed. The precipitate was suspended with 2 mL of boric acid buffer, and then 0.1 mg of H9-VHH was added, and 1% BSA blocking solution was added. The mixture was placed in a 3D rotator at room temperature, rotated overnight, and then ultrasonically dispersed. After centrifugation and removal of the supernatant again the next morning, the precipitate was resuspended in 2 mL of boric acid marker diluent containing 1% BSA to obtain a fluorescent probe, which was stored in a 4°C refrigerator away from light for later use.

[0089] H9-D7-VHH was diluted to 0.4 mg / mL using coating diluent, and goat anti-chicken IgY was diluted to 0.5 mg / mL. They were sprayed onto the NC membrane at a rate of 1 μL / cm using a dot film sprayer as T line and C line, respectively, and placed in a 37°C forced air drying oven overnight.

[0090] The prepared fluorescent probe was diluted 3 times using a labeling diluent with added plant pigments, and chicken IgY-coupled microspheres (with pigments, the labeling method was the same as the H9-VHH labeling method) were added and mixed, and then sprayed on the binding pad at a rate of 2 μL / cm and placed in a 37°C air drying oven away from light for more than 4 hours.

[0091] The prepared sample pad, conjugate pad, NC membrane coated with C-line and T-line, and absorbent pad are sequentially pasted on the PVC base plate. The upper and lower ends of the conjugate pad are cross-linked with the sample pad and NC membrane by 1 to 2 mm, respectively. Cut it into 4.12 mm wide strips, put it into the card shell, press it tightly, seal it and place it at 4°C. Put it in a tin foil bag, put in a desiccant and seal it for storage; the pasting position and detection principle are as follows Figure 5 shown.

[0092] 2. Detection limit test

[0093] The SEB standard was diluted 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 each concentration was tested 3 times. The detection curve of the method was drawn with SEB concentration as the horizontal axis and sample potency ratio (T / C value) as the vertical axis, as shown in the figure. Figure 6 shown. Figure 6 The detection limit was shown to be 0.041 ng / mL and the visual detection limit was 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 to 1000 ng / mL, and each concentration was repeated 3 times to verify the specificity of the method under the optimized conditions. The detection results of each toxin are shown in Figure 2. Figure 7 shown. Figure 7 It shows that the interfering biological toxins have little or no effect on the detection of SEB, so the test strip prepared by the present invention shows high specificity for SEB.

[0096] 4. Stability

[0097] The stability of the immunochromatographic test strips was determined by using a thermal stability accelerated test. The prepared test strips were sealed and placed in a 37°C electric blast drying oven. The fluorescence intensity of different concentrations of SEB was tested 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 showed that in the 30-day accelerated test, the T / C of 50ng / mL and 1ng / mL SEB was controlled within 10% of the initial detection value ( Figure 8 ). According to the Arrhenius formula, stable storage at 37°C for at least 30 days is equivalent to storage at 4°C for one year. Therefore, the test paper prepared by the present invention can be stably stored 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 strips was evaluated by a recovery experiment. Three positive spiked samples (45, 15, 5 ng / mL) of different concentrations were prepared in skim milk using SEB protein standards. Each spiked concentration of the same batch was measured six times to calculate the intra-batch difference, and each spiked sample of different batches was measured three times to calculate the inter-batch difference. The average sample recovery of different concentrations and the intra-batch and inter-batch differences of the test strips were obtained to evaluate the accuracy and precision of the method. Before the test, skim milk was diluted 5 times as a diluent. The results are shown in Table 3.

[0100] The results in Table 3 show that the intra-batch recovery of the test strips in skim milk samples is 94.81% to 118.4%, the inter-batch recovery is 97.77% to 113.69%, the intra-batch coefficient of variation is less than 11.73%, and the inter-batch coefficient of variation is less than 12.28%. This shows that the TRFICA established in the present invention has good accuracy and practicality in SEB detection of 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 nanoantibodies. The process is as follows:

[0105] (1) Immobilization of capture antibody: H9-VHH was diluted with 1× coating solution (Chengdu Weirui Biotechnology Co., Ltd., Chengdu, China), added to each well of the ELISA plate at 100 μL, and placed at 4°C overnight. The next day, the liquid was discarded, and the ELISA plate was washed three times with 350 μL PBST using a plate washer.

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

[0107] (3) Incubation of toxins: SEB was diluted to 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19, 0.097, and 0.048 ng / mL using 5% BSA, and 100 μL was added to each well. Each concentration was repeated for 3 wells, and the wells were placed in a shaking incubator at 37°C at 1200 r / min for 20 min. After incubation, the wells were washed 3 times.

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

[0109] (5) Incubation with colorimetric antibody: dilute SA-HPR with 5% BSA, add 100 μL to each well, place in a shaking incubator at 37°C, 1200 rpm, shake for 20 min. Wash three times after incubation.

[0110] (6) Color development: Add TMB, 100 μL per well, incubate in dark for 20 min, and add 50 μL sulfuric acid stop solution to terminate the reaction. 450 Read the value.

[0111] Test results such as Fig. 9 The horizontal axis is the logarithm of SEB concentration, and the vertical axis is 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 Greater than 0.99. The A value at 0.78 ng / mL in the set SEB concentration gradient450 The value was still greater than 2.1 times of negative, therefore, the detection limit of the established ELISA was 0.78 ng / mL.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Staphylococcal enterotoxin B nanobody, It is characterized in that Including H9-VHH and H9-D7-VHH; The amino acid sequence of the 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 the 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; The amino acid sequence of the 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 the 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.

2. The Staphylococcal enterotoxin B nanobody according to claim 1, It is characterized in that The amino acid sequence of the H9-VHH is shown in SEQ ID NO.15; the amino acid sequence of the H9-D7-VHH is shown in SEQ ID NO.

16.

3. A gene encoding the Staphylococcal enterotoxin B nanobody according to claim 2, It is characterized in that The nucleotide sequence encoding the H9-VHH is shown in SEQ ID NO.17; the nucleotide sequence encoding the H9-D7-VHH is shown in SEQ ID NO.

18.

4. A recombinant plasmid, It is characterized in that The recombinant plasmid contains the gene according to claim 3.

5. A recombinant microorganism, It is characterized in that The recombinant microorganism is obtained by transducing the recombinant plasmid described in claim 4 into Escherichia coli.

6. Use of the recombinant plasmid according to claim 4 or the recombinant microorganism according to claim 5 in the preparation of Staphylococcal enterotoxin B nanobodies.

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

8. A test strip for detecting Staphylococcal enterotoxin B, It is characterized in that The test strip comprises a PVC bottom plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane and absorbent paper are overlapped in sequence from left to right; The conjugate pad contains microsphere-labeled H9-VHH and chicken IgY; the nitrocellulose membrane is coated with a detection line and a quality control line according to the chromatography direction; the detection line is coated with H9-D7-VHH, and the quality control line is coated with sheep anti-chicken IgY.

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

10. The test strip according to claim 8, It is 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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