Nanometer antibody for detecting staphylococcus aureus enterotoxin B and application thereof

Abstract

The invention relates to the technical field of antigen detection, in particular to a nano antibody for detecting staphylococcus aureus enterotoxin B and application of the nano antibody. The nano antibody comprises an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The application comprises purification, detection and removal of the staphylococcus aureus enterotoxin B. The two SEB nanometer antibodies obtained through screening have high specificity and high affinity with the SEB, SEB detection based on the two nanometer antibodies has high specificity and sensitivity, and the SEB nanometer antibodies have important value in the field of staphylococcus aureus enterotoxin detection.

Description

Technical Field

[0001] The present invention relates to the technical field of antigen detection, and in particular to a nano antibody for detecting Staphylococcus aureus enterotoxin B and an application thereof. Background Art

[0002] Staphylococcus aureus is one of the most common foodborne pathogens and can cause severe staphylococcal food poisoning (SFP). Staphylococcus aureus enterotoxins (SEs) can cause nausea, severe vomiting and abdominal cramps, and occasionally diarrhea after a short incubation period. Its molecular weight is between 19-30kD and is resistant to high temperatures and protease hydrolysis. So far, a total of 29 different SEs and enterotoxin-like substances (SELs) have been reported, and the classic SEs serotypes include SEA, SEB, SEC, SED and SEE. Ingestion of 20-100 ng of SEB will lead to the release of a large number of proinflammatory cytokines, leading to SFP and even inducing severe toxic shock syndrome (TSS).

[0003] At present, the main methods for detecting SEs include biological detection, genetic molecular biology detection, mass spectrometry detection and immunological detection. The detection method specified in the national standard is enzyme-linked immunosorbent assay (ELISA), which has the advantages of high throughput, easy operation, high sensitivity, etc., and can directly detect SEs in samples. In addition, compared with other technologies for detecting SEs, the ELISA method is not easily interfered by complex matrices, does not involve ethical issues, and is not easily contaminated to produce false positives. Several existing commercial SEs immunological detection kits can detect 5 classic enterotoxins at the same time, and the antibodies (mainly IgG) are mainly derived from mouse monoclonal antibodies or rabbit polyclonal antibodies. For example, the enzyme-linked immunosorbent assay kit for the total amount of Staphylococcus aureus enterotoxin jointly developed by China Agricultural University and Beijing Weideweikang Biotechnology Co., Ltd. uses monoclonal antibodies obtained by immunizing mice with recombinant Staphylococcus aureus classic enterotoxins.

[0004] Traditional monoclonal antibodies for detecting enterotoxins are obtained by immunizing animals. The screening process is time-consuming, costly, and requires skilled operators and specialized cell culture facilities. In particular, the crystallizable fragment (Fc) region of the monoclonal antibody can bind to the surface protein A (SpA) of Staphylococcus aureus, which makes the ELISA method for detecting enterotoxins prone to false positives. Nanobodies can avoid the problem of false positives because they lack the Fc segment. Currently available kits for Staphylococcus aureus enterotoxins include: Ridascreen from Germany ® set, 3M of the United States @ TECRA ® Staph enterotoxins, Identification Test 3Etc. However, these kits all have the problem of overlap between SEB and SEC detection.

[0005] Nanobodies have many advantages: Nanobodies are more inclined to bind to concave epitopes and can bind to antigenic epitopes that traditional antibodies cannot bind to; the extension of complementarity determining region 3 (CDR3) and the conservative disulfide bonds make nanobodies have higher thermal stability; the hydrophilic amino acids in the framework region 2 (FR2) of nanobodies increase the water solubility of nanobodies and reduce the aggregation ability; they are easy to express and purify in prokaryotes, reduce production costs, break through the limitations of in vivo antibody preparation, and shorten the development cycle. The nanobody VHH fragment does not contain the Fc region and theoretically will not bind to the Staphylococcus aureus SpA protein and produce cross-reactions. Therefore, it is necessary to screen nanoantibody pairs with good specificity against SEB for the immunological detection of Staphylococcus aureus enterotoxin B. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a nano antibody for detecting Staphylococcus aureus enterotoxin B and application thereof.

[0007] The present invention screens out a pair of anti-SEB nanoantibodies with good specificity, uses capture antibody AntiSEB Nb3 and detection antibody AntiSEB Nb6 as an antibody pair, constructs a double antibody sandwich method, and can achieve specific detection of SEB. The detection antibody AntiSEB Nb6 is specific and only recognizes SEB, but not Staphylococcus aureus enterotoxin SEC, thereby ensuring the high specificity of the diagnostic result.

[0008] The capture antibody AntiSEB Nb3 and the detection antibody AntiSEB Nb6 recognized different epitopes of SEB; among them, AntiSEB Nb6 recognized the amino acid sequence region that had no homology between SEB and SEC, so the double antibody sandwich method established based on this antibody had no cross-reaction to SEC. In addition, both AntiSEB Nb3 and AntiSEB Nb6 had high affinity for SEB, and the extension of CDR3 and the conserved disulfide bond made the nanobody have high thermal stability, which was not only easy to store, but also conducive to the establishment of subsequent experimental methods.

[0009] In a first aspect, the present invention provides an anti-SEB nanobody (AntiSEB Nb3), wherein the nanobody has at least one of the following CDRs: i) the heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO.1; ii) heavy chain CDR2 at positions 51 to 58 as shown in SEQ ID NO.1; iii) heavy chain CDR3 at positions 97 to 117 as shown in SEQ ID NO.1.

[0010] Furthermore, the nanobody has an amino acid sequence as shown in SEQ ID NO.1; preferably, the nanobody is encoded by a nucleotide sequence as shown in SEQ ID NO.3.

[0011] The present invention further provides another anti-SEB nanobody (AntiSEB Nb6), wherein the nanobody has at least one of the following CDRs: i) the heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO.2; ii) heavy chain CDR2 at positions 51 to 57 as shown in SEQ ID NO.2; iii) heavy chain CDR3 at positions 96 to 113 as shown in SEQ ID NO.2.

[0012] Furthermore, the nanobody has an amino acid sequence as shown in SEQ ID NO.2; preferably, the nanobody is encoded by a nucleotide sequence as shown in SEQ ID NO.4.

[0013] As shown in SEQ ID NO.1 amino acid sequence: QVQLVESGGRLVQPGGSLSLSCAASGATFSSYAMAWFRRTPGQEREFVAHITWSGESTHYASSVKGRFTISRDNAKNTLSLHMNNLEPEDTGVYYCASEGNPYYSHYPDISPVRFSSWGQGTLVTVSS.

[0014] As shown in SEQ ID NO.2 amino acid sequence: QVQLVESGGGLVQTGGSLRLSCAASGDTTLDYYYIGWFRQGPGKEHEGVSCISSTGSTTQYTESVKGRFTISRDNAKKSVYLQMSGLRPEDTAVYYCAAFFVDPRVTTINIQRVCSMSYDSRGQGTQVTVSS.

[0015] The nucleotide sequence shown in SEQ ID NO.3: CAGGTGCAGCTGGTAGAGTCTGGGGGAAGATTGGTGCAGCCTGGGGGCTCTCTGAGTCTCTCCTGTGCAGCTTCCGGAGCCACCTTTAGTTCTTATGCCATGGCCTGGTTCCGCCGGACTCCAGGGCAGGAGCGTGAGTTTGTAGCACATATTACATGGAGTGGCGAGAGTACTCACTATGCAAGCTCTGTG AAGGGCCGCTTCACCATCTCCAGAGACAACGCCAAGAACACACTGTCGCTACACATGAACAACCTGGAACCTGAGGACACGGGCGTTTATTGTGCTTCAGAAGGGAACCCCTACTATAGCCACTACCCGGACATAAGTCCCGTGCGCTTCAGTTCCTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCA.

[0016] The nucleotide sequence shown in SEQ ID NO4: CAGGTGCAGCTGGTAGAGTCTGGAGGAGGGTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCGTCTGGAGACACCATCAACAGTTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCTATTAGCTGGAGTGATAATCCTTACTATACAGACTCC GTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAAGACACAGCCGTTTATTACTGCGCCCGAGGAAAAGCGAAATTTTACATCAGACTAACTGATGACGAGTATACTGACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0017] In a second aspect, the present invention provides a nucleic acid for encoding the aforementioned Nanobody.

[0018] In a third aspect, the present invention provides a biomaterial, wherein the biomaterial comprises the aforementioned nucleic acid, and the biomaterial is an expression cassette, a vector or a transgenic cell.

[0019] The transgenic cells described in the present invention do not include transgenic cells that have the ability to independently develop into a complete individual, that is, do not include plant varieties and animal varieties.

[0020] In a fourth aspect, the present invention provides a kit comprising the aforementioned nanoantibody, or the aforementioned nucleic acid, or the aforementioned biological material.

[0021] In a fifth aspect, the present invention provides a nanobody combination comprising the two aforementioned nanobodies.

[0022] Furthermore, in the nanoantibody combination, AntiSEB Nb3 is a capture antibody and AntiSEB Nb6 is a detection antibody.

[0023] In a sixth aspect, the present invention provides the use of the aforementioned nanoantibody, or the aforementioned nucleic acid, or the aforementioned biomaterial, or the aforementioned nanoantibody combination in the preparation of a diagnostic reagent, a diagnostic kit or a drug.

[0024] Furthermore, the drug is used for purification, detection or removal of Staphylococcus aureus enterotoxin B.

[0025] Furthermore, based on the nanoantibody combination, Staphylococcus aureus enterotoxin B was detected by a double antibody sandwich method.

[0026] The present invention has the following beneficial effects: The present invention screens out two Staphylococcus aureus enterotoxin B-specific antibodies, using AntiSEB Nb3 as a capture antibody and AntiSEB Nb6 as a detection antibody. The AntiSEB Nb6 antibody provided by the present invention solves the crossover problem between SEB and SEC, and significantly improves the specificity of detecting Staphylococcus aureus enterotoxin B. Both AntiSEB Nb3 and AntiSEB Nb6 have high affinity with SEB and have high sensitivity when detecting SEB.

[0027] The nano-antibody combination provided by the present invention can be used in detection techniques such as enzyme-linked immunosorbent assay (ELISA), western blot, colloidal gold method, etc. for Staphylococcus aureus enterotoxin B, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is the SDS-PAGE identification diagram of the purified SEB protein provided in Example 1 of the present invention; wherein M is a marker.

[0030] Figure 2 The elimination screening product provided in Example 1 of the present invention was diluted by 10 6 The colonies grown after infecting TG1.

[0031] Figure 3 This is the result of expressing AntiSEB Nb3 and AntiSEB Nb6 using the prokaryotic expression system provided in Example 1 of the present invention.

[0032] Figure 4 This is the SDS-PAGE identification result of the purified Nanobody provided in Example 1 of the present invention.

[0033] Figure 5 It is the binding ability of the purified AntiSEB Nb3 provided in Example 2 of the present invention to SEB protein.

[0034] Figure 6 It is the binding ability of the purified AntiSEB N6 provided in Example 2 of the present invention to SEB protein.

[0035] Figure 7 This is the western blot result of AntiSEB Nb3 provided in Example 2 of the present invention against 9 enterotoxins of Staphylococcus aureus.

[0036] Figure 8 This is the western blot result of AntiSEB Nb6 provided in Example 2 of the present invention against 9 enterotoxins of Staphylococcus aureus.

[0037] Fig. 9 This is the affinity fitting curve of the affinity between AntiSEB Nb3 and SEB provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0040] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example:

[0041] Example 1 The present invention provides SEB nanobodies, which are obtained by screening through the following process: 1. SEB protein expression and purification The present invention extracts DNA from strain DC53060 stored in the Chinese Center for Disease Control and Prevention, and amplifies enterotoxin by PCR. seb The PCR amplification conditions were as follows: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and 72°C for 5 min, for a total of 35 cycles. seb The PCR product size was 720 bp. Bm I and Hind The PCR product and expression vector pET28a(+) were digested with enzyme III, and the recombinant plasmid was transformed into Escherichia coli BL21(DE)3 after ligation. After successful ligation, the recombinant plasmid was induced to express with 1mM isopropyl-β-d-thiogalactopyranoside (IPTG). The expressed SEB protein was purified with HisTrap HP column and identified by SDS-PAGE (e.g. Figure 1 As shown in Figure 2, SEB protein was successfully expressed, and a high-purity soluble protein with a molecular weight of 32kD was obtained. The concentration of SEB protein was determined using a BCA protein kit, and a total of 4mg of protein was obtained as an antigen for screening antibodies.

[0042] 2. Nanobody library selection Recombinant SEB protein antibodies were screened from the natural nanoantibody phage display library constructed earlier. First, the ELISA plate was coated with recombinant SEB protein (4 μg / well in the first round, 3 μg / well in the second round, 3 μg / well in the third round, 2 μg / well in the fourth round, and 1 μg / well in the fifth round). 8 replicate wells were set for antigen and 4 replicate wells for blank control (coating solution) were set at 4°C overnight. The coating solution was discarded, and the plate was washed 6 times with PBST. 200-300 μl of blocking solution PBS (containing 2% skimmed milk powder) was added to each well, and the plate was blocked at 37°C for 1 hour. After washing, the phage display library was diluted to 10 with the blocking solution. 12 -10 13pfu, add 100μl phage library to each well and place at 37℃ for 1h. Wash 9 times with PBST (0.1% (v / v) in the first round, 0.1% (v / v) in the second round, 0.2% (v / v) in the third round, 0.3% (v / v) in the fourth round, and 0.3% (v / v) in the fifth round) and wash once with PBS. Add 150μl 0.1M glycine (pH=2.2) to each well and elute for 20min at room temperature, then add 1MTris (pH=8.0) solution for rapid neutralization. The eluted phage infects Escherichia coli TG1 cells in the logarithmic growth phase, shake slowly at 37℃ and 180rpm for 1h. Add about 10 12 pfu of M13KO7 helper phage was placed at 37°C for 40 min, and then ampicillin at a final concentration of 50ug / ml and kanamycin at 25ug / ml were added. The cells were cultured overnight at 220rpm and 30°C. The cells were centrifuged at 12000rpm for 20min, and 1 / 4 volume of PEG / NaCl solution was added to the supernatant. The cells were precipitated on ice for more than 4h. 20μl of the phage suspension was taken to calculate the titer, and clones that bound to SEB were screened through five rounds of panning ( Figure 2 ), the product titer was measured after the fifth round of panning, and the fifth round output was calculated to be 3.59×10 9 pfu.

[0043] 3. Phage-ELISA Phage-ELISA was used to analyze the response of different clones to SEB. Single colonies grown on the above plates were randomly selected and cultured in 96-well deep-well plates (containing LB medium) at 37°C until OD 600 Reach 0.6. Add about 10 12 pfu of M13KO7, incubate at 30°C overnight. Coat the ELISA plate with recombinant SEB (0.5μg / well) protein, take 100μl of the phage display library from the deep-well plate and add it to the ELISA plate, incubate at 37°C for 1h. After washing the plate, add 1:10000 diluted HRP-labeled mouse anti-M13 phage antibody and incubate at 37°C for 1h. After washing the plate, add 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution to each well and measure the absorbance at 450nm. Select clones with significant differences in absorbance between the positive wells and the control wells for sequencing.

[0044] 4. Construction of prokaryotic expression vector to express nanobodies First, the enriched nanobody fragments were amplified by PCR under the following conditions: 95°C for 5 min, 95°C for 30 s, 53°C for 30 s, 72°C for 45 s, 72°C for 5 min, for a total of 35 cycles. The AntiSEB Nb3 PCR product was 384 bp in size, and its sequence was shown in SEQ ID NO.3; the AntiSEB Nb6 PCR product was 372 bp in size, and its sequence was shown in SEQ ID NO.4.

[0045] Plasmid pET-28a(+) and VHH amplified fragment were Bm I and Hind III double enzyme digestion, reaction conditions: 37°C, reaction for 2h. The ligation product was transformed into E. coli BL21(DE)3, and then cultured on LB plates containing 50μg / ml kanamycin at 37°C for 12h, and single clones were screened for protein expression and expression verification.

[0046] Monoclonal clones of two nanobodies were selected and induced to express with 0.5 mM isopropyl-β-d-thiogalactoside (IPTG), as Figure 3 The secretion of AntiSEB Nb3 and AntiSEB Nb6 is shown in FIG. The expressed nanoantibody protein supernatant was purified using a HisTrapHP column and identified by SDS-PAGE to obtain a highly pure soluble protein. Figure 4 As shown, the molecular weight of AntiSEB Nb3 is 19kD, and the sequence is shown in SEQ ID NO.1; the molecular weight of AntiSEB Nb6 is 19kD, and the sequence is shown in SEQ ID NO.2. The concentration of the purified protein was determined using a BCA protein kit. 4mg and 3mg of AntiSEB Nb3 and AntiSEB Nb6 proteins were obtained, respectively, for subsequent nanobody evaluation.

[0047] Example 2 In this example, the Nanobody provided in Example 1 was subjected to functional evaluation, and the process was as follows: 1. Evaluation of the binding ability of nanoantibodies to SEB protein Western blot experiments were used to evaluate the ability of purified nanoantibodies to bind to SEB protein. The SEB obtained after SDS-PAGE in step 1 above was transferred to a PVDF membrane and blocked with skim milk powder for 1 h at room temperature. After washing, it was incubated with purified nanoantibodies at room temperature for 1 h, detected with a 1:5000 dilution of HRP-labeled rabbit anti-camelid antibody, and developed with DAB substrate. AntiSEB Nb3 (1 μg / ml) (as Figure 5 as indicated) and AntiSEB Nb6 (1 μg / ml) (as indicated) Figure 6 as shown).

[0048] 2. Cross-reaction between nanobodies and other enterotoxins The SEB obtained after SDS-PAGE in step 1 of Example 1 and the SEA, SEC, SED, SEE, SEG, SEH, SEK, and SEQ proteins stored in the laboratory were transferred to a PVDF membrane and blocked with skim milk powder for 1 h at room temperature. After washing, they were incubated with nanobodies with binding activity at room temperature for 1 h, detected with a 1:5000 diluted HRP-labeled rabbit anti-camelid antibody, and developed with a DAB substrate. AntiSEB Nb3 (1 μg / ml) (as Figure 7 as indicated) and AntiSEB Nb6 (1 μg / ml) (as indicated) Figure 8 The AntiSEB Nb3 of the present invention can effectively identify the recombinant protein SEB and can also identify the recombinant protein SEC, and its band is located at 32kD, indicating that the AntiSEB Nb3 antibody can simultaneously detect the recombinant SEB protein and the recombinant SEC protein (as shown). Figure 7 The AntiSEB Nb6 of the present invention can effectively identify the recombinant protein SEB, and its band is located at 32kD, and has no cross reaction with other recombinant enterotoxins SEA, SEC, SED, SEE, SEG, SEH, SEK, and SEQ proteins, indicating that the AntiSEB Nb6 antibody has good specificity for the recombinant SEB protein (as shown in Figure 8 as shown).

[0049] 3. Determine the best antibody pair by chessboard titration Nine nanoantibodies with binding activity were labeled with HRP. The titers of the detection antibodies were determined by an indirect method, and the titers of AntiSEB HRP-Nb1 were finally determined to be 1:64,000; AntiSEB HRP-Nb2 was 1:1,000; AntiSEBHRP-Nb3 was 1:16,000; AntiSEB HRP-Nb4 was 1:1,000; AntiSEB HRP-Nb5 was 1:32,000; AntiSEB HRP-Nb6 was 1:64,000; AntiSEB HRP-Nb7 was 1:1,000; and AntiSEBHRP-Nb8 was 1:1,000. The unlabeled nanoantibody was used as a capture antibody to coat the ELISA plate (2 μg / well), incubated at 4°C overnight, and after washing, 5% skim milk powder was used to block at 37°C for 1h; after washing, 2 μg of antigen was added to each well and blocked at 37°C for 1h; after washing, HRP-labeled antibody was added as a detection antibody and blocked at 37°C for 1h; after washing, 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well to measure the absorbance at 450nm. The antibody pair with significant difference in absorbance between the positive well and the control well was selected as the candidate antibody pair for constructing the enzyme-linked immunosorbent double antibody sandwich method. The best antibody pair screened by the present invention is AntiSEBNb3 as the capture antibody and AntiSEB HRP-Nb6 as the detection antibody.

[0050] 4. AntiSEB Nb3 antibody and SEB affinity test experiment: Biolayer Interferometry (BLI) was used to detect the affinity of the antibody. The instrument model for this experiment was OCTET RH16, and the AR2G sensor was selected. The buffer system was PBST+0.1% BSA. AntiSEB Nb3 with a concentration of 10ug / mL was fixed on the sensor surface, and the SEB antigen was diluted to 300nM, 150nM, 75nM, 37.5nM, and 18.8nM in sequence and loaded onto the sensor. The real-time kinetic data of the binding and dissociation of the antigen and the antibody at each concentration was recorded, and the curve of the response signal changing with time was recorded. The sensor surface was rinsed with a specific regeneration buffer to release the bound SEB antigen so that multiple binding / dissociation cycles could be performed. The experimental data was exported and analyzed, and the kinetic model was fitted. The analysis software Octet BLI Discovery 13.0 was used to calculate the binding rate constant (kon) of the antibody and the antigen as 1.27E+05 and the dissociation rate constant (koff) as 3.03E-04. The affinity constant (KD) calculated according to the formula KD=koff / kon was 2.38E-09, indicating that AntiSEB Nb3 has a high affinity with SEB.

[0051] The results are shown in the following table and Fig. 9 As shown: Table 1 Affinity test results of AntiSEB Nb3 and SEB

[0052] 5. Specificity and sensitivity Based on the above results, the present invention determines the double antibody sandwich method using AntiSEB Nb3 as the capture antibody and AntiSEB HRP-Nb6 as the detection antibody.

[0053] 1. After verification by specific experiments, this method does not cross-react with common foodborne strains such as Vibrio parahaemolyticus, Salmonella, Bacillus, Escherichia coli O157:H7, Listeria monocytogenes and Shigella flexneri type 2a during the detection process, and has high specificity.

[0054] 2. After sensitivity experiment verification, the detection limit (LOD) of this method for detecting Staphylococcus aureus enterotoxin B (SEB) can reach 5.57 ng / mL.

[0055] 3. The present invention tested 24 strains containing only SEB and 24 strains containing only SEC. All strains containing SEB were detected, and all strains containing SEC were negative, showing 100% sensitivity and specificity.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-SEB nanobody, characterized in that: The Nanobody has at least one of the following CDRs: i) the heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO.1; ii) heavy chain CDR2 at positions 51 to 58 as shown in SEQ ID NO.1; iii) heavy chain CDR3 at positions 97 to 117 as shown in SEQ ID NO.

1.

2. The Nanobody according to claim 1, characterized in that The nanobody has the amino acid sequence shown in SEQ ID NO.

1.

3. An anti-SEB nanobody, characterized in that: The Nanobody has at least one of the following CDRs: i) the heavy chain CDR1 at positions 26 to 33 as shown in SEQ ID NO.2; ii) heavy chain CDR2 at positions 51 to 57 as shown in SEQ ID NO.2; iii) heavy chain CDR3 at positions 96 to 113 as shown in SEQ ID NO.

2.

4. The Nanobody according to claim 3, characterized in that The nanobody has the amino acid sequence as described in SEQ ID NO.

2.

5. A nucleic acid, characterized in that The nucleic acid is used to encode the Nanobody according to any one of claims 1 to 4.

6. A biomaterial, characterized in that The biological material comprises the nucleic acid according to claim 5, and the biological material is an expression cassette, a vector or a transgenic cell.

7. A kit, characterized in that: Comprising the nanobody according to any one of claims 1 to 4, or the nucleic acid according to claim 5, or the biomaterial according to claim 6.

8. A nanobody combination, characterized in that: Includes the Nanobody of claim 1 or 2, and the Nanobody of claim 3 or 4.

9. Use of the nanobody according to any one of claims 1 to 4, or the nucleic acid according to claim 5, or the biomaterial according to claim 6, or the nanobody combination according to claim 8 in the preparation of a diagnostic reagent, a diagnostic kit or a drug.

10. The use according to claim 9, characterized in that: The medicine is used for purifying, detecting or removing Staphylococcus aureus enterotoxin B.

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