A single-chain antibody and its application in adsorbing aflatoxin

Abstract

The present application provides a single-chain antibody and its application in adsorbing aflatoxin. The amino acid sequences of the heavy-chain CDRs of the single-chain antibody are SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4; the amino acid sequences of the light-chain CDRs of the single-chain antibody are SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

Description

Field of the Invention

[0001] This application belongs to the field of antibodies. Specifically, this application provides a single-chain antibody and its application in adsorbing aflatoxin. Background Art

[0002] Mycotoxins are a series of secondary metabolites produced by various fungi. Among the approximately 10,000 fungi that have been isolated and identified, about 5% can produce toxic complexes, and nearly 50 mycotoxins can cause toxicity and carcinogenicity to humans and animals by inhibiting the synthesis of DNA, RNA, proteins, and various enzymes or by damaging cell structures. Mycotoxin contamination is widespread in all links of the growth, harvesting, storage, processing, circulation, and sales of human food and livestock feed, posing a great threat to the health of humans and livestock. According to reports by the Food and Agriculture Organization of the United Nations, a large proportion of the world's food is wasted due to mycotoxin contamination every year.

[0003] Among mycotoxins, aflatoxin B1 is the most harmful to humans and livestock. The detection and removal of AFB1 in food and feed have become one of the research hotspots for ensuring public safety and livestock health. On the one hand, sensitive detection of AFB1 requires antibodies with good effect, simple preparation, and stability; on the other hand, antibodies

[0004] In the past decade, the methods for removing AFB1 from food and feed mainly include: physical adsorption method, chemical method, and biological enzyme detoxification method. However, these methods have problems such as poor stability, easy loss of nutrients, generation of secondary toxic substances, high cost, and harsh storage conditions. Therefore, developing stable, efficient, non-toxic, and environmentally friendly removal methods using new biotechnology is an important development direction for AFB1 removal. Summary of the Invention

[0005] On the one hand, this application provides a single-chain antibody. The amino acid sequences of the heavy-chain CDRs of the single-chain antibody are SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4; the amino acid sequences of the light-chain CDRs of the single-chain antibody are SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0006] Further, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is SEQ ID NO.1, and the amino acid sequence of the light-chain variable region of the single-chain antibody is SEQ ID NO.5.

[0007] Further, the single-chain antibody is composed of a heavy-chain variable region, a linker peptide, and a light-chain variable region arranged in sequence from the amino terminus to the carboxyl terminus.

[0008] Further, the amino acid sequence of the linker peptide is SEQ ID NO.9.

[0009] Further, the amino acid sequence of the single-chain antibody is SEQ ID NO. 10.

[0010] On the other hand, the present application provides a gene encoding the above single-chain antibody, and the gene contains the nucleic acids shown in SEQ ID NO. 11 and SEQ ID NO. 12.

[0011] Further, the gene contains the nucleic acid shown in SEQ ID NO. 14.

[0012] On the other hand, the present application provides a method for preparing the above single-chain antibody, and the method includes the step of culturing a host cell, and the host cell contains the above gene.

[0013] On the other hand, the present application provides the use of the above single-chain antibody in adsorbing aflatoxin.

[0014] Further, the application is used for adsorbing aflatoxin in food or feed.

[0015] Further, the aflatoxin is aflatoxin B1

[0016] On the other hand, the present application provides the use of the above single-chain antibody in preparing a kit for detecting aflatoxin B1.

[0017] Further, the kit is an ELISA kit or a colloidal gold immunochromatography kit.

[0018] The above host cells can be selected from various yeasts, Escherichia coli, Bacillus subtilis, and cell-free expression systems, etc. Those skilled in the art can also select a suitable commercial vector according to the host and perform operations such as insertion according to the operation instructions.

[0019] In the case of knowing the CDR region or variable region of an antibody, those skilled in the art can routinely design parts such as the framework region and design the corresponding gene sequence according to the host preference. Brief Description of the Drawings

[0020] Figure 1 It is the detection result of serum antibody titer;

[0021] Figure 2 It is the electrophoresis analysis of the PCR amplification products of rabbit VH, VLκ and VLλ fragments;

[0022] Figure 3A Amino acid sequence analysis of the scFv heavy chain antibody fragment containing the correct reading frame;

[0023] Figure 3B Amino acid sequence analysis of the scFv light chain antibody fragment containing the correct reading frame;

[0024] Figure 4 For SDS-PAGE analysis of antibody expression;

[0025] Figure 5 For SDS-PAGE analysis of antibody purification results;

[0026] Figure 6 For Western Blot identification of the purified antibody. Specific implementation mode

[0027] Example 1 Animal immunization

[0028] Antigen preparation:

[0029] The active ester method was used to conjugate the AFB1 hapten with BSA and OVA to prepare the immunogen and the coating antigen. Among them, AFB1-BSA was used as the immunogen and AFB1-OVA was used as the coating antigen.

[0030] Animal immunization:

[0031] The aflatoxin B1-BSA conjugate in Example 1 was used for immunization. Four New Zealand rabbits were immunized, 150 μg / rabbit, once every two weeks for 4 times. It was recorded as day 0 before immunization. On day 1, it was mixed with Freund's complete adjuvant for the first immunization. On days 14, 28, and 42, it was mixed with Freund's incomplete adjuvant for the second, third, and fourth immunizations respectively. 2 mL of peripheral blood was collected on days 0, 35, and 49 respectively for serum titer detection.

[0032] The antiserum titer was detected by indirect ELISA. The specific steps of indirect ELISA are as follows:

[0033] (1) Dilute aflatoxin B1-OVA with PBS coating solution to the required concentration, mix well and add it to the strip, 100 μL per well, and incubate overnight in a refrigerator at 4°C.

[0034] Coating antigen: Aflatoxin B1-OVA;

[0035] Coating concentration: 2 μg / mL, 100 μL / well;

[0036] Coating buffer: Phosphate buffer (PBS, pH 7.4).

[0037] (2) After coating, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate in a constant temperature incubator at 37°C for 1 h. Take out the enzyme-labeled plate, discard the internal solution, and wash the plate once.

[0038] (3) Dilute the antiserum 3-fold at 1:500, 100 μL per well, and incubate in a constant temperature incubator at 37°C for 1 h.

[0039] (4) Take out the enzyme-labeled plate, discard the internal liquid, wash the plate 3 times, and add 100 μL of diluted enzyme-labeled secondary antibody (goat anti-rabbit, 1:50,000) to each well. Incubate in a constant temperature incubator at 37 °C for 1 h.

[0040] (5) Take out the enzyme-labeled plate, discard the internal liquid, wash the plate 4 times, add 100 μL of TMB chromogenic solution to each well first, and incubate at 37 °C for 15 min.

[0041] (6) Add 100 μL of 1 M HCl solution to each well to terminate the reaction. Immediately read the absorbance at 450 nm on an enzyme-labeled instrument. The dilution corresponding to the well with an OD value greater than 2.1 times the OD value of the set negative control is defined as the titer of the sample.

[0042] The ELISA assay results showed that after 4 immunizations, a certain immune response occurred. The titers of the anti-serum of 4 rabbits against aflatoxin B1-OVA were all greater than 1093.5K-fold dilution, meeting the experimental standards for downstream construction of the phage display library ( Figure 1 ).

[0043] Example 2 Construction of phage-ScFV library

[0044] Isolate rabbit anticoagulated PBMC from 20 mL of peripheral blood collected, extract the total RNA of the sample using TRIzol, and reverse transcribe to obtain cDNA. Using cDNA as a template, the scFv antibody gene fragment ( Figure 2 ) was obtained by PCR amplification, and NotI and SfiI restriction enzyme sites were introduced at its 5' end and 3' end respectively. After double digestion with NotI / SfiI, it was inserted into the phagemid vector pCantab5E digested with the same enzymes, and electrotransformed into E. coli TG1 to obtain the M13 phage display library displaying scFv. The library capacity was 2.0×10 9 . Randomly pick 30 clones from the library for DNA sequencing analysis, and a total of 22 correct sequences ( Figure 3A and Figure 3B ) were obtained, and the correct rate of the library was approximately 73.30%.

[0045] Example 3 Enrichment screening of phage antibody library

[0046] Construction of phage antibody library:

[0047] Take 100 μL of the preserved E. coli TG1 transformed bacterial liquid library, add it to 25 mL of 2×YT liquid medium (ampicillin 100 μg / mL), and culture it in a shaker at 37 °C until OD 600nmAfter reaching 0.4, an appropriate amount of helper phage M13KO7 was added (at a ratio of Escherichia coli count / phage count = 1 / 20). After mixing evenly, it was first placed at 37°C and left static for 30 min, then placed in a shaker at 37°C and 150 r / min for 30 min. The culture solution was centrifuged at 8000 r / min for 15 min. After discarding the supernatant, the precipitate was resuspended in 50 mL of 2×YT liquid medium (ampicillin 100 μg / mL, kanamycin 70 μg / Ml) and cultured overnight in a shaker at 37°C and 150 r / min.

[0048] The next day, the culture solution was centrifuged at 10000 r / min for 20 min at 4°C. The supernatant was transferred to another sterile centrifuge tube. 5 mL of pre-cooled PEG / NaCl solution was added, and after mixing evenly, it was placed on ice for precipitation for 1 h. Then it was centrifuged at at 10000 r / min for 30 min at 4°C to remove the supernatant. The precipitate was resuspended with 600 μL of PBS buffer. The resuspended solution was centrifuged again at 12000 r / min for 10 min at 4°C, and the supernatant was transferred to another sterile 1.5 mL centrifuge tube. This supernatant is the primary phage antibody library.

[0049] Determination of the concentration of the phage antibody library:

[0050] Take 10 μL of the primary phage antibody library and perform gradient dilution with PBS buffer. Take 100 μL of the diluted solution and mix it with 900 μL of E.coil TG1 OD 600nm = 0.4. Gently shake at 37°C for 30 min. Take 100 μL of the culture solution and spread it on a 2×YT solid plate (ampicillin 100 μg / mL), and culture it overnight at 37°C. Calculate the concentration of the phage antibody library based on the number of colonies.

[0051] Screening and enrichment of the anti-AFB1 ScFV phage antibody library:

[0052] Dilute the antigen AFB1-OVA to 10 μg / mL, add 100 μL of the above diluted solution to each well, and incubate overnight at 4°C. Block the ELISA plate with PBST solution containing 5% skim milk. Adjust the titer of the recombinant phage obtained in the previous step to 10 12 cfu / mL, add 100 μL to each well, and incubate at 37°C for 2 h. Wash 5 times with PBST and PBS respectively. Add 200 μL of Gly-HCl buffer at pH 2.2 to each well, incubate at 37°C with humidity for 6 min for elution. During the elution process, pipette several times to ensure complete elution. Add an appropriate amount of Tris-HCl buffer to the eluted phage to neutralize it to pH 7.0. Immediately collect the eluate and infect E.coil TG1 OD 600nm= 0.4, and placed in a shaker at 37°C with gentle shaking for 30 min. Take 10 μL of the eluate and add it to 90 μL of 2×YT liquid medium, then spread it on a 2×YT solid plate (ampicillin 100 μg / mL), and incubate it upside down at 37°C overnight. The next day, record the number of colonies on the plate, and based on the number of colonies, estimate the number of phages eluted. The remaining bacterial solution is cultured again to expand, and the phages are precipitated using the PEG / NaCl solution to enter the next round of panning. This experiment was repeated 2 times for the screening step, and the concentration of the antigen AFB1-OVA during the screening process was 10 μg / mL. The screening and enrichment results are shown in Table 1. After the first round of screening, the titer of the eluted scFv decreased to 3.60×10 5 cfu / mL, and the phage-scFv captured in the second round of screening was 1.80×10 6 cfu / mL, and it reached 1.98×10 6 cfu / mL in the third round, with a 5.5-fold enrichment.

[0053] Table 1 Enrichment screening of anti-AFB1 ScFV

[0054]

[0055] Example 4 Preliminary identification of positive ScFV phage clones

[0056] A total of 940 monoclonal colonies were picked from the resistance plates for measuring the eluted phage concentration in the second and third rounds and inoculated into 10 96-well culture plates. 200 μL of 2×YT liquid medium (ampicillin 100 μg / mL) was pre-added to each well and cultured overnight at 37°C on a shaker at 150 r / min. The next day, 2 μL was taken from each well and inoculated into another 96-well plate at 37°C. Each well contained 200 μL of 2×YT liquid medium (ampicillin 100 μg / mL) and was cultured at 37°C at 150 r / min for 2 h. An appropriate amount of helper phage M13KO7 solution (with the ratio of Escherichia coli number / phage number = 1 / 20) was added to each well to infect E. coil TG1, and the mixture was cultured with shaking at 37°C at 150 r / min for 1 h. The culture solution was centrifuged at 5000 r / min for 10 min, and the supernatant was discarded. The precipitate was resuspended in 198 μL of 2×YT liquid medium (ampicillin 100 μg / mL, kanamycin 70 μg / mL) and cultured overnight at 37°C at 150 r / min. The next day, the culture solution was centrifuged at 5000 r / min for 10 min, and 80 μL of phage supernatant was taken from each well for Phage ELISA detection. A total of 940 bacteria were randomly picked from the plates for measuring the concentration in the second and third rounds for phage ELISA. AFB1-OVA at 2 μg / mL was used as the coating antigen, OVA at 10 μg / mL was used as the non-specific antigen, and PBS was used as the negative control. The judgment criterion was that the OD450 value of the tested antibody / OD450 value of the negative antibody > 2.1, and the OD450 value of the tested antibody > 0.5. Finally, 38 positive clones were obtained (Table 2).

[0057] Table 2 Detection results of positive phages and OVA Phage ELISA

[0058]

[0059] Note: Bold is the negative control.

[0060] Example 5 Sequence analysis of positive phage antibodies

[0061] Gene sequence analysis:

[0062] The 38 positive clones were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. According to the sequencing results, the nucleic acid sequences obtained by using NCBI Blast alignment were analyzed for homology with other antibody genes, and a total of 15 unique sequences were obtained.

[0063] Competitive detection of free small molecules:

[0064] Fifteen unique clones were subjected to free small molecule competition assays. The results are shown in Table 3. The OD values of the 15 unique clones decreased compared to those in the Phage Elisa, indicating that the screened ScFV antibodies had competition against free small molecules and could specifically bind to the small molecule AFB1.

[0065] Table 3 Free small molecule competition assays

[0066]

[0067]

[0068] Note: Bold indicates the PBS negative control

[0069] Example 6 Prokaryotic expression and purification of single-chain antibodies

[0070] Antibody expression:

[0071] Clone 2-1 was selected for prokaryotic expression. The plasmid containing the antibody gene was transformed into Arctic-Express competent cells, and then evenly spread onto an LB plate (containing 50 μg / mL kanamycin sulfate), and then incubated overnight in a 37 °C incubator in an inverted position. Monoclonal colonies were picked from the transformed plate and inoculated into 4 mL of LB medium (containing 50 μg / mL kanamycin sulfate). When the culture reached an OD600 of 0.5 - 0.8, IPTG with a final concentration of 0.2 mM was added to the test tube culture solution, and then induced expression was carried out at 15 °C and 37 °C respectively. The induced culture solution was centrifuged at 12,000 rpm for 5 min, the supernatant was removed, the precipitate was resuspended with PBS solution, and finally SDS-PAGE loading buffer was added and the sample was heated at 100 °C for 10 min, and then centrifuged to take the supernatant for electrophoresis. The whole bacteria were lysed by ultrasonic treatment with 20 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-100, 1 mM DTT, and 1 mM PMSF. The supernatant and precipitate were analyzed by SDS-PAGE. Through the analysis of whole bacteria ultrasonic lysis, no obvious expression was observed in the induced supernatant, and it was expressed as inclusion bodies ( Figure 4 ).

[0072] Antibody purification:

[0073] The inclusion bodies were washed with 50 mM Tris (pH 8.0), 300 mM NaCl containing 1% Triton X-100, 2 mM EDTA, and 5 mM DTT, and then the inclusion bodies were dissolved with a buffer of 50 mM Tris (pH 8.0), 300 mM NaCl, 8 M urea, and 20 mM imidazole to equilibrate the Ni-IDA column at the same time. Finally, the target protein was eluted with an equilibration buffer containing different concentrations of imidazole, and each elution fraction was collected for SDS-PAGE analysis and detection( Figure 5 ). Western Blot was used to analyze and identify the purified protein( Figure 6 ).

[0074] Purified antibody detection:

[0075] The purified antibody (starting from 12.5 μg / mL, 2-fold dilution) was detected with ELISA wells coated with aflatoxin OVA conjugate (2.5 μg / mL / well), and the purified antibody (starting from 12.5 μg / mL, 2-fold dilution) + 50 μL / well of aflatoxin B1 (20 μg / mL) was used for competitive detection with ELISA wells coated with aflatoxin OVA conjugate (2.5 μg / mL / well). The results showed that the purified antibody could bind to the aflatoxin OVA conjugate (Table 4), and there was competition with the small molecule aflatoxin B1 (Table 5).

[0076] Table 4 Indirect ELISA detection of prokaryotic expression protein (aflatoxin OVA conjugate)

[0077]

[0078] Table 5 Indirect competitive ELISA detection of prokaryotic expression protein

[0079]

[0080] Note: The bold is the PBS negative control.

[0081] Example 7 Preliminary preparation and application of colloidal gold test strip

[0082] Zhongke Keyou 20 nm colloidal gold particles were selected; the labeling concentration of the antibody was 15 ng / mL and the pH was 8.0; ZC-J6 gold label pad from Shanghai Jieyi was selected; 1 mg / mL of aflatoxin B1-BSA was used to coat the T line, and 1 mg / mL of goat anti-mouse IgG was used to coat the C line; the aflatoxin B1 colloidal gold test strip was assembled.

[0083] The specificity was verified using a variety of high-concentration mycotoxin solutions, and the sensitivity was judged using a 30% methanol solution of aflatoxin B1 with gradient concentrations.

[0084] It has good specificity for fumonisin 1, fumonisin 2, ochratoxin, and zearalenone toxin reactions; the detection sensitivity is at the level of 0.5 - 1 ng / mL (the color development at 0.5 ng / mL is already very faint); if further optimized with reagents (such as nanogold modification, particle size) and materials (NC membrane, gold label pad), the sensitivity is expected to be further improved.

[0085] Example 8 Antibody 2 - 1 Related Sequences

[0086] Amino acid sequence of the variable region of the antibody heavy chain, SEQ ID NO.1:

[0087] PVVGGVRGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEWIGIISSSDSTYCASWAKGRFTIS

[0088] KTSTTVDLKITSPTTEDTATYFCARGFDLWGQGTLVTVSSGQPKAPSV CDR - H1 amino acid sequence, SEQID NO.2:

[0089] GFSLSSYA CDR - H2 amino acid sequence, SEQ ID NO.3:

[0090] ISSSDST CDR - H3 amino acid sequence, SEQ ID NO.4:

[0091] ARGFDL

[0092] Amino acid sequence of the variable region of the antibody light chain, SEQ ID NO.5:

[0093] ELVLTQSPSLSASLDTTARLTCTLSTGYSVGSFVIAWYQQVPGRPPRYLLTYYTEEIKDQASGVHSCFS

[0094] GSKDDSANAGVLTISGLQPEDEADYYCATPHGSGNNFHYVFGGGTQLTVT CDR - L1 amino acid sequence, SEQ ID NO.6:

[0095] TGYSVGSFV CDR - L2 amino acid sequence, SEQ ID NO.7:

[0096] YYTEEIK CDR - L3 amino acid sequence, SEQ ID NO.8:

[0097] ATPHGSGNNFHYV

[0098] Linker peptide amino acid sequence, SEQ ID NO.9:

[0099] GGGGSGGGGSGGGGS

[0100] Antibody amino acid sequence, SEQ ID NO.10:

[0101] PVVGGVRGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEWIGIISSSDSTYCASWAKGRFTIS

[0102] KTSTTVDLKITSPTTEDTATYFCARGFDLWGQGTLVTVSSGQPKAPSVGGGGSGGGGSGGGGSELVLTQ

[0103] SPSLSASLDTTARLTCTLSTGYSVGSFVIAWYQQVPGRPPRYLLTYYTEEIKDQASGVHSCFSGSKDDS

[0104] ANAGVLTISGLQPEDEADYYCATPHGSGNNFHYVFGGGTQLTVT

[0105] Heavy chain nucleotide sequence, SEQ ID NO.11:

[0106] CCAGTCGTTGGAGGAGTCCGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTC

[0107] TCTGGATTCTCCCTCAGTAGCTATGCAATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGG

[0108] ATCGGAATCATTAGTAGTAGTGATAGCACATACTGCGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC

[0109] AAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGT

[0110] GCCAGAGGGTTTGACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCA

[0111] TCAGTC

[0112] Light chain nucleotide sequence, SEQ ID NO.12:

[0113] GAGCTCGTGCTGACTCAGTCGCCCTCCCTGTCTGCGTCTCTGGACACAACGGCCAGACTCACCTGCACC

[0114] CTGAGCACTGGCTACAGTGTTGGCAGTTTTGTAATTGCCTGGTACCAGCAGGTGCCAGGGAGGCCTCCC

[0115] AGGTATCTCCTGACCTACTACACAGAAGAAATTAAAGACCAGGCCTCCGGGGTGCACAGCTGCTTCTCT

[0116] GGATCCAAGGATGACTCGGCCAATGCAGGCGTCCTAACCATCTCTGGGCTGCAGCCCGAGGACGAGGCC

[0117] GACTATTACTGTGCTACACCTCACGGTAGTGGGAACAACTTCCATTATGTGTTTGGCGGAGGGACCCAG

[0118] CTGACCGTCACA

[0119] Linker peptide nucleotide sequence, SEQ ID NO.13:

[0120] GGTGGCGGAGGGAGTGGGGGAGGCGGTTCTGGCGGAGGTGGGTCG

[0121] Antibody nucleotide sequence, SEQ ID NO.14:

[0122] CCAGTCGTTGGAGGAGTCCGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTC

[0123] TCTGGATTCTCCCTCAGTAGCTATGCAATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGG

[0124] ATCGGAATCATTAGTAGTAGTGATAGCACATACTGCGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC

[0125] AAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGT

[0126] GCCAGAGGGTTTGACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCA

[0127] TCAGTCGGTGGCGGAGGGAGTGGGGGAGGCGGTTCTGGCGGAGGTGGGTCGGAGCTCGTGCTGACTCAG

[0128] TCGCCCTCCCTGTCTGCGTCTCTGGACACAACGGCCAGACTCACCTGCACCCTGAGCACTGGCTACAGT

[0129] GTTGGCAGTTTTGTAATTGCCTGGTACCAGCAGGTGCCAGGGAGGCCTCCCAGGTATCTCCTGACCTAC

[0130] TACACAGAAGAAATTAAAGACCAGGCCTCCGGGGTGCACAGCTGCTTCTCTGGATCCAAGGATGACTCG

[0131] GCCAATGCAGGCGTCCTAACCATCTCTGGGCTGCAGCCCGAGGACGAGGCCGACTATTACTGTGCTACACCTCACGGTAGTGGGAACAACTTCCATTATGTGTTTGGCGGAGGGACCCAGCTGACCGTCACA。

[0132] As described above, it is only a preferred specific embodiment of the present invention. However, the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A single-chain antibody that binds to aflatoxin B1, characterized in that: The amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO.

5.

2. The single-chain antibody according to claim 1, wherein the single-chain antibody consists of a heavy chain variable region, a connecting peptide and a light chain variable region arranged in sequence from the amino terminus to the carboxyl terminus. 3 . The single-chain antibody according to claim 1 , wherein the amino acid sequence of the single-chain antibody is SEQ ID NO.

10.

4. The gene encoding the single-chain antibody according to claim 3, characterized in that The gene comprises the nucleic acid shown in SEQ ID NO.

14.

5. The method for preparing the single-chain antibody according to claim 3, characterized in that: The method comprises the step of culturing a host cell comprising the gene according to claim 4.

6. Use of the single-chain antibody according to any one of claims 1 to 3 in adsorbing aflatoxin B1.

7. The use according to claim 6, which is used for adsorbing aflatoxin B1 in food or feed.

8. Use of the single-chain antibody according to any one of claims 1 to 3 in the preparation of a kit for detecting aflatoxin B1.

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