CN117347621A - Method for detecting aflatoxin B1 by using protein mimic antigen-nano antibody - Google Patents
Method for detecting aflatoxin B1 by using protein mimic antigen-nano antibody Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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Abstract
The invention provides a method for detecting aflatoxin B1 by using a protein mimic antigen-nano antibody. The method is to use green fluorescent protein as a simulation antigen for detecting aflatoxin B1. The method uses protein to replace AFB1-BSA and other conjugates as antigens for detection, wherein eGFP is used as a simulated antigen, has low detection IC50 value, is obviously superior to AFB1-BSA, and can better meet detection requirements. In addition, the protein can be subjected to large-scale expression and purification through a prokaryotic expression system, is easy to obtain and low in cost, and can better meet the requirements of AFB1 on environmental protection, low cost and high sensitivity detection.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a method for detecting aflatoxin B1 by using a protein simulated antigen-nano antibody.
Background
Enzyme-linked immunoassay (ELISA) is a method commonly used for molecular detection, and the principle is that specific binding of antigen and antibody is utilized to identify a molecule to be detected, and a signal generated by further enzymatic reaction is used for quantitatively detecting a specific substance in a solution. The method has strong specificity and high sensitivity. The ELISA technology can be used for detecting macromolecules and small molecules, including proteins, antibodies, hormones, medicines and the like. The sandwich method, the indirect method, the competition method, and the like can be classified according to kinds and variations.
Enzyme linked immunosorbent assay technology is a common means for large-scale screening of aflatoxin B1 (AFB 1) at present. The principle of detecting AFB1 by using the traditional ELISA technique is that firstly, a layer of antigen (AFB 1-BSA) obtained by coupling proteins such as specific AFB1 and bovine serum albumin BSA is coated on an ELISA plate, a sample or a standard substance to be detected is added into an orifice plate, the AFB1 in the sample or the standard substance and the antigen fixed on the orifice plate are allowed to be combined in competition with a monoclonal antibody specifically recognizing the AFB1, unbound antibody is removed by washing, an ELISA secondary antibody recognizing monoclonal antibody is added, washing and detection is carried out by enzymatic reaction, and finally, the content of the AFB1 in the sample is calculated according to a standard curve and the detection value of the sample to be detected. This traditional method is cumbersome in steps, requires the use of multiple commercial antibodies, and is costly and has a high false positive rate.
In order to solve the defects of the traditional enzyme-linked immunoassay technology, a one-step bioluminescence enzyme-linked immunoassay technology is developed, and the technology uses a specific nanobody-nano luciferase complex which is fused and expressed in a prokaryotic system to replace the traditional monoclonal antibody and enzyme-labeled secondary antibody, so that the steps are simple, the cost is low and the sensitivity is higher. However, these methods all require coating with a conjugate of AFB1 with other proteins such as BSA (AFB 1-BSA) as an antigen. Taking AFB1-BSA as an example, this antigen is obtained by first preparing AFB1 in high purity, then activating it by a chemical reagent such as carbodiimide, and reacting it with BSA under appropriate conditions. The preparation process is complex, the cost is high, AFB1 needs to be used as a component, and the AFB1 is a chemical extremely harmful to the environment and human beings and has strong carcinogenicity.
Disclosure of Invention
In order to overcome the defect, a method for detecting AFB1 based on a protein mimic antigen-nanobody as a core reagent is invented.
The first object of the invention is to provide the application of the green fluorescent protein in detecting aflatoxin B1.
Preferably, the green fluorescent protein is sfGFP or eGFP, the amino acid sequence of the sfGFP is shown as SEQ ID NO.1, and the amino acid sequence of the eGFP is shown as SEQ ID NO. 2.
The second object of the present invention is to provide a method for detecting aflatoxin B1, which uses green fluorescent protein as a simulation antigen for detecting aflatoxin B1.
Preferably, green fluorescent protein is used as a simulated antigen, and is combined with nano luciferase-nano antibody for detecting aflatoxin B1.
Preferably, the green fluorescent protein specifically binds to a nano-luciferase-nanobody.
Preferably, the green fluorescent protein is sfGFP or eGFP, the amino acid sequence of the sfGFP is shown as SEQ ID NO.1, and the amino acid sequence of the eGFP is shown as SEQ ID NO. 2.
Preferably, the nano luciferase-nano antibody is Nluc-NB28, and the amino acid sequence of the Nluc-NB28 is shown in SEQ ID NO. 3.
Preferably, the method comprises the following steps: and (3) taking green fluorescent protein as a detection antigen, adding a sample to be detected and a nano luciferase-nano antibody into a solid phase carrier coated with the detection antigen, washing after full reaction, adding a fluorescent substrate, measuring bioluminescence intensity, and calculating the content of aflatoxin B1 according to a luminous intensity value.
The third object of the invention is to provide a kit for detecting aflatoxin B1, which takes green fluorescent protein as a detection antigen and nano luciferase-nano antibody as a detection antibody.
The fourth object of the present invention is to provide the use of the above method or the above kit for detecting aflatoxin B1.
The invention has the advantages that:
the method uses protein to replace AFB1-BSA and other conjugates as antigens for detection, wherein eGFP is used as a simulated antigen, has low detection IC50 value, is obviously superior to AFB1-BSA, and can better meet detection requirements. In addition, the protein can be subjected to large-scale expression and purification through a prokaryotic expression system, is easy to obtain and low in cost, and can better meet the requirements of AFB1 on environmental protection, low cost and high sensitivity detection.
Drawings
FIG. 1 is a graph showing the activity of different nanoluciferase-nanobody fusion proteases and binding assays using GFP as a mimetic antigen.
FIG. 2 is a comparison of the detection of AFB1 and its use with AFB1-BSA as antigen using GFP as a mimetic antigen to establish a bioluminescent ELISA assay.
Detailed Description
In order to realize the technical invention, the invention adopts the following technical scheme:
the corresponding amino acid sequence is searched from NCBI database or literature, DNA sequence is designed according to amino acid, and the DNA sequence is delivered to gene synthesis company for synthesis. Green Fluorescent Protein (GFP) and nano luciferase-nano antibody fusion protein (Nluc-NB 28) expression plasmids are generated by a genetic engineering method, and a bioluminescence enzyme-linked immunoassay method based on GFP protein simulated antigen is established for detecting AFB1 based on the proteins. Simultaneously, independent nano luciferase Nluc and fusion proteins (G8-Nluc, nluc-NB 26) of various nano luciferases and anti-AFB 1 nano antibodies are generated by a genetic engineering method, and are used for comparing analysis with the Nluc-NB28 to identify the specificity of the Nluc-NB28 in the analysis of AFB1.
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1: construction of expression vectors
The DNA sequence of the target fragment is as follows:
the green fluorescent protein sfGFP is shown as SEQ ID NO.7, the green fluorescent protein eGFP is shown as SEQ ID NO.8,
Nluc-NB28 is shown as SEQ ID NO.9, nluc-NB26 is shown as SEQ ID NO.10, G8-Nluc is shown as SEQ ID NO.11, and Nluc is shown as SEQ ID NO. 12.
The above sequences were each synthesized by commercial company (Soy silicon based biotechnology Co., ltd.) and genes were inserted into PET.M.3C vector (Soy silicon based biotechnology Co., ltd., cat. No. hat.M.3C: G080-2) using BamH1 and Ecor1 cleavage sites to obtain recombinant plasmids. E.coli DH5 alpha competent cells were transformed with the recombinant plasmid, plated on LB medium plates containing 100. Mu.g/mL ampicillin resistance, and cultured at 37℃for 16 hours to obtain single colonies with uniform growth. The monoclonal is picked and cultured in 50ml culture medium at 37 ℃ overnight, colony PCR verification is carried out on the monoclonal by adopting the general primers T7 and T7-ter of the pet.M.3C vector, and plasmids are extracted after positive clones are obtained for sequencing verification. The recombinant plasmid after sequencing verification is used for subsequent experiments.
Example 2: expression of the protein of interest
S1, respectively converting recombinant plasmids with correct sequence into E.coli BL21 competent cells. 1. Mu.L of recombinant plasmid was added to competent cells, and the mixture was subjected to ice bath for about 30 minutes, followed by heat shock at 42℃for 90 seconds and ice bath for 5 minutes.
S2, 200 mu L of LB medium containing amp (100 mu g/mL, the same applies below) with resistance is added, the culture is carried out at 220rpm and 37 ℃ for about one hour, bacterial liquid is coated on a LB medium plate with corresponding resistance, and the culture is carried out overnight in a constant temperature incubator at 37 ℃.
S3, picking single colonies with good states on the resistance plate, inoculating the single colonies into 5mL LB culture medium containing amp resistance, and culturing at 220rpm and 37 ℃ overnight. The cultured bacterial liquid is transferred into 1L LB medium containing amp resistance, and is cultured at 220rpm and 37 ℃ until OD is about 0.6-0.8. The culture medium was cooled to 16℃by standing at 4-8℃for a period of time, and the protein expression was induced by adding IPTG (LB medium: about 0.3mM final IPTG concentration), and the induction was carried out at 220rpm at 16℃for 18 hours.
Example 3: purification of proteins of interest
S1, centrifuging the induced bacterial liquid at 4000rpm for 15min, discarding the supernatant, and re-suspending the bacterial cells by using 25-30ml binding buffer (20 mM Tris-HCl,500mM NaCl,5mM Imidazole,pH 7.9).
S2, adding a general protease inhibitor 150 mu LPMSF (100 mM dissolved in isopropanol) into the resuspended bacterial liquid, and carrying out ultrasonic waves of 20KHz and 130W, and carrying out ultrasonic waves for 5s and 5s, wherein the total time is 15 minutes, so as to crush the bacterial cells.
S3.4 ℃, centrifuging the crushed thalli at 15000rpm for 30 minutes, and respectively running the supernatant and the sediment after centrifugation to determine whether the protein is soluble.
S4, pouring the supernatant obtained in the step 3 into a Ni affinity column at the temperature of 4 ℃, fully stirring and uniformly mixing, and then stirring and uniformly mixing every 10 minutes to fully combine the protein and the affinity column, wherein the total time is about half an hour.
S5, after combining for a period of time, flowing down the supernatant, washing the affinity column with binding buffer for 2 times
S6, eluting with 5-10mL of the solution buffer (20 mM Tris-HCl,500mM NaCl,1M Imidazole,pH 7.9) for 10 minutes and collecting the eluate.
S7, further purifying the protein by gel filtration chromatography (AKTA protein purifier, hiLoad 16/600Superdex 200pg chromatographic column), and identifying protein expression and purification by SDS-PAGE.
S8, concentrating the high-purity target protein component by using a 3kDa ultrafiltration tube for later use.
Example 4: different nano-luciferase-nano-antibody fusion protease activity assays
S1, buffer solution preparation
S2, substrate configuration: 250 μg of coelengterazine-H (CTZ-H) was dissolved in 613 μl of solvent (85% methanol, 15% glycerol) to give a 1mM final concentration mother liquor.
S3, color development liquid (100 mu L)
S4, testing activities of different nano-luciferases and nano-antibody fusion proteases
Purifying to obtain Nluc-NB28, nluc-NB26 and G8-Nluc, diluting three nano luciferase-nano antibody fusion proteins to 0.1nM, 1nM, 10nM and 100nM by using PBS, adding 50 μl/hole into an ELISA plate, adding CTZ-h substrate color development solution, adding 50 μl/hole, and measuring the change of bioluminescence intensity with concentration by using an ELISA instrument.
The results of the enzyme activity assay showed a corresponding trend from low to high concentration of protein fluorescence values, thus determining that the Nluc-NB28, nluc-NB26, G8-Nluc and Nluc proteins had good enzyme activity (FIG. 1).
Example 5: GFP is used as simulated antigen coating to determine the combination of nano luciferase-nano antibody fusion protein
(1) Binding comparison of different GFP variants
The mock antigens 0. Mu.g/mL GFP (control), 5. Mu.g/mL sfGFP and 5. Mu.g/mL eGFP (experimental) were individually coated onto 96-well plates with PBS buffer (pH=7.4), 100. Mu.L of each well, and allowed to bind overnight at 4 ℃. The next day, the well plate was spun dry, PBST washed three times, and 3% (m/v, the same applies below) skimmed milk powder (300. Mu.L/well) was added and reacted in an incubator at 37℃for 1 hour. PBST was washed five times, 50. Mu.L of 4. Mu.M, 1. Mu.M, 0.1. Mu.M, and 0. Mu.M Nluc-NB28 protein were added to each well, after which the well plates were placed at 37℃for binding for 30 minutes, and the well plates were washed five times with PBST, 100. Mu.L of a color-developing solution was added to each well, and the bioluminescence signal intensity was measured using an enzyme-labeled instrument.
(2) Binding comparison of different antibodies
The mock antigen 5. Mu.g/mL sfGFP, 5. Mu.g/mL eGFP was coated onto 96-well plates with 100. Mu.L of PBS buffer (pH=7.4) and allowed to bind overnight at 4 ℃. The next day, the well plate was spin-dried, PBST washed three times, 3% skimmed milk powder (300. Mu.L/well) was added, and the reaction was carried out in a 37℃incubator for 1 hour. PBST was washed five times, 50. Mu.L of PBS and 50. Mu.L of 4. Mu.M, 3. Mu.M, 2. Mu.M, 1.5. Mu.M, 1. Mu.M, 0.4. Mu.M, 0.1. Mu.M Nluc (control group), nluc-NB28, G8-Nluc or Nluc-NB26 protein (experimental group) were each added, after which the well plate was placed at 37℃for binding for 30 minutes, PBST was washed five times, 100. Mu.L of a color-developing solution was added to each well, and the bioluminescence signal intensity was detected using an enzyme-labeled instrument.
(3) Results
The results of the experiment are shown in FIG. 1, and indicate that both sfGFP and eGFP are capable of specifically binding to Nluc-NB28, and not to G8-Nluc, nluc-NB26 and Nluc.
Example 6: GFP is used as a simulated antigen coating, and nano luciferase-nano antibody fusion protein is used for detecting AFB1
The mock antigens 5. Mu.g/mL sfGFP, 5. Mu.g/mL eGFP and 5. Mu.g/mL AFB1-BSA (control) were each coated with 96-well plates, 100. Mu.L each, and allowed to bind overnight at 4℃in PBS buffer (pH=7.4). The next day, the well plate was spin-dried, PBST washed three times, 3% skimmed milk powder (300. Mu.L/well) was added, and the reaction was carried out in a 37℃incubator for 1 hour. PBST was washed five times with 50. Mu.L of 100ng/mL, 75ng/mL, 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1ng/mL, 0.5ng/mL, 0.1ng/mL AFB1 standard, followed by 50. Mu.L of Nluc-NB28 protein, after which the well plate was placed at 37℃for binding for 30 minutes, PBST was washed five times with 100. Mu.L of color development liquid per well, and the change in bioluminescence signal intensity with different concentrations of AFB1 standard was detected using an enzyme-labeled instrument.
The results show that the combination of two GFP and Nluc-NB28 can be used for the detection of AFB1, wherein the eGFP as a simulated antigen has a low detection IC50 value of 0.482ng/mL, is obviously superior to the AFB1-BSA, and can better meet the detection requirement (figure 2).
SEQ ID NO.1 (Green fluorescent protein Superfold GFP, sfGFP)
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSEQ ID NO.2 (Green fluorescent protein enhanced GFP, eGFP)
MTSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSEQ ID NO.3 (nanoluciferase-nanobody fusion protein Nluc-NB 28)
VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGSGGSGGSGGSMQLQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYTDSVKGRFTINRDNAKNTVYLQMNSLKPEDTAVYYCAAGFWSGNYYRTPDYWGQGTQVTVSSLESEQ ID NO.4 (nanoluciferase-nanobody fusion protein Nluc-NB 26)
VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGSGGSGGSGGSMQLQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAVVNWSGRRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYNCAAGKWDGSYYGAPDYWGQGTQVTVSSEPKTPKPQDGQAGSEQ ID NO.5 (nanoluciferase-nanobody fusion protein G8-Nluc)
MAQLQLVESGGGLVQAGDSLRLSCAASGRTGTIYGMGWFREAPGKEREFVATLWWTVGAPYYADSVKGRFTISRDNDKNTVYLQMNSLKPEDTATYYCALDNRRSYVDYHSVSEYDYWGQGTQVTVSSEPKTPKPQDAASGAEFAAARVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAVDKLAAALESEQ ID No.6 (nano luciferase Nluc)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASEQ ID No.7 (sfGFP nucleotide sequence)
ATGTCCAAAGGTGAAGAACTGTTTACTGGCGTTGTTCCGATTCTGGTTGAACTGGACGGCGACGTTAACGGTCACAAGTTCAGCGTTCGTGGTGAAGGTGAGGGTGACGCGACCAACGGCAAACTGACTCTGAAATTCATCTGTACCACCGGTAAACTGCCGGTTCCATGGCCGACTCTGGTGACTACCCTGACCTACGGCGTACAGTGCTTCAGCCGTTATCCGGACCATATGAAACGTCACGATTTCTTCAAAAGCGCAATGCCGGAGGGTTATGTCCAGGAACGTACTATCAGCTTCAAAGATGACGGTACTTATAAAACCCGTGCGGAAGTTAAATTCGAAGGTGATACTCTGGTGAATCGTATTGAACTGAAAGGCATTGATTTCAAAGAGGATGGCAACATCCTGGGCCACAAACTGGAATACAATTTTAACTCCCATAACGTGTACATCACCGCGGACAAACAGAAAAACGGCATCAAAGCTAACTTCAAAATCCGTCACAACGTTGAAGATGGTTCTGTGCAGCTGGCTGACCACTATCAGCAGAACACTCCTATCGGTGATGGTCCGGTTCTGCTGCCGGATAACCACTACCTGTCCACCCAGTCCGTTCTGAGCAAAGACCCTAACGAAAAACGTGACCACATGGTGCTGCTGGAATTTGTAACCGCGGCTGGCATCACTCACGGTATGGATGAACTGTATAAGSEQ ID NO.8 (eGFP nucleotide sequence)
ATGACTAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAGGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAASEQ ID NO.9 (Nluc-NB 28 nucleotide sequence)
GTGTTCACTCTGGAAGATTTCGTGGGTGATTGGCGCCAGACTGCAGGTTACAATCTGGATCAGGTTCTGGAACAGGGCGGCGTCTCCTCTCTGTTCCAAAACCTGGGCGTTTCTGTTACTCCGATCCAACGTATCGTCCTGTCTGGTGAAAACGGCCTGAAAATTGACATCCACGTGATTATTCCGTACGAAGGTCTGAGCGGTGATCAGATGGGCCAGATTGAAAAAATTTTCAAGGTTGTTTATCCGGTTGACGACCACCACTTTAAAGTTATCCTGCACTATGGTACGCTGGTTATCGACGGTGTCACCCCGAACATGATCGATTATTTTGGTCGTCCGTACGAAGGTATCGCGGTTTTCGACGGCAAAAAAATCACGGTGACCGGCACCCTGTGGAACGGTAACAAAATCATCGACGAACGTCTGATCAACCCGGACGGTTCCCTGCTGTTCCGTGTTACCATCAACGGCGTCACCGGTTGGCGTCTGTGCGAACGTATCCTGGCGGGTGGTTCTGGCGGCTCCGGCGGTAGCGGTGGCTCTATGCAACTGCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAAGCAGGTGGTTCTCTGCGTCTGTCTTGTGCTGCTTCTGGTCGCACCTTCTCCAGCTACGCAATGGGTTGGTTCCGTCAAGCACCAGGTAAAGAACGTGAGTTCGTTGCAGCTATCTCCTGGAGCGGTGGTTCCACTTATTATACTGATTCCGTTAAAGGCCGTTTCACTATCAACCGCGACAACGCTAAAAACACCGTATATCTGCAGATGAACTCCCTGAAACCGGAAGATACTGCTGTGTACTATTGCGCGGCTGGCTTCTGGTCTGGTAACTACTACCGTACCCCGGACTACTGGGGTCAGGGTACCCAGGTTACCGTGTCCAGCCTGGAASEQ ID NO.10 (Nluc-NB 26 nucleotide sequence)
GTGTTCACTCTGGAAGATTTCGTGGGTGATTGGCGCCAGACTGCAGGTTACAATCTGGATCAGGTTCTGGAACAGGGCGGCGTCTCCTCTCTGTTCCAAAACCTGGGCGTTTCTGTTACTCCGATCCAACGTATCGTCCTGTCTGGTGAAAACGGCCTGAAAATTGACATCCACGTGATTATTCCGTACGAAGGTCTGAGCGGTGATCAGATGGGCCAGATTGAAAAAATTTTCAAGGTTGTTTATCCGGTTGACGACCACCACTTTAAAGTTATCCTGCACTATGGTACGCTGGTTATCGACGGTGTCACCCCGAACATGATCGATTATTTTGGTCGTCCGTACGAAGGTATCGCGGTTTTCGACGGCAAAAAAATCACGGTGACCGGCACCCTGTGGAACGGTAACAAAATCATCGACGAACGTCTGATCAACCCGGACGGTTCCCTGCTGTTCCGTGTTACCATCAACGGCGTCACCGGTTGGCGTCTGTGCGAACGTATCCTGGCGGGTGGTTCTGGCGGCTCCGGCGGTAGCGGTGGCTCTATGCAGCTGCAGCTGGTTGAATCTGGTGGTGGTCTGGTACAAGCTGGTGGCAGCCTGCGTCTGAGCTGTGCAGCATCCGGTCGTACTTTCTCTTCTTACGCGATGGGCTGGTTCCGTCAGGCTCCTGGTAAAGAACGTGAATTTGTTGCGGTGGTTAACTGGAGCGGTCGTCGTACCTACTACGCAGACTCTGTTAAAGGTCGTTTCACCATTTCTCGTGATAACGCAAAAAATACCGTGTATCTGCAGATGAACTCTCTGAAACCGGAAGACACCGCCGTTTACAACTGCGCTGCTGGCAAATGGGATGGTAGCTACTACGGCGCACCAGATTATTGGGGTCAGGGCACCCAGGTTACCGTCTCCTCTGAACCTAAAACCCCGAAACCTCAGGACGGTCAGGCAGGTSEQ ID NO.11 (G8-Nluc nucleotide sequence)
ATGGCTCAGCTGCAGCTGGTAGAATCTGGTGGTGGTCTGGTACAAGCTGGCGATTCTCTGCGCCTGTCCTGTGCTGCGTCTGGTCGTACCGGCACCATTTACGGTATGGGTTGGTTCCGTGAAGCTCCGGGCAAGGAACGTGAGTTCGTGGCTACCCTGTGGTGGACCGTTGGTGCTCCGTACTACGCGGACTCCGTGAAAGGTCGTTTCACGATCTCCCGTGACAACGATAAAAACACCGTATATCTGCAAATGAACAGCCTGAAACCGGAAGATACCGCTACCTACTACTGCGCCCTGGACAACCGCCGCAGCTACGTAGACTATCATTCCGTGTCCGAATATGACTACTGGGGTCAGGGTACTCAGGTTACCGTGTCCTCTGAACCGAAAACCCCGAAACCGCAGGATGCAGCTTCCGGTGCCGAATTTGCTGCAGCTCGTGTGTTCACTCTGGAAGATTTCGTGGGTGATTGGCGCCAGACTGCAGGTTACAATCTGGATCAGGTTCTGGAACAGGGCGGCGTCTCCTCTCTGTTCCAAAACCTGGGCGTTTCTGTTACTCCGATCCAACGTATCGTCCTGTCTGGTGAAAACGGCCTGAAAATTGACATCCACGTGATTATTCCGTACGAAGGTCTGAGCGGTGATCAGATGGGCCAGATTGAAAAAATTTTCAAGGTTGTTTATCCGGTTGACGACCACCACTTTAAAGTTATCCTGCACTATGGTACGCTGGTTATCGACGGTGTCACCCCGAACATGATCGATTATTTTGGTCGTCCGTACGAAGGTATCGCGGTTTTCGACGGCAAAAAAATCACGGTGACCGGCACCCTGTGGAACGGTAACAAAATCATCGACGAACGTCTGATCAACCCGGACGGTTCCCTGCTGTTCCGTGTTACCATCAACGGCGTCACCGGTTGGCGTCTGTGCGAACGTATCCTGGCGGTGGATAAACTGGCAGCGGCGCTGGAASEQ ID NO.12 (Nluc nucleotide sequence)
ATGGTGTTCACTCTGGAAGATTTCGTGGGTGATTGGCGCCAGACTGCAGGTTACAATCTGGATCAGGTTCTGGAACAGGGCGGCGTCTCCTCTCTGTTCCAAAACCTGGGCGTTTCTGTTACTCCGATCCAACGTATCGTCCTGTCTGGTGAAAACGGCCTGAAAATTGACATCCACGTGATTATTCCGTACGAAGGTCTGAGCGGTGATCAGATGGGCCAGATTGAAAAAATTTTCAAGGTTGTTTATCCGGTTGACGACCACCACTTTAAAGTTATCCTGCACTATGGTACGCTGGTTATCGACGGTGTCACCCCGAACATGATCGATTATTTTGGTCGTCCGTACGAAGGTATCGCGGTTTTCGACGGCAAAAAAATCACGGTGACCGGCACCCTGTGGAACGGTAACAAAATCATCGACGAACGTCTGATCAACCCGGACGGTTCCCTGCTGTTCCGTGTTACCATCAACGGCGTCACCGGTTGGCGTCTGTGCGAACGTATCCTGGCG。
Claims (10)
1. The application of the green fluorescent protein in detecting aflatoxin B1.
2. The use according to claim 1, wherein the green fluorescent protein is sfGFP or eGFP, the sfGFP having the amino acid sequence shown in SEQ ID No.1 and the eGFP having the amino acid sequence shown in SEQ ID No. 2.
3. A method for detecting aflatoxin B1 is characterized in that green fluorescent protein is used as a simulation antigen for detecting aflatoxin B1.
4. The method of claim 3, wherein the green fluorescent protein is used as a mimetic antigen in combination with a nano-luciferase-nanobody for detection of aflatoxin B1.
5. The method of claim 4, wherein the green fluorescent protein specifically binds to a nano-luciferase-nanobody.
6. The method according to any one of claims 3 to 5, wherein the green fluorescent protein is sfGFP or eGFP, the amino acid sequence of sfGFP is shown in SEQ ID No.1, and the amino acid sequence of eGFP is shown in SEQ ID No. 2.
7. The method of claim 4 or 5, wherein the nano-luciferase-nanobody is a Nluc-NB28, and the amino acid sequence of the Nluc-NB28 is shown in SEQ ID No. 3.
8. A method according to claim 3 or 4, comprising the steps of: and (3) taking green fluorescent protein as a detection antigen, adding a sample to be detected and a nano luciferase-nano antibody into a solid phase carrier coated with the detection antigen, washing after full reaction, adding a fluorescent substrate, measuring bioluminescence intensity, and calculating the content of aflatoxin B1 according to a luminous intensity value.
9. A kit for detecting aflatoxin B1 is characterized in that green fluorescent protein is used as a detection antigen, and nano luciferase-nano antibody is used as a detection antibody.
10. Use of the method of claim 3 or the kit of claim 9 for detecting aflatoxin B1.
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