CN114989309A - Nano antibody for rapidly detecting various aflatoxins and application of nano antibody in detection of prepared vegetables - Google Patents
Nano antibody for rapidly detecting various aflatoxins and application of nano antibody in detection of prepared vegetables Download PDFInfo
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- 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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- 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
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
Abstract
The invention provides a nano antibody for rapidly detecting a plurality of aflatoxins and application thereof, wherein the nano antibody targets the epitopes of a plurality of common aflatoxins such as aflatoxin B1(AFB1), aflatoxin M1(AFM1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2) and the like. A contrast test proves that the nano antibody is more sensitive than a commercial monoclonal antibody in aflatoxin detection, the nano antibody is combined with a colloidal gold labeling technology, the sensitivity is high, and the detection limit for detecting PRV can reach less than 10 ng/mL. Strong specificity and less cross reaction. The colloidal gold test strip developed for the first time realizes rapid detection, shortens the originally time-consuming immune identification test into rapid detection finished in a few minutes, and realizes the primary application of rapid detection of aflatoxin.
Description
Technical Field
The invention relates to the field of protein antibodies and detection thereof, in particular to a nano antibody for rapidly detecting various aflatoxins and application thereof.
Background
Aflatoxins are mainly secondary metabolites secreted by aspergillus flavus and aspergillus parasiticus, and are natural toxic compounds which can cause various damages to people and livestock. Aflatoxins are difuranocoumarol derivatives with similar chemical structures, and more than 20 aflatoxins are found at present and mainly comprise aflatoxins B1(AFB1), B2(AFB2), G1(AFG1), G2(AFG1), M1(AFM1) and the like. The aflatoxin B1 has the highest toxicity, which is 10 times that of potassium cyanide and 68 times that of arsenic trioxide. Aflatoxin B1 was classified by the cancer research institute of the world health organization as one of the strongest known carcinogenic chemicals, i.e., group i carcinogens, as early as 1993. Various aflatoxins are mainly found in mildewed peanuts, grains, nuts and rice. It is the first of the toxicity because of the severe damage to human and livestock livers. They are commonly found in various animal feeds and human foods. Feeding livestock and poultry with contaminated feed causes contamination of meat, milk and products thereof, so care must be taken to examine food raw materials particularly susceptible to contamination by aflatoxins, and aflatoxins in foods processed with these raw materials, such as peanuts, walnuts, pistachios, almonds, peach kernels and plum kernels, shredded coconut, sesame and various foodstuffs. The eating of contaminated food by human beings can cause acute poisoning, liver necrosis and hemorrhage, and chronic poisoning can cause liver cancer. Meanwhile, the livestock and poultry fed by the polluted feed can reduce the production rate of the livestock and poultry, slow the weight gain and cause great economic loss.
In recent years, a number of detection methods for aflatoxin have been studied, and these methods can be roughly classified into Thin Layer Chromatography (TLC), liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). The thin layer chromatography is the first detection method in GB5009.22-2003, A method for measuring aflatoxin B1 in food, but the method is not specific and easily causes other fluorescent substances in a sample to interfere with the measurement. The liquid chromatography has strong specificity, but the experimental process is complicated, the loss of the measured substance is easily caused, the required instruments and equipment are expensive, and the popularization is reduced. The enzyme linked immunosorbent assay is one of the more common methods for measuring aflatoxin at present, and the method provides a TLC method which is used in the national standard before for measuring aflatoxin. The aflatoxin is measured by a TLC method, the steps are multiple, the time consumption is long, the used reagents are various, and only semi-quantification can be realized. In the operation process of the experiment, an experimenter needs to be in direct contact with the toxin for a long time, and the method is not suitable for detecting a large quantity of samples and cannot realize rapidness. The ELISA method has high sensitivity and high analysis speed, simplifies the steps of extraction and purification, only needs 2 hours in the whole experiment, can determine a plurality of samples at one time, and has the unique advantage of ELISA, but because the method utilizes the specific binding reaction of an antigen and an antibody and the biological, physical or chemical amplification of the antibody and a marker on the antigen to qualitatively and quantitatively detect the ultra-trace residues, the similarity of similar molecules and the immunodetection methods such as ELISA and the like easily generate certain cross reactivity on other aflatoxins or chemical substances with similar structures, thereby influencing the accuracy of the experiment.
Single domain antibodies contain only the variable region of the heavy chain (VHH), which is of small molecular weight and can be isolated and screened from camelids by molecular cloning. The single domain antibody not only has the specificity and reactivity of the traditional antibody, but also has higher stability, solubility, permeability and the like. However, a satisfactory single domain antibody for various aflatoxins is still lacking in the prior art, and although various nano antibodies for aflatoxins exist in the prior art, the single property antibodies are mostly limited by the preparation method, and the effect of realizing various detections by one antibody cannot be realized. In particular, in practical applications, during food testing, the tester generally does not care which of aflatoxins B1(AFB1), B2(AFB2), G1(AFG1), G2(AFG1) and M1(AFM1) the contaminant is, but directly rejects the aflatoxins. Therefore, the development of a novel broad-spectrum single-domain antibody which effectively aims at various aflatoxins is urgently needed in the field, a high-efficiency detection method is established, and a test basis and a candidate material are provided for the development of a corresponding detection reagent.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for quickly and specifically detecting various aflatoxins, so as to solve the problems in the prior art.
In order to achieve the above purpose, the invention provides an optimized aflatoxin epitope fusion polypeptide, which comprises a plurality of common aflatoxin epitopes such as aflatoxin B1(AFB1), aflatoxin M1(AFM1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2), and the like, and the three-dimensional structure of the epitope fusion polypeptide is optimized, so that the epitopes are exposed in a high-level structure and named as AFBMG, and the amino acid sequence of the epitope fusion polypeptide is as follows: SAEEHAVSDPEHPGPSDDVMAISAPDGEGDWPLYRLNPWSDGALDARTAVRAMAPSSWRVATERATAEVALGQRVLTSFRPHSARVLARDREHVCVRCYSLKPGLCGAVYVQNTFIEGQLGDNGTAVFSE (SEQ ID No. 1).
Further, the invention provides a Nano antibody capable of identifying aflatoxin with high accuracy and sensitivity, the Nano antibody targets epitopes of a plurality of common aflatoxins such as aflatoxin B1(AFB1), aflatoxin M1(AFM1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2), and is named as Nano-AF, and the amino acid sequence of the Nano antibody is as follows: QLQLGGSGGGLVQPGGLVQACAASGSTFAGFRAMGWRQAFRQAREFVAAITWSGASTYYTDSVKGRFTISRDNAKNTVYLQMNNLESLKPEDTAVYYCAGFMYYASNYCRYWGQGTQVTVSSQGTQVKPQD (SEQ ID No. 2).
Further, the invention provides a preparation method of the NANO antibody NANO-AF, which comprises the following steps:
(1) construction of recombinant eukaryotic expression vectors: connecting a DNA sequence of the coding NANO antibody NANO-AF to an expression vector through PCR amplification and enzyme digestion to obtain a positive plasmid;
(2) and (2) transforming the positive plasmid in the step (1) into a host cell, and inducing and expressing the NANO antibody NANO-AF.
Further, the expression vector may be a eukaryotic expression vector or a prokaryotic expression vector, and preferably, the expression vector is a eukaryotic expression vector, and the expression vector may be selected from the group consisting of: pCRII, pCR3, and pcDNA3.1(Invitrogen, San Diego, CA), pB SII (Stratagene, La Jolla, CA), pET 15(Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N1(Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO90/14363), and pFastBacDual (Gibco-BRL, Grand Island, NY), and the like.
The host cell is e.coli, yeast or eukaryotic cell, preferably the host cell is eukaryotic cell, which may be selected from e.g.: CHO of Chinese hamster ovary cells, COS of monkey kidney cells, HEK-293 of human embryonic kidney cells, HELA of human cervical cancer cells, etc.
Furthermore, the invention provides a rapid detection test strip for aflatoxin, which adopts a mode of combining the NANO antibody NANO-AF and the colloidal gold test strip to rapidly detect aflatoxin, thereby realizing rapid detection, shortening the original time-consuming immune identification test for a plurality of hours into rapid detection finished in a few minutes, and realizing the primary application of the NANO antibody NANO-AF in the aspect of rapid detection.
Further, the test strip for rapidly detecting aflatoxin provided by the invention comprises a bottom plate, a water absorption packing paper, an NC membrane, a gold pad and a sample chromatography pad, wherein the water absorption packing paper, the NC membrane, the gold pad and the sample chromatography pad are sequentially pasted on the bottom plate from top to bottom, and a NANO antibody NANO-AF marked by colloidal gold is arranged on the gold pad;
the NC membrane is provided with a detection area and a quality control area which are separated from each other, the detection area is sprayed with aflatoxin coated antigen, and the quality control area is sprayed with an antibody which is specifically combined with the NANO antibody NANO-AF marked by colloidal gold.
Further, the invention provides a preparation method of the test strip for rapidly detecting aflatoxin,
1) purifying the NANO antibody NANO-AF, and dialyzing for later use;
2) labeling a NANO antibody NANO-AF by colloidal gold;
3) BSA blocking reaction;
4) after centrifugation, washing the precipitate by PBS containing 1% BSA;
5) spraying a gold mark pad after redissolution;
6) coating an NC film;
7) drying at room temperature and low humidity; sequentially adhering absorbent paper, an NC membrane, a gold pad and a sample chromatography pad from top to bottom on a bottom plate, and cutting for later use;
8) the colloidal gold test strip is subjected to application test, the detection limit of the colloidal gold test strip is obtained to be in a grade of less than 10ng/mL, and the requirement of rapidly detecting the aflatoxin can be met.
Advantageous effects
The optimized aflatoxin epitope fusion polypeptide comprises a plurality of common aflatoxin epitopes such as aflatoxin B1(AFB1), aflatoxin M1(AFM1), aflatoxin B2(AFB2), aflatoxin G1(AFG1) and aflatoxin G2(AFG2), and the optimized aflatoxin epitopes are all in a spiral structure, so that the epitopes can be completely exposed, the antibody can be conveniently identified and prepared, and the subsequent operation is convenient;
the aflatoxin nano antibody provided by the invention can effectively identify aflatoxin B1, can identify aflatoxin B2, G1, G2 and M1, can be widely applied to a food detection process, does not need to detect for many times, realizes the effect of detecting all identifications at one time, and saves manpower, material resources and financial resources;
the aflatoxin nano antibody provided by the invention has clear sequence and simple structure, can be applied to various detections on aflatoxin, and can effectively reduce the interference of other components such as organic reagents and the like in a sample extracting solution, thereby improving the detection accuracy; and can be directly expressed and purified by a eukaryotic or prokaryotic expression system, can effectively reduce the production cost of the antibody, and is suitable for marketization operation.
Drawings
FIG. 1 is a 3D epitope mimic diagram of epitope fusion polypeptide of aflatoxin
FIG. 2 is a cross-sectional structure of a test strip, wherein 1 is a PVC base plate, 2 is a sample chromatographic pad, 3 is a gold pad, 4 is an NC membrane, and 5 is absorbent pad paper.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Example 1 screening and design of epitope fusion polypeptides for aflatoxins
Firstly, homology analysis is carried out on linear simulation epitopes of a plurality of common aflatoxins known in the prior literature, such as aflatoxin B1(AFB1), aflatoxin M1(AFM1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2), an antigen fragment candidate region is screened by a biological analysis means, partial amino acid residues in the antigen fragment candidate region are replaced or optimized according to bioinformatics analysis (see figure 1), and finally the amino acid sequence of the epitope fusion polypeptide is determined to be SAEEHAVSDPEHPGPSDDVMAISAPDGEGDWPLYRLNPWSDGALDARTAVRAMAPSSWRVATERATAEVALGQRVLTSFRPHSARVLARDREHVCVRCYSLKPGLCGAVYVQNTFIEGQLGDNGTAVFSE (SEQ ID No. 1).
An epitope fusion polypeptide expression cassette is constructed by using an overlap PCR method, is connected to PET-28a (purchased from Invitrogen company, a product number A11499) by using double enzyme digestion, a single clone is picked to identify the insertion direction, a plasmid with the correct insertion direction is sent to the Invitrogen company for sequencing, and the plasmid with the correct sequencing is named as PET-28 a-AFBMG. And (3) expressing the PET-28a-AFBMG recombinant positive plasmid, and purifying by a nickel column chromatography after SDS-PAGE verification to obtain the soluble fusion protein.
Example 2 immunopanning procedure for native Single Domain antibodies directed against epitope fusion polypeptide AFBMG
(1) Amplifying the established natural single-domain antibody phage library: adding 2 XYT culture medium into 100 mul of glycerol bacterial library, adding 20MOI auxiliary phage when OD600 is 0.5, standing for 30min, centrifuging, re-suspending the precipitate with 2 XYT culture medium, culturing for 1h, adding antibiotic, culturing for 16h, centrifuging, precipitating the supernatant with precooled PEG-NaCl (1/4 vol), and re-suspending with 1mL PBS to obtain the amplified single domain antibody library;
(2) immune tube panning: coating an immune tube with a biotin-labeled and purified epitope fusion polypeptide AFBMG (immune protein library) at 50 mu g/tube overnight, removing a coating solution, washing for 3 times, blocking for 2h with 2mL BSA (1%), washing for 3 times with PBST (basic antibody test), adding 100 mu L of the single-domain antibody library amplified in the step (1) as a primary antibody, acting for 2h at 37 ℃, washing for 3 times with PBST, eluting with Glycine-HCI (PH2.2), adjusting the eluent to PH 7.4 with Tris-HCI, and obtaining a first round of natural single-domain antibody library after elutriation;
(3) amplifying the 1 st round natural single-domain antibody library obtained in the step (2) according to the step (1) to obtain a1 st round natural single-domain antibody heavy suspension library, then repeating the step (2) of immune tube panning, only adding 100 mu L of amplified 1 st round natural single-domain antibody heavy suspension library as a primary antibody, and finally obtaining a panning 2 nd round natural single-domain antibody library; (4) amplifying the 2 nd round natural single-domain antibody library obtained in the step (3) according to the step (1) to obtain a 2 nd round natural single-domain antibody heavy suspension library, then repeating the step (2) of immune tube panning, only adding 100 mu L of amplified 2 nd round natural single-domain antibody heavy suspension library as a primary antibody, and finally obtaining a panning 3 rd round natural single-domain antibody library.
EXAMPLE 3 ELISA identification of Individual clones
(1) Panning for single positive clonal shake amplification for antibody expression: the 3 rd round natural single-domain antibody library selected above was inoculated into 2 XYT medium, OD 600nm When the OD600 is equal to 0.5, 20MOI auxiliary phage is added, the sediment is resuspended in 2 XYT medium and is cultured for 30min, 40 single colonies are selected and inoculated in 2 XYT medium the next day, when the OD600 is equal to 0.5, 20MOI auxiliary phage is added, the sediment is resuspended in 2 XYT medium and is cultured again, and IPTG is added for induction expression for 8 h.
(2) And (3) ELISA identification: an epitope fusion polypeptide AFBMG (concentration of 1 ng/. mu.L) which is labeled by biotin and purified 100. mu.L/well is coated on an ELISA plate overnight, the coating solution is removed and then washed for 3 times, 200. mu.L/well BSA (3%) is used for blocking for 2h, PBST is washed for 3 times, a single-domain antibody library which is respectively amplified in the step of 100. mu.L/well (1) is added as a primary antibody (the library construction carrier belt M13), the single-domain antibody library acts for 2h at 37 ℃, PBST is washed for 3 times, a secondary antibody is added with M13-HRP, and an OD450nm value is detected after termination, and the result is interpreted: the test piece was judged to be positive if it was 3 times higher than the control OD450nm value.
Example 4 sequencing identification of Positive clones
The clones that were positive in ELISA detection in example 3 were extracted with plasmids from the corresponding bacterial solutions and sequenced using the universal primers for the plasmid vector. Analyzing the gene sequence of each clone strain according to the sequence alignment software MEGA 6.0, regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain, and regarding the strains with different sequences as different clone strains, and finally obtaining the single domain antibody specific to the epitope fusion polypeptide AFBMG. The gene is named Nano-AF, and the amino acid sequence is as follows: QLQLGGSGGGLVQPGGLVQACAASGSTFAGFRAMGWRQAFRQAREFVAAITWSGASTYYTDSVKGRFTISRDNAKNTVYLQMNNLESLKPEDTAVYYCAGFMYYASNYCRYWGQGTQVTVSSQGTQVKPQD (SEQ ID No. 2).
EXAMPLE 5 specific identification of Positive clones
Identifying specificity of the Nano-AF single-domain antibody by Western blot: A. protein electrophoresis: storing five different standard substance stock solutions of AFB1, AFB2, AFG1, AFG2 and AFM 1; standard solutions of ochratoxin A, fusarium graminearum toxin (zearalenone), T-2 toxin, deoxynivalenol (DON, vomitoxin) and Nivalenol (NIV); salmonella O antigen, enterohemorrhagic Escherichia coli O157: H7 Lipopolysaccharide (LPS) antigen, Listeria monocytogenes surface protein LMO1847 antigen, norovirus capsid protein antigen and vibrio parahaemolyticus FlaE protein are respectively diluted to 60 mu g by 6 x SDS protein electrophoresis loading buffer solution, boiled for 5min at 100 ℃, pre-dyed Marker 2 mu L, subjected to protein electrophoresis concentrated gel 80v and separated gel 120 v. B. Film transfer: placing the gel on a nitrocellulose membrane (NC membrane), placing 3 Whatman 3mm filter papers on the upper and lower sides respectively, and soaking the above materials in a membrane-transfer electrophoresis buffer for 15min to remove the air bubbles remained on the filter membrane. The electrotransfer device is sequentially arranged, 3 pieces of filter paper, gel, NC membrane and 3 pieces of filter paper are sequentially placed on the negative electrode plate to ensure that each layer is accurately aligned (from bottom to top), bubbles between layers are removed, the position is marked, and the anode plate is closed. 2mA/cm 2 Constant current 2h conditionAnd (5) carrying out film transfer. And C, identifying Western blot: sealing with 5% skimmed milk powder at 4 deg.C overnight; adding purified His-tagged Nano-AF single-domain antibody (5% skimmed milk diluted at a ratio of 1: 3000), and incubating the membrane for 3h at room temperature; washing membrane with PBST (Tween 1 ‰) for 10min for three times; adding goat anti-His Dy800 antibody (diluted with 5% skimmed milk at a ratio of 1: 20000), and incubating at room temperature in dark for 40 min; finally, the Nano-AF single-domain antibody can be specifically combined with AFB1, AFB2, AFG1, AFG2 and AFM15 aflatoxins and does not have cross reaction with other toxins or pathogens by scanning and identifying by using 700 and 800 fluorescence channels of Odyssey. Therefore, the Nano-AF single domain antibody is a high-specificity Nano antibody aiming at aflatoxin, and can be applied to the research and development of a detection reagent for specifically recognizing aflatoxin.
Example 6 Assembly of Nano-AF Single-Domain antibody colloidal gold test strip
Preparing a Nano-AF single-domain antibody colloidal gold test strip by using a conventional preparation method, wherein in brief, the test strip comprises a bottom plate, water absorption packing paper, an NC membrane, a gold pad and a sample chromatography pad, wherein the water absorption packing paper, the NC membrane, the gold pad and the sample chromatography pad are sequentially pasted on the bottom plate from top to bottom (see figure 2), and a Nano antibody NANO-AF marked by colloidal gold is arranged on the gold pad;
the NC membrane is provided with a detection area and a quality control area which are separated from each other, the detection area is sprayed with aflatoxin coated antigen, and the quality control area is sprayed with an antibody which is specifically combined with the NANO antibody NANO-AF marked by colloidal gold.
1) Cross validation
In order to verify whether the test strip can carry out corresponding detection in practical application occasions such as food detection in large-scale spot inspection and the like and verify the practical effect of the test strip, three parts of mildewed peanuts, mildewed corns, fresh peanuts, fresh corns, commodity vegetable oil and feed blank samples are respectively detected by the test strip, and the test result shows that the test strip only shows positive to the mildewed peanuts and the mildewed corns and the rest is negative. The result shows that the test strip can be applied to the detection of aflatoxin in the actual detection process.
2) Sensitivity test
In order to verify the sensitivity of the nano antibody screened by the application, the epitope fusion polypeptide AFBMG is subjected to volume fixing until the concentration is 1mg/mL, and is diluted in a ratio of 1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000 respectively, and then the detection is carried out, and the steps are repeated for three times. The detection result shows that when the dilution ratio is 1:100000 (namely the lowest detection limit is 10 ng/mL), the positive result can be still detected, and the sensitivity is very high.
3) Stability test
4 ℃ storage stability test: the prepared Nano-AF single-domain antibody colloidal gold test strip and the drying agent are packaged together in an aluminum foil bag in a sealing mode, 2 strips are taken out every two months in a refrigerator at the temperature of 4 ℃, epitope fusion polypeptide AFBMG standard series solutions with visible detection limit concentration are detected, and stability test results (including the existence of detection lines and quality control lines, the definition of strips, the degree of gold-labeled antibodies placed in gold-labeled pads, the sensitivity of the test strip and the like) are observed. The result proves that the test strip can still keep good detection effect after being stored for more than 8 months at 4 ℃.
The test proves that the Nano-AF single-domain antibody colloidal gold test strip has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like, and is wide in detection range, low in false positive rate and reliable in detection result. When the Nano-AF single-domain antibody colloidal gold test strip is used, the sample pretreatment time is short, and the detection limit of the standard substance is 10 ng/mL. The detection method is suitable for market food sampling detection and sample detection; a large number of samples can be detected in a short time, and a large number of negative samples can be excluded. Because the sample treatment is simple and easy, and the detection does not need expensive instruments and equipment, the method is suitable for popularization and use in primary inspection units.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
New Wanfu food Co Ltd in Qingdao (110)
< 120 > nano antibody for rapidly detecting various aflatoxins and application thereof in detection of prepared vegetables
〈160〉 2
〈170〉 PatentIn version 3.3
〈210〉 1
〈211〉 130
〈212〉 protein
Antigen epitope fusion polypeptide AFBMG of < 213 >
〈400〉SAEEHAVSDPEHPGPSDDVMAISAPDGEGDWPLYRLNPWSDGALDARTAVRAMAPSSWRVATERATAEVALGQRVLTSFRPHSARVLARDREHVCVRCYSLKPGLCGAVYVQNTFIEGQLGDNGTAVFSE
〈210〉 2
〈211〉 131
〈212〉 protein
Nano-antibody Nano-AF of < 213 > aflatoxin
〈400〉QLQLGGSGGGLVQPGGLVQACAASGSTFAGFRAMGWRQAFRQAREFVAAITWSGASTYY
TDSVKGRFTISRDNAKNTVYLQMNNLESLKPEDTAVYYCAGFMYYASNYCRYWGQGTQVTVSSQGTQVKPQD
Claims (4)
1. An optimized epitope fusion polypeptide of aflatoxin comprises epitopes of a plurality of common aflatoxins, such as aflatoxin B1(AFB1), aflatoxin M1(AFM1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2), and the amino acid sequence of the epitope fusion polypeptide is as follows: SAEEHAVSDPEHPGPSDDVMAISAPDGEGDWPLYRLNPWSDGALDARTAVRAMAPSSWRVATERATAEVALGQRVLTSFRPHSARVLARDREHVCVRCYSLKPGLCGAVYVQNTFIEGQLGDNGTAVFSE (SEQ ID No. 1).
2. The Nano antibody can identify aflatoxin with high accuracy and sensitivity, targets epitopes of a plurality of common aflatoxins such as aflatoxin B1(AFB1), aflatoxin M1(AFM1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2) and the like, and is named as Nano-AF, and the amino acid sequence of the Nano antibody is as follows: QLQLGGSGGGLVQPGGLVQACAASGSTFAGFRAMGWRQAFRQAREFVAAITWSGASTYYTDSVKGRFTISRDNAKNTVYLQMNNLESLKPEDTAVYYCAGFMYYASNYCRYWGQGTQVTVSSQGTQVKPQD (SEQ ID No. 2).
3. A test strip for rapidly detecting aflatoxin is characterized by comprising a bottom plate, a water absorption packing paper, an NC membrane, a gold pad and a sample chromatography pad, wherein the water absorption packing paper, the NC membrane, the gold pad and the sample chromatography pad are sequentially pasted on the bottom plate from top to bottom, and a NANO antibody NANO-AF marked by colloidal gold is arranged on the gold pad; the NC membrane is provided with a detection area and a quality control area which are separated from each other, the detection area is sprayed with aflatoxin coated antigen, and the quality control area is sprayed with an antibody which is specifically combined with the NANO antibody NANO-AF marked by colloidal gold.
4. The optimized aflatoxin epitope fusion polypeptide of claim 1, the nanobody of claim 2 or the rapid test strip of claim 3, for detecting aflatoxin, wherein the application is aflatoxin detection of prepared vegetables.
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