CN103059101B - Antigenic mimic epitope of aflatoxin (AF) B1 and application thereof - Google Patents
Antigenic mimic epitope of aflatoxin (AF) B1 and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and relates to an antigenic mimic epitope of aflatoxin B1. The amino acid sequence of the antigenic mimic epitope is CDPRKHIHC. The antigenic mimic epitope of aflatoxin B1 can replace an expensive AFB1 standard substance with strong toxicity and be applied to an immunological detection of AFB1 as a competitive antigen or a solid phase envelope antigen. The antigenic mimic epitope has immunoreaction characteristics similar to natural AFB1 molecules, and has good effects. Harm to human health caused by AFB1 is reduced, the cost is saved, and the antigenic mimic epitope of AFB1 has great application value.
Description
Technical field
The invention belongs to biological technical field, be specifically related to AFB
1antigenic epitope and application thereof.
Background technology
AFB
1(aflatoxin B
1, AFB
1) be the secondary metabolite mainly being produced by flavus and Aspergillus parasiticus, be extensively present in the farm crop such as peanut, cottonseed, corn, wheat and rice.AFB
1people and animal livers tissue are had to destruction, when serious, can cause liver cancer even dead, within 1993, delimited as I class carcinogens by the cancer research mechanism of WHO.Therefore, AFB in food
1detection significant for human health.
At present, detect AFB in food
1method mainly contain the methods such as high performance liquid chromatography, gas-chromatography, thin-layer chromatography and immunology detection, immunological detection method with its advantage such as highly sensitive, easy to detect, with low cost at AFB
1detection in be widely used.But, in the process of setting up immunological detection method, must use AFB
1standard substance are that raw material is prepared competition antigen or solid-phase coating antigen, AFB
1not only expensive but also have extremely strong carinogenicity, the health to testing staff and environment cause great threat, thereby have restricted to a certain extent application and the popularization of immunological detection method.In view of this, people start to adopt antiidiotypic antibody and antigenic epitope technology to realize substituting of harmful small-molecule substance standard substance, and make some progress.The principal feature of phage display peptide library technology is can effectively filter out and the phage-displayed polypeptides of target target body specific combination, this technology exploring the binding site that interacts between acceptor and part, seek the bioactive ligand molecular of high-affinity, the aspect such as development of exploring agnoprotein matter space structure epi-position, new generation vaccine is widely used.
The present invention is by using phage display peptide library technology, from peptide storehouse, filter out can with target molecule (anti-AFB
1monoclonal antibody) polypeptide (antigenic epitope) of specific binding, this antigenic epitope has and natural A FB
1the immune response characteristic of molecular mimicry, by the AFB obtaining
1antigenic epitope, to replace the AFB of expensive and strong toxicity
1standard substance, and be applied to AFB as competition antigen or solid-phase coating antigen
1immunology detection.
Summary of the invention
The present invention is with anti-AFB
1monoclonal antibody is target molecule, and target molecule solid-phase coating, on enzyme plate, is dropped into respectively to phage random and shows ring seven, dodecapeptide storehouse, carries out respectively affine elutriation, has obtained two kinds of AFB
1antigenic epitope (each a kind of ring seven peptide and dodecapeptide).Their aminoacid sequence is as follows:
AFB
1antigenic epitope 1: the AFB screening from phage random ring seven peptide storehouse
1antigenic epitope (polypeptide), its aminoacid sequence is: CDPRKHIHC.
AFB
1antigenic epitope 2: the AFB screening from phage random dodecapeptide storehouse
1antigenic epitope (polypeptide), its aminoacid sequence is: VPYSPHYFERMI.
The nucleotide sequence that the invention still further relates to the above-mentioned antigenic epitope aminoacid sequence of coding, is preferably GAT CCG CGT AAG CAT ATT CAT or GTT CCG TAT TCG CCT CAT TAT TTT GAG CGT ATG ATT.
In above-mentioned antigenic epitope (polypeptide) structure, capitalization English letter represents respectively the one of 21 kinds of known natural L-type amino-acid residues or its D-type isomer, be that C represents cysteine residues, D represents asparagicacid residue, P represents proline residue, R represents arginine residues, K represents lysine residue, and H represents histidine residues, and I represents Isoleucine residue, V represents α-amino-isovaleric acid residue, Y represents tyrosine residues, and S represents serine residue, and F represents phenylalanine residue, E represents glutaminic acid residue, and M represents methionine residues.Two cysteine residues of the N-terminal of mimic epitopes 1 and C-terminal form intramolecular disulfide bond, are ring texture.
The AFB that the present invention mentions
1antigenic epitope (polypeptide) can be prepared in a large number by the recombinant expressed mode of phage amplification, chemosynthesis or genetically engineered.Phage amplification refers to that displaying is had to AFB
1the phage of antigenic epitope (polypeptide), the mode increasing by biology, amount reproduction is produced and is shown there is AFB
1the bacteriophage particles of antigenic epitope (polypeptide).Chemosynthesis refers to according to the mimic epitopes 1 of announcing, the aminoacid sequence of mimic epitopes 2, carries out polypeptide synthesize by the mode of chemically synthesized polypeptide.The recombinant expressed mode of genetically engineered refers to the gene of coding simulation epi-position 1 or mimic epitopes 2, by being cloned into expression vector, carries out AFB with the form of polypeptide-fusion rotein
1a large amount of preparations of antigenic epitope.
The invention still further relates to described AFB
1the application of antigenic epitope in immunology detection is analyzed.The type of immunology detection comprises the immune analysis type of detection based on Ag-Ab specific reaction such as Enzyme-linked Immunosorbent Assay detection, colloidal gold immunochromatographimethod, immunodotting hybridization.
AFB of the present invention
1antigenic epitope (CDPRKHIHC, VPYSPHYFERMI), in the time of application, can, synthetic mimic epitopes for immunology detection analysis, have AFB by the displaying obtaining of increasing by phage
1the bacteriophage particles of antigenic epitope (polypeptide) is directly used in analyzing and testing, certainly, and also can be by AFB
1antigenic epitope scales off and replaces AFB from phage
1standard substance carry out immunology detection analysis.
Also relate to AFB
1antigenic epitope application in immunology detection is analyzed with solid phase antigen or competition antigen.
Also relate to AFB
1antigenic epitope detects the application in analyzing as solid phase antigen at colloidal gold immunochromatographimethod.
Aforementioned AFB
1antigenic epitope can be replaced the AFB of expensive and strong toxicity
1standard substance, and be applied to AFB as competition antigen or solid-phase coating antigen
1immunology detection, this antigenic epitope has and natural A FB
1the immune response characteristic of molecular mimicry, effect is very good.
The invention has the beneficial effects as follows: AFB of the present invention
1antigenic epitope can be replaced the AFB of expensive and strong toxicity
1standard substance, and be applied to AFB as competition antigen or solid-phase coating antigen
1immunology detection, this antigenic epitope has and natural A FB
1the immune response characteristic of molecular mimicry, effect is very good.Reduce AFB
1to the harm of HUMAN HEALTH, save cost, there is very high using value.
Brief description of the drawings
Fig. 1 is the indirect competitive ELISA typical curve of setting up with AFB1 antigenic epitope 1.Sensing range 4.0-31.3 ng/mL, IC
50be 11.7 ng/mL.
Fig. 2 is the indirect competitive ELISA typical curve of setting up with AFB1 antigenic epitope 2.Sensing range 4.6-50.1 ng/mL, IC
50be 15.2 ng/mL.
Embodiment
Embodiment 1. AFB
1affine elutriation and the qualification thereof of antigenic epitope
1) AFB
1the affine elutriation of antigenic epitope: concrete grammar is: dilute anti-AFB with 10 mM PBS (pH 7.4)
1monoclonal antibody, and with the coated 96 hole enzyme plates of final concentration 100 μ g/mL, 4 DEG C of overnight incubation.Within second day, wash after 10 times with TBST (50 mM NaCl, pH 7.5 comprises 0.1% Tween-20 (v/v)), add 4 DEG C of 300 μ l confining liquids (3% BSA-PBS) to hatch 2 hours.After 2 hours, abandon confining liquid, with TBST washing 5 times, every hole adds 100 μ l phage peptide libraries, and (phage display ring seven peptide storehouse or dodecapeptide storehouse, purchased from NEB company, with 10 times of dilution phage stostes of TBS, approximately 1.0 × 10
11pfu), 22-26 DEG C of oscillatory reaction 1 hour.Discard unconjugated phage, with TBST washing 10 times, in conjunction with 0.2 M Glycine-HCl (pH 2.2) wash-out for upper phage, and use immediately 15 μ l 1 M Tris-HCl (pH 9.1) neutralizations.Get 10 μ l wash-out bacteriophages and survey titres, remaining grows to logarithm early stage for infecting 20 mL
e. colieR2738 bacterial strain increases.The 3rd day with PEG/NaCl deposition and purification phage, and measures the titre of phage after amplification.
In the elutriation process of taking turns at second, third, coated anti-AFB
1monoclonal anti bulk concentration is respectively 75 μ g/mL and 50 μ g/mL, and TBST concentration used is 0.25% and 0.5%, and all the other steps are the same.
2) qualification of positive phage clones: measure random 20 phage spots of picking in the flat board of phage titre from third round elutriation, carry out the amplification of phage, adopt indirect enzyme-linked immunosorbent absorption detection method (Indirect Enzyme Linked immunoasorbent assay, I-ELISA) carry out the qualification of positive phage clones, concrete grammar is: first, dilute anti-AFB with 10 mM PBS (pH 7.4)
1monoclonal antibody, 10 μ g/mL are coated with 96 hole enzyme plates, 4 DEG C of overnight incubation.Within second day, wash after 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), use the PBS that contains 3% skim-milk to seal, hatch 1 hour for 37 DEG C; Drop into 100 μ l phage spot amplification liquid (1.0 × 10
11pfu),, using original phage peptide library as negative control, hatch 1 hour for 37 DEG C; The anti-100 μ l of the anti-M13 phage two of HRP mark that add 1:5000 doubly to dilute, hatch 1 hour for 37 DEG C; Add 100 μ l tmb substrate liquid, lucifuge colour developing 5min, microplate reader (Thermo Scientific Multiskan FC) reads the absorption value at 450 nm places.Choose OD
450be greater than the positive clone of phage clone of 2 times of negative controls.
3) AFB
1the qualification of antigenic epitope: adopt the method for indirect competitive ELISA to carry out AFB
1the qualification of antigenic epitope, concrete grammar is: dilute anti-AFB with 10 mM PBS (pH 7.4)
1monoclonal antibody, 10 μ g/mL coated elisa plates, 4 DEG C of overnight incubation; Within second day, wash after 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), use the PBS that contains 3% skim-milk to seal, hatch 1 hour for 37 DEG C; Drop into 50 μ l and be accredited as positive phage clone (1.0 × 10 through indirect ELISA
11pfu) and 50 μ l AFB
1standard substance (concentration range is 0-20 ng/ml), hatch 1 hour for 37 DEG C; Add the anti-100 μ l of anti-M13 phage two of 1:5000 dilution HRP mark, hatch 1 hour for 37 DEG C; Add 100 μ l tmb substrate liquid, lucifuge colour developing 5min, reads OD
450, can be in conjunction with anti-AFB
1monoclonal antibody, and can be by AFB
1the phage that standard substance are blocked, is accredited as AFB
1antigenic epitope.
Embodiment 2. AFB
1determining of the order-checking of antigenic epitope encoding gene and aminoacid sequence thereof
By having the phage of AFB1 antigenic epitope to increase through indirect competitive ELISA qualification displaying, extract the DNA sequencing template of phage.Concise and to the point process is as follows: carry out phage amplification, after the first step is centrifugal, 800 μ l are proceeded to a new centrifuge tube containing phage supernatant.Add 200 μ l PEG/NaCl precipitation phages.After centrifugal, precipitation is resuspended in to 100 μ l iodide damping fluid (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), add 250 μ l dehydrated alcohols precipitations DNA, precipitate (DNA sequencing template) by 70% washing with alcohol again after centrifugal.Precipitation is finally resuspended in 20 μ l aqua sterilisas, gets 2 μ l and carries out agarose gel electrophoresis analysis; Get 5 μ L phage templates and carry out DNA sequencing, its-96 gIII sequencing primer is: 5 '-
hOcCC TCA TAG TTA GCG TAA CG-3 '.Can obtain AFB according to DNA sequencing result and password sublist
1the aminoacid sequence of antigenic epitope: CDPRKHIHC and VPYSPHYFERMI.
Embodiment 3. AFB
1antigenic epitope is the application in ELISA as competition antigen
(1) sample extraction
Take 5g sample (cereal and relevant food thereof), add methyl alcohol-PBS solution of 25 milliliter of 60 %, 200 rpm vibrations 5 minutes; After extracting solution is filtered with No. 1 filter paper of whatman, get 1 milliliter of filtrate and add 4 milliliters of PBS(phosphate buffered saline buffers, pH=7.2) mix after, be sample extracting solution, stand-by.
(2) coated and sealing
Dilute anti-AFB with 10 mM PBS (pH 7.4)
1monoclonal antibody, 10 μ g/mL coated elisa plates, 4 DEG C of overnight incubation.Second day with after PBST (10 mM PBS, 0.05% Tween-20 (v/v)) washing 3 times, seals with the PBS that contains 3% skim-milk, hatches after 1 hour for 37 DEG C, washes plate 6 times stand-by with PBST.
(3) foundation of typical curve
Take out the lath of handling well through step (2), every hole is dropped into respectively 50 μ l and is shown there is AFB
1the phage (1.0 × 10 of antigenic epitope
11and 50 μ l AFB of a series of different concns pfu)
1standard substance, hatch 1 hour for 37 DEG C.Add the anti-M13 phage two of 1:5000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450.With AFB
1concentration logarithm is X-coordinate, and combination rate (adds AFB
1the OD in hole
450/ do not add AFB
1the OD in hole
450× 100%) be ordinate zou, set up indirect competition typical curve.Result display standard curve is S-type, and linear dependence is better, sensing range 4.0-31.3 ng/mL, IC
50be 11.7 ng/mL(Fig. 1).Similarly, set up indirect competitive ELISA typical curve with mimic epitopes 2, sensing range 4.6-50.1 ng/mL, IC
50be 15.2 ng/mL(Fig. 2).
(4) detection of sample
Take out the lath of handling well through step (2), every hole is dropped into respectively 50 μ l and is shown there is AFB
1the phage (1.0 × 10 of antigenic epitope
11pfu) and testing sample extracting solution, hatch 1 hour for 37 DEG C.Add the anti-M13 phage two of 1:5000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450, calculations incorporated rate, and according to typical curve, AFB in the sample of retrodicting out
1content.
Embodiment 4. AFB
1antigenic epitope is the application in ELISA as solid phase antigen
(1) sample extraction
Take 5g sample (cereal and relevant food thereof), add methyl alcohol-PBS solution of 25 milliliter of 60 %, 200 rpm vibrations 5 minutes; After extracting solution is filtered with No. 1 filter paper of whatman, get 1 milliliter of filtrate and add 4 milliliters of PBS(phosphate buffered saline buffers, pH=7.2) mix after, be sample extracting solution, stand-by.
(2) coated and sealing
Dilute and show there is AFB with 10 mM PBS (pH 7.4)
1the phage (2.0 × 10 of antigenic epitope
11pfu), 100 microlitres are coated in enzyme plate, 4 DEG C of overnight incubation.Second day with after PBST (10 mM PBS, 0.05% Tween-20 (v/v)) washing 3 times, seals with the PBS that contains 3% skim-milk, hatches after 1 hour for 37 DEG C, washes plate 6 times stand-by with PBST.
(3) foundation of typical curve
Take out the lath of handling well through step (2), the anti-AFB of 50 μ l is dropped into respectively in every hole
150 μ l AFB of monoclonal antibody (0.5 ng/ml) and a series of different concns
1standard substance, hatch 1 hour for 37 DEG C.Add the sheep anti-mouse igg two of 1:2000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450.With AFB
1concentration logarithm is X-coordinate, and combination rate (adds AFB
1the OD in hole
450/ do not add AFB
1the OD in hole
450× 100%) be ordinate zou, set up indirect competition typical curve.
(4) detection of sample
Take out the lath of handling well through step (2), the anti-AFB of 50 μ l is dropped into respectively in every hole
150 μ l AFB of monoclonal antibody (0.5 ng/ml) and a series of different concns
1standard substance, hatch 1 hour for 37 DEG C.Add the sheep anti-mouse igg two of 1:2000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450.With AFB
1concentration logarithm is X-coordinate, and combination rate (adds AFB
1the OD in hole
450/ do not add AFB
1the OD in hole
450× 100%) be ordinate zou, set up indirect competition typical curve.
Embodiment 5. AFB
1antigenic epitope is the application in highly-pathogenic avian influenza as solid phase antigen
(1) sample extraction
Take 5g sample (cereal and relevant food thereof), add methyl alcohol-PBS solution of 25 milliliter of 60 %, 200 rpm vibrations 5 minutes; After extracting solution is filtered with No. 1 filter paper of whatman, get 1 milliliter of filtrate and add 4 milliliters of PBS(phosphate buffered saline buffers, pH=7.2) mix after, be sample extracting solution, stand-by.
(2) dot matrix of phage and control line
Dilute and show there is AFB with 10 mM PBS (pH 7.4)
1the phage (2.0 × 10 of antigenic epitope
11pfu), phage is lined to (aperture 0.2-0.45 micron) on nitrocellulose filter with dot matrix instrument or micropipet, as detection line; By anti-the sheep anti-mouse igg of the HRP mark of 0.5 mg/ml two, line on same nitrocellulose filter and (be positioned at the top of detection line, apart from being greater than 5 millimeters) with dot matrix instrument or micropipette, as control line.
(3) colloid gold label AFB
1antibody
By AFB
1antibody dropwise adds in colloidal gold solution (pH=8.2), stir while dripping, after 30 minutes, getting 1% PEG adds in above-mentioned solution, continue to stir the 10% BSA solution that adds 1/10th volumes after 15 minutes, stir after 15 minutes, leave standstill 30 minutes, after centrifugal, remove supernatant, obtain the AFB of colloid gold label
1antibody-solutions.
(4) assembling of colloidal-gold detecting-card
By the AFB of colloid gold label
1antibody point is sprayed on glue gold pad upper (1.0 ug/ml), by sample pad, glue gold pad, dot matrix nitrocellulose filter and the blotter of detection line and control line assemble, be cut into test strip, pack in test card stand-by.
(5) detection of sample
Sample extracting solution is added in sample pad, leave standstill 10 minutes, if contain AFB in sample
1and exceed the detection threshold of colloidal gold test, do not develop the color in detection line region, and the colour developing of control line region; If do not contain AFB in sample
1and lower than the detection threshold of colloidal gold test, detection line region colour developing, also develop the color in control line region.If do not develop the color in control line region, show that test strip lost efficacy.
Embodiment 5 AFB
1a large amount of preparations of antigenic epitope
(1) mode increasing with phage
Having the phage of AFB1 antigenic epitope to be added to 20 ml inoculations displaying has in the culture of ER 2738,37 degree 220 rpm shaking culture 4.5 h.Culture is proceeded in another centrifuge tube, and 4 DEG C of 10000 centrifugal 10 min of rpm, proceeds to the top of supernatant 80 % in one fresh tube, adds the PEG/NaCl of 1/6 volume, leaves standstill 120 min at 4 DEG C.4 DEG C of 10000 centrifugal PEG/NaCL of rpm leaves standstill solution 15 min.Abandon supernatant, of short durationly suck residual supernatant liquor after centrifugal.Add 1mL TBS to carry out resuspended, be phage amplification liquid.
(2) with AFB
1the mode of antigenic epitope-fusion rotein is prepared
A. the external source encoding gene of pcr amplification AFB1 antigenic epitope
PCR reaction system: (50 μ L)
10 × Pyrobest Buffer (Mg2+ plus) 5 μL
dNTP Mixture (each for 2.5 mM) 4 μL
M13KE insert extension primer (10 mM） 1 μL
-96 gIII sequencing primer (10 mM) 1 μL
Phage DNA template 1 μ L
Pyrobest DNA Polymerase 0.5 μL
Sterilizing ddH2O 37.5 μ L
PCR reaction conditions:
95℃ 5 min
Then 95 DEG C of 30 sec, 55 DEG C of 30 sec, 72 DEG C of 40sec, 72 DEG C of 10 min totally 30 cycles.
Adopt PCR product to reclaim test kit purifying PCR product, trace dna quantitative instrument is quantitative.The coding gene sequence of AFB1 antigenic epitope 1 is GAT CCG CGT AAG CAT ATT CAT; The coding gene sequence of AFB1 antigenic epitope 2 is: GTT CCG TAT TCG CCT CAT TAT TTT GAG CGT ATG ATT.
B. the double digestion of external source encoding gene and expression vector
Adopt respectively the external source code gene of ACC65I and Eag I enzyme and expression vector (MBP fusion rotein can be expressed by pMAl-pIII, NEB company) to carry out double digestion.
C. enzyme is cut connection and the conversion of after product
By plasmid pMal-PIII and object fragment with 1: 10(mol ratio) mix, connect 12 h in 16 DEG C of water-baths, get 10 μ L and connect products and add in 100 μ L competent cell TB1, fully mix.After ice bath 30 min, 42 DEG C of water-bath heat shock 90 s, after ice bath 5 min, add immediately 600 μ L LB liquid mediums, 37 DEG C, 200 rpm cultivate 1 h, centrifugal 2 min of 10000 rpm, suck supernatant and leave and take approximately 200 μ L, coat in LB-A solid (Ampr) substratum, 37 DEG C of incubated overnight, obtain positive colony.
AFB
1the expression of antigenic epitope-MBP fusion rotein
By positive colony of above-mentioned acquisition, choose a single colony inoculation in 5 mL LB-A from flat board, in 0.2% sucrose, 37 DEG C, 220 r/min, shaking culture is spent the night, overnight culture is inoculated in to the LB-A of 50 mL by 1 % inoculum size (v/v), in 0.2 % sucrose medium, inoculate respectively 3 bottles, 37 DEG C, 220 r/min shaking culture, in the time that culture bacterial concentration OD600 reaches 0.6, to add in three bottles of cultures IPTG to final concentration be 0.2 mmol/L, 220 r/min shaking culture, by inductor (PEG solution) in 4 DEG C, 4000 g, centrifugal 20 min collect bacterial sediment, abandon supernatant.Re-suspended cell is in 400 mL 30 mM Tris-HCl, 20% sucrose, pH 8.0 (80 mL/g wet cell weight), add EDTA to 1 mM, under room temperature, shake 5-10 min, 8000 g, 4 DEG C, centrifugal 20 min, abandon supernatant, the 5 mM MgSO4 that precipitation is resuspended in 400 ml precoolings, shake 10 min on ice, 8000 g, 4 DEG C, centrifugal 20 min, retain supernatant, in supernatant liquor, add 8 mL 1 M Tris-HCl, pH 7.4, obtains AFB1 antigenic epitope-MBP fusion rotein.
SEQUENCE LISTING
<110> University Of Nanchang
The antigenic epitope of <120> aflatoxin B1 and application thereof
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213> artificial sequence
<400> 1
Cys Asp Pro Arg Lys His Ile His Cys
1 5
<210> 2
<211> 12
<212> PRT
<213> artificial sequence
<400> 2
Val Pro Tyr Ser Pro His Tyr Phe Glu Arg Met Ile
1 5 10
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<400> 3
gatccgcgta agcatattca t 21
<210> 4
<211> 36
<212> DNA
<213> artificial sequence
<400> 4
gttccgtatt cgcctcatta ttttgagcgt atgatt 36
Claims (7)
1. AFB
1antigenic epitope, its aminoacid sequence is CDPRKHIHC, N end and C end halfcystine pass through disulfide linkage Cheng Huan.
2. the Nucleotide of antigenic epitope described in coding claim 1.
3. Nucleotide as claimed in claim 2, its sequence is GAT CCG CGT AAG CAT ATT CAT.
4. the preparation method of antigenic epitope as claimed in claim 1, is characterized in that preparing in a large number by the recombinant expressed mode of phage amplification, chemosynthesis or genetically engineered; Described phage amplification refers to that displaying is had to AFB
1the phage of antigenic epitope, the mode increasing by biology, amount reproduction is produced and is shown there is AFB
1the bacteriophage particles of antigenic epitope; Described chemosynthesis refers to according to mimic epitopes aminoacid sequence, carries out polypeptide synthesize by the mode of chemically synthesized polypeptide; The recombinant expressed mode of described genetically engineered refers to the gene of coding simulation epi-position, by being cloned into expression vector, carries out AFB with the form of polypeptide-fusion rotein
1a large amount of preparations of antigenic epitope.
5. the application of antigenic epitope in immunology detection is analyzed described in claim 1.
6. application as claimed in claim 5, is characterized in that AFB
1antigenic epitope application in immunology detection is analyzed with solid phase antigen or competition antigen.
7. application as claimed in claim 5, is characterized in that AFB
1antigenic epitope detects application in analysis as solid phase antigen at colloidal gold immunochromatographimethod.
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| CN201310621194.2A CN103848895B (en) | 2012-12-21 | 2012-12-21 | The linear dodecapeptide antigenic epitope of aflatoxin B1 and its application |
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| Phage-displayed peptides that mimic a¯atoxin B1 in serological reactivity;K. Thirumala-Devi等;《Journal of Applied Microbiology》;20020315;第90卷(第3期);330-336 * |
| 邓省亮等.采用噬菌体展示技术筛选黄曲霉毒素B，模拟抗原表位.《卫生研究》.2007,第36卷(第1期),59-62. * |
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