CN113899907A - Method for efficiently screening aflatoxin green prevention and control material in one step and application thereof - Google Patents

Method for efficiently screening aflatoxin green prevention and control material in one step and application thereof Download PDF

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CN113899907A
CN113899907A CN202111041426.8A CN202111041426A CN113899907A CN 113899907 A CN113899907 A CN 113899907A CN 202111041426 A CN202111041426 A CN 202111041426A CN 113899907 A CN113899907 A CN 113899907A
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aft
yjfzp008
aflatoxin
solution
green prevention
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CN113899907B (en
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张奇
马飞
岳晓凤
李培武
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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    • G01—MEASURING; TESTING
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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • 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
    • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to a method for efficiently screening aflatoxin green prevention and control materials in one step and application thereof. The content of an indicator molecule AFT-YJFZP008 of a test treatment group and a control group of the aflatoxin green prevention and control material is measured, the inhibition rate of the green prevention and control material to the indicator molecule AFT-YJFZP008 is calculated, the aflatoxin green prevention and control material is efficiently screened according to the inhibition rate result, and the amino acid sequence of the indicator molecule AFT-YJFZP008 is shown as SEQ ID NO. 1. The method can be used for screening the aflatoxin green prevention and control material which can inhibit the synthesis of aflatoxin sources and the growth of toxigenic bacteria thereof in one step, is easy to operate, strong in practicability and easy to popularize and apply, and has important significance for developing aflatoxin microorganism prevention and control technology and guaranteeing food safety.

Description

Method for efficiently screening aflatoxin green prevention and control material in one step and application thereof
Technical Field
The invention relates to a method for efficiently screening aflatoxin green prevention and control materials in one step and application thereof.
Background
The aflatoxin has strong toxicity and great harm, is a pollutant which pollutes most food types, is one of the most serious countries in the world with aflatoxin pollution, generally presents a pollution aggravating trend in recent years, and seriously threatens food safety and people health. Aflatoxin is a kind of mycotoxin with the highest toxicity in nature, wherein aflatoxin B1 is a class I carcinogen identified by International Agency for Research on Cancer (IARC), has caused many cases of poisoning of human and livestock, and is one of the main causes of high incidence of liver Cancer cases. According to the statistics of the Web of Science retrieval data in the last 5 years: the aflatoxin-polluted food and raw material variety exceeds 110, and the aflatoxin-polluted food and raw material is high in the first place of pollutants. Therefore, screening of the aflatoxin green prevention and control material has important significance for promoting development and application of aflatoxin green prevention and control technology.
The existing aflatoxin green prevention and control material screening method is summarized, and the green prevention and control material with better effect is evaluated and screened by detecting the content of the processed aflatoxin and comparing the content difference of the aflatoxin. However, there are two important questions that are not resolved about the green prevention and control material screened by the conventional method: (1) does the green prevention and control material have the effect of inhibiting the growth of aspergillus flavus? (2) Can the green control material block toxin synthesis from the source of aflatoxin biosynthesis? To answer the above two important questions, on one hand, it is still necessary to further determine the bacteriostatic effect of the green prevention and control material on the growth of aflatoxin-producing bacteria through bacteriostatic tests, and on the other hand, it is still necessary to determine the aflatoxin biosynthesis pathway, such as a series of precursor substances.
How can one-step efficient screening of aflatoxin green prevention and control materials be achieved?
Aiming at the bottleneck problem, an inventor team successfully discovers an aflatoxin toxigenicity indicator molecule through years of attack and relationship research, wherein the content of the indicator molecule is positively correlated with the aflatoxin toxigenicity of strains for different toxigenicity strains cultured under the same toxigenicity condition. Further research also finds that the content of the indicator molecule is in positive correlation with the production of the strain in a certain culture time for the same strain of aflatoxin toxigenic bacteria. Through further research, the source of the aflatoxin synthesis pathway is also inhibited as long as the generation of the indicator molecule is inhibited. On the basis, the method for efficiently screening the aflatoxin green prevention and control material is successfully invented, and the method is applied to efficiently screening the effective green prevention and control material which can inhibit the synthesis of aflatoxin from the source and can also inhibit the growth of aflatoxin toxigenic bacteria, and provides an important support for the efficient green prevention and control of aflatoxin.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the method for efficiently screening the aflatoxin green prevention and control material in one step and the application thereof, and the method is applied to efficiently screening the effective green prevention and control material which can inhibit the synthesis of aflatoxin from the source and can also inhibit the growth of toxigenic bacteria of the aflatoxin in one step, and is easy to popularize and apply.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the method provides a one-step method for efficiently screening the aflatoxin green prevention and control material, measures the content of indicator molecules AFT-YJFZP008 of a candidate aflatoxin green prevention and control material test treatment group and a reference group, calculates the inhibition rate of the green prevention and control material on the indicator molecules AFT-YJFZP008, and efficiently screens the aflatoxin green prevention and control material according to the inhibition rate result of the indicator molecules AFT-YJFZP008, wherein the amino acid sequence of the indicator molecules AFT-YJFZP008 is shown as SEQ ID NO. 1. The higher the inhibition rate is, the better the effects of inhibiting the synthesis of aflatoxin sources and inhibiting the growth of toxigenic bacteria are, and therefore the aflatoxin green prevention and control material with better effect is screened out.
According to the scheme, the aflatoxin green prevention and control material test treatment group and the control group can be realized by the following steps: mixing peanut kernel powder, aspergillus flavus toxigenic bacteria spores and a series of candidate green prevention and control materials, and culturing for a certain time in a culture medium to obtain a test treatment group, wherein the culture medium is used for replacing the candidate green prevention and control materials, and culturing for a certain time to obtain a control group.
According to the scheme, the peanut kernel powder can be but is not limited to a powdery product prepared by shelling and grinding varieties such as Zhonghua No. 6 and Luhua No. 8.
According to the scheme, the aspergillus flavus toxigenic bacteria spore can be but is not limited to 5 multiplied by 105And the aspergillus flavus toxigenic bacteria spore liquid of cfu/mL. The aspergillus flavus toxigenic bacteria refers to aspergillus flavus toxigenic bacteria capable of producing aflatoxin, such as standard aspergillus flavus toxigenic strain 3.4408, and the like, but is not limited to the aspergillus flavus toxigenic bacteria.
According to the scheme, the culture medium can be a liquid Sabouraud's culture medium or other culture media with similar effects.
According to the scheme, the mixed culture in the culture medium can be cultured for a certain time, but is not limited to 24 hours or more.
The quantitative detection of the aflatoxin indicator molecule concentration can be realized by adopting the amino acid sequence of the indicator molecule AFT-YJFZP008 or a partial sequence thereof, preparing an antibody corresponding to the protein through a conventional antibody preparation process, realizing the quantitative detection of the indicator molecule peptide, and realizing the quantitative detection of the indicator molecule peptide in one-to-one correspondence relationship with the indicator molecule peptide through other detection technical means, so as to achieve the detection, wherein the partial sequence refers to a part of a complete sequence in one-to-one correspondence relationship with the aflatoxin indicator molecule protein.
Specifically, the content of the indicator molecule AFT-YJFZP008 can be realized by, but is not limited to, the following steps:
a, after the treatment group and the control group are cultured for a certain time, fully homogenizing the culture to prepare a solution to be detected, adding the solution to an enzyme-labeled plate hole with the bottom coated with a nano antibody or a monoclonal antibody of an indicator molecule AFT-YJFZP008, reacting, and washing the plate;
b, adding an indicator molecule AFT-YJFZP008 polyclonal antibody for reaction, and washing the plate;
c, adding horseradish peroxidase labeled antibody which has a binding reaction with the polyclonal antibody of the indicator molecule AFT-YJFZP008, reacting, and washing the plate;
d, adding a color development solution for reaction; adding a stop solution, reading by an enzyme-linked immunosorbent assay and calculating the content of AFT-YJFZP008 in the solution to be detected;
replacing the solution to be detected with the pure solution of the indicator molecule AFT-YJFZP008 with series concentration and serving as a standard substance, making a standard curve, and calculating the content of the indicator molecule AFT-YJFZP008 according to the reading of an enzyme-labeling instrument.
The inhibition rate of the green prevention and control material to AFT-YJFZP008 can be calculated by the following method: the inhibition ratio is (content of control AFT-YJFZP 008-content of treatment AFT-YJFZP 008)/content of control AFT-YJFZP008 x 100%.
According to the scheme, the animal source of the polyclonal antibody of AFT-YJFZP008 is different from the animal source of the nano antibody or the monoclonal antibody of AFT-YJFZP008, and the nano antibody or the monoclonal antibody and the rabbit source polyclonal antibody can be prepared and obtained by directly using an indicator molecule AFT-YJFZP008 as an antigen, and specifically:
the nano antibody or monoclonal antibody of the indicator molecule AFT-YJFZP008 can be obtained by the following method: AFT-YJFZP008 is used as an immune antigen, an alpaca or Balb/c mouse is immunized by a conventional mode, and the preparation method can be developed by a known conventional technical scheme of a nano antibody or a mouse-derived monoclonal antibody;
the polyclonal antibody of the indicator molecule AFT-YJFZP008 can be obtained by the following method: AFT-YJFZP008 is used as an immune antigen, a test rabbit such as a New Zealand white rabbit is immunized in a conventional mode, and a known conventional polyclonal antibody preparation technical scheme is utilized to develop and obtain a rabbit source polyclonal antibody of an aflatoxin indicator molecule AFT-YJFZP 008;
the horseradish peroxidase-labeled antibody which is in binding reaction with the polyclonal antibody of the indicator molecule AFT-YJFZP008 is a horseradish peroxidase-labeled goat anti-rabbit antibody, and can be directly purchased from commercial products.
The ELISA coating buffer solution is a conventional carbonate buffer solution, and the preparation method comprises the following steps: weighing NaHCO31.465g、Na2CO30.795g, adding deionized water to the solution until the volume is up to 500 mL.
The ELISA color developing solution refers to ELISA conventional hydrogen peroxide and TMB color developing solution.
The stop solution is 2mol/L sulfuric acid aqueous solution of ELISA conventional chromogenic stop solution, and the preparation method comprises the following steps: adding 44 mL of concentrated sulfuric acid into 300mL of deionized water, stirring until the mixture is cooled, and finally metering to 400 mL.
The invention provides an application of a one-step method for efficiently screening an aflatoxin green prevention and control material in screening of the aflatoxin green prevention and control material.
According to the scheme, the aflatoxin green prevention and control material comprises but is not limited to aflatoxin green prevention and control materials such as plant essential oil, citral derivative bionic materials and the like.
Once the aflatoxin-based toxigenicity-producing indicator molecule AFT-YJFZP008 peptide is inhibited, the source of an aflatoxin biosynthesis pathway is also inhibited; (2) for the same toxigenic strain, the toxigenic indicator molecule AFT-YJFZP008 peptide of the aflatoxin is in positive correlation with the growth amount of the strain, so that an effective green prevention and control material which can inhibit synthesis of the aflatoxin from the source and growth of the aflatoxin toxigenic strain is successfully invented by one-step efficient screening, can be applied to screening of aflatoxin green prevention and control materials such as plant essential oil, citral derivative bionic materials and the like, and provides an important support for efficient green prevention and control of the aflatoxin.
The invention has the beneficial effects that:
1. the method can be used for screening the aflatoxin green prevention and control material which can simultaneously inhibit the synthesis of aflatoxin sources and the growth of toxigenic bacteria in one step, 2, the method is easy to operate, strong in practicability and easy to popularize and apply, and 3, the method has important significance for developing aflatoxin microorganism prevention and control technology and protecting food safety.
Detailed Description
The initial acquisition of the indicator molecule AFT-YJFZP008 was carried out using the discovery method:
(1) culturing Aspergillus flavus strain with high virulence producing ability to obtain strain culture and extracellular secretion protein mixture; then breaking the cells of the strain culture to obtain an intracellular protein mixture; combining the extracellular secretion protein mixture and the intracellular protein mixture, and adding carbodiimide for coupling to obtain an aspergillus flavus antigen;
(2) immunizing an animal to be tested with the aspergillus flavus antigen to obtain a nano antibody library or a monoclonal antibody library;
(3) obtaining protein combined solution of the aspergillus flavus strains with different virulence productions, detecting the proteins of the aspergillus flavus strains with different virulence productions by using the antibodies in the antibody library obtained in the step (2), and obtaining a series of detection signals;
(4) finding out a nano antibody with a detection signal showing positive correlation with the virulence production of the aspergillus flavus strain, namely an aspergillus flavus strain virulence indicator molecule antibody, and finding out a protein corresponding to the aspergillus flavus strain virulence indicator molecule antibody, namely the found aspergillus flavus strain virulence indicator molecule.
In the scheme, the aspergillus flavus strain with strong virulence producing capacity in the step (1) is separated and identified from the nature by a conventional method or obtained by artificial modification, and the identification result of the strain with strong virulence producing capacity by an NY/T2311-2013 standard method is not less than 10 mug/kg.
The aspergillus flavus strains with different virulence generating capacities in the step (3) are not less than 3 strains, and the virulence generating capacity is identified by an NY/T2311-2013 standard method, and the results are presented in at least 3 levels of high, medium and low.
The culture medium adopted in the culture of the aspergillus flavus strain with strong virulence is a Chao's culture medium or other nutrients for normal growth of the aspergillus flavus, the culture time is not less than 12 hours, and the culture environment temperature is 15-35 ℃.
The cell disruption of the strain culture is carried out by a conventional liquid nitrogen grinding method or a cell disruptor and the like.
The amount of the carbodiimide is 0.005-0.1 g per 1.0mL of combined extracellular secretion protein mixture and intracellular protein mixture.
The coupling reaction is carried out for 2-6 h at 15-37 ℃, and is carried out overnight at 4-10 ℃.
The immunization is a conventional immunization mode and is used for inoculating the aspergillus flavus antigen. The test animal is a white mouse or alpaca or other test animals with similar effects.
According to the scheme, the antibody preparation process refers to a conventional nano antibody preparation technical process or a conventional hybridoma monoclonal antibody preparation technical process based on cell fusion.
According to the scheme, the detection of the proteins of the aspergillus flavus strains with different virulence degrees is realized by adopting a conventional Western Blot technical process, namely, the proteins of the aspergillus flavus strains with different virulence degrees are transferred onto a nitrocellulose membrane, and then the antibodies in the antibody library are used for detection by a direct method or an indirect method, or other technical processes with similar effects are adopted.
According to the above scheme, the above direct method is that the antibody in the antibody library is coupled with a signal material by a conventional method, and then undergoes an immunological binding reaction with the corresponding protein transferred onto the nitrocellulose membrane.
According to the above scheme, the indirect method is that the antibody in the antibody library is firstly subjected to immunological binding reaction with the corresponding protein transferred to the nitrocellulose membrane, and then the second antibody and the signal material conjugate are subjected to immunological binding reaction with the antibody bound to the nitrocellulose membrane.
The signal material in the detection is horseradish peroxidase or colloidal gold or fluorescent material or other materials with similar effects. The detection signal is a chromogenic reaction signal or a spot signal or a fluorescent signal.
The indicator molecule AFT-YJFZP008 antibody can be prepared by using all peptide segments or partial peptide segments of the aflatoxin indicator molecule AFT-YJFZP008 antibody through a conventional antibody preparation technical process after knowing the whole sequence of the aflatoxin indicator molecule AFT-YJFZP008 antibody.
The culture medium is prepared according to the following formula: 3% (w/v) sucrose, 0.3% (w/v) NaNO3, 0.1% (w/v) K2HPO4, 0.05% (w/v) MgSO4 & 7H2O, 0.05% (w/v) KCl, 0.001% (w/v) FeSO4, pH6.5, and prepared to obtain a Chaudhur medium. Randomly selecting 10 strains of Aspergillus flavus distribution, toxigenicity and infection research in typical peanut producing areas in China-Master academic thesis of Chinese academy of agricultural sciences, Zhang apricot by author, page 33-published toxigenic fungus strains HLJ-1, HeNZY-2, HuBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-108-19, JSnnt-1, HuNdx-7, HBHA-8-17 and the like, respectively inoculating the strains into the Czochralski culture medium, culturing for 5 days at 28 ℃ and 200rpm/min, fully homogenizing by a conventional method, crushing cells, and purifying by a conventional protein purification system, protein electrophoresis, immune affinity and other methods to obtain the indicating AFT-YJF008 ZP. Test results show that AFT-YJFZP008 can be prepared in all the strains of the toxigenic fungi, under the same culture conditions, AFT-YJFZP008 prepared by HBHA-8-17 is the most abundant, and AFT-YJFZP008 prepared by HLJ-1 is the least abundant.
The immunoaffinity method is that a nano antibody or a monoclonal antibody of an indicator molecule AFT-YJFZP008 is used for preparing an immunoaffinity column by a conventional method, and then the immunoaffinity column is enriched and purified from aflatoxin toxigenic fungal cell disruption solution by an immunoaffinity method and is obtained by dissolving with deionized water. Specifically, the aflatoxin toxigenic fungal cell disruption solution can be diluted by using a sample loading solution, filtered by using conventional filter paper, continuously added into the immunoaffinity column, when the basic flow is exhausted, the column body is washed by using conventional eluent of the immunoaffinity column, finally eluted by using glycine buffer solution with pH 2.2 or 70% methanol aqueous solution, the solution is timely removed by using a conventional ultrafiltration centrifugal method after the eluent is collected, and then the protein remained in the ultrafiltration centrifugal tube is dissolved out from the ultrafiltration centrifugal tube by using sterile water, so that the aqueous solution of the indicator molecule AFT-YJFZP008 can be obtained.
Example 1 preparation of Nanobody of Aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZP008
AFT-YJFZP008 is used as an immune antigen, an alpaca or Balb/c mouse is immunized by a conventional mode, and the preparation method can be developed by a known conventional technical scheme of preparing a nano antibody or a mouse-derived monoclonal antibody.
Dissolving AFT-YJFZP008 obtained by the preparation in a conventional PBS buffer solution or physiological saline until the concentration is not lower than 0.1mg/mL, mixing and emulsifying the solution with Freund complete adjuvant in the same volume, immunizing alpaca by back subcutaneous or intradermal multipoint injection, then boosting the immunity for 1 time every 2-4 weeks, and replacing the Freund complete adjuvant with Freund incomplete adjuvant when boosting the immunity. Monitoring the immune effect by adopting a conventional ELISA process until the serum titer of the alpaca does not rise any more, then performing operations of venous blood sampling, total RNA extraction, cDNA synthesis, VHH gene amplification, VHH gene fragment recovery, VHH gene and double enzyme digestion treated pCANTAB 5E (his) vector connection, connection product electrotransformation, construction of a nano antibody gene bank, nano antibody gene bank rescue and the like on the immune alpaca according to a method of patent document CN103866401A, and finally obtaining the rescued nano antibody gene bank.
The AFT-YJFZP008 obtained by the preparation is fixed on a solid phase carrier such as a 96-hole enzyme label plate according to the gradient of 8 mug/hole, 2 mug/hole, 0.5 mug/hole and 0.1 mug/hole, 2-4 times of panning is carried out on the rescued nano antibody gene bank by the method of reference patent document CN103866401A, the antibody generated by each phage clone is identified by AFT-YJFZP008 and indirect non-competitive ELISA, the phage corresponding to the positive result is a phage positive clone, and the nano antibody is prepared by the positive clone in a conventional mode of nano antibody preparation, namely the nano antibody of the AFT-YJFZP008, is used for further application and research work, preferably, the nano antibody with strong specificity and high affinity is characterized by the ELISA method.
Example 2 preparation of monoclonal antibody against Aflatoxin toxigenic bacteria producing virulence indicator molecule AFT-YJFZP008
AFT-YJFZP008 is used as an immune antigen, an alpaca or Balb/c mouse is immunized by a conventional mode, and the preparation method can be developed by a known conventional technical scheme of preparing a nano antibody or a mouse-derived monoclonal antibody.
Dissolving AFT-YJFZP008 obtained by the preparation in a conventional PBS buffer solution or physiological saline until the concentration is not lower than 0.1mg/mL, mixing and emulsifying the solution with Freund complete adjuvant in the same volume, performing back subcutaneous or intradermal multipoint injection on BALB/c mice, performing booster immunization for 1 time every 2-4 weeks, and replacing Freund complete adjuvant with Freund incomplete adjuvant during booster immunization. Monitoring the immune effect by adopting a conventional ELISA process until the serum titer of a BALB/c mouse does not rise, then separating spleen cells of an immune mouse, fusing the spleen cells with murine myeloma cells SP2/0, and selectively culturing hybridoma cells by using a semisolid culture medium according to the method of patent document CN103849604A, and after white spots of a needle tip grow on the semisolid culture medium, respectively picking the white spots to a 96-well culture plate internally provided with a conventional hybridoma culture medium, thereby obtaining a monoclonal hybridoma resource library.
The monoclonal antibody obtained from the culture supernatant of the monoclonal hybridoma is obtained by the method of patent document CN103849604A, the AFT-YJFZP008 obtained by the above preparation is fixed on a solid phase carrier such as a 96-well enzyme label plate according to gradients of 8 mug/well, 2 mug/well, 0.5 mug/well and 0.1 mug/well, each monoclonal antibody is identified by an indirect non-competitive ELISA program, and a positive clone is selected to obtain the AFT-YJFZP008 monoclonal antibody which is used for further application and research work, preferably the AFT-YJFZP008 monoclonal antibody with strong specificity and high affinity.
Example 3 preparation of specific Rabbit-derived polyclonal antibody of Aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZP008
AFT-YJFZP008 is used as an immune antigen, a test rabbit such as a New Zealand white rabbit and the like is immunized by a conventional mode, and the test rabbit can be obtained by developing by utilizing a known conventional rabbit polyclonal antibody preparation technical scheme.
The AFT-YJFZP008 obtained by the preparation method is directly used as an antigen, a solution with the concentration of not less than 0.1mg/mL and Freund's complete adjuvant are mixed and emulsified in equal volume, the New Zealand white rabbits are boosted for 1 time every 2-4 weeks by a back subcutaneous or intradermal multipoint injection mode, and the Freund's complete adjuvant is replaced by the Freund's incomplete adjuvant during boosting. The immune effect is monitored by adopting a conventional ELISA process, and after the serum titer of the immune animal does not rise any more, the serum of the immune animal is prepared by a conventional method, namely the rabbit source polyclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZP 008.
Example 4 establishment of an immune rapid detection method for aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZP008
The basic operation procedure of an immune rapid detection method of AFT-YJFZP008, namely a double-antibody sandwich indirect non-competitive ELISA method: coating a nano antibody of AFT-YJFZP008 in an ELISA plate, and washing the plate; adding sealing liquid for sealing, and washing the plate; adding AFT-YJFZP008 or a solution to be detected for reaction, and washing the plate; adding AFT-YJFZP008 rabbit source polyclonal antibody for reaction, and washing the plate; adding a goat anti-rabbit antibody labeled by horseradish peroxidase for reaction, and washing the plate; adding color development liquid for reaction; adding stop solution, reading by a microplate reader and calculating the result. The following work is all done using this basic procedure.
Determination of antibody concentration: a chessboard titration method is adopted to carry out a plurality of parallel experiments simultaneously, the nano-antibodies with different concentrations are adopted to coat, in addition, the rabbit source polyclonal antibody is set to have different concentrations, finally, a proper working concentration of the nano-antibodies and the rabbit source polyclonal antibody is determined according to results, namely, the concentrations of the two antibodies corresponding to a point with an OD450nm value approximate to 1.0 are selected under the principle of saving the using amount of the antibodies, ELISA result researches show that other concentrations of the nano-antibodies and the rabbit source polyclonal antibody can also realize detection, one proper coating concentration of the nano-antibodies is 2.2 mug/mL, and one proper concentration of the rabbit source polyclonal antibody is 2.8 mug/mL.
Determination of optimal antibody coating conditions: coating is the first step of ELISA method research, and the quality of the coating effect has a very critical influence on the ELISA result. In order to determine the influence of different coating conditions on the detection result, three different conditions of 4 ℃ coating overnight, 37 ℃ constant-temperature coating for 2h and 37 ℃ constant-temperature coating for 1h are selected to be coated in the hole, and the detection result shows that the three coating modes can realize coating, and the coating overnight at 4 ℃ is the optimal coating condition.
Determination of the optimal blocking agent: after the antibody is coated, in order to avoid the unoccupied sites of the wells of the enzyme label interfering with the subsequent steps of ELISA, some inert protein, i.e., blocking agent, needs to be used to occupy the sites, and the improper blocking agent can be non-specifically combined with the secondary antibody, so as to cause false positive. The research adopts three different sealing agents of 3% BSA/PBST, 3% skimmed milk powder/PBST and 5% skimmed milk powder/PBST for sealing, and research results show that although the three sealing agents can achieve the purpose of sealing in different degrees, the sealing effect of the 5% skimmed milk powder/PBST is the best sealing agent.
Determination of the closure time: the research sets three different sealing durations of 1h, 2h and 3h for sealing at the constant temperature of 37 ℃, and the detection result shows that the value of the positive hole OD450nm value/the negative hole OD450nm value is the largest under the setting of the constant temperature sealing of 2h at 37 ℃, and the value of the positive sample OD450nm is more than 1, so the constant temperature sealing of 2h at 37 ℃ is the optimal sealing time.
Determination of the reaction time of the rabbit polyclonal antibody: the research respectively adopts three reaction durations of 30min, 50min and 1h for constant temperature reaction at 37 ℃, and the detection result shows that: the reaction time is the optimal reaction time of the rabbit polyclonal antibody when the reaction is carried out for 50min at the constant temperature of 37 ℃.
Drawing an ELISA standard curve of aflatoxin toxigenicity-producing indicator molecule AFT-YJFZP 008: AFT-YJFZP008 molecules are respectively diluted to 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30 and 300ng/mL, 200 mu L of the AFT-YJFZP molecules are put into each hole, and an ELISA standard curve of the AFT-YJFZP008 molecules is drawn by adopting the optimal conditions. The correlation coefficient of the double-antibody sandwich enzyme-linked immunosorbent assay method established under the optimal optimization condition reaches 0.99, and the detection limit of AFT-YJFZP008 molecules can reach 0.05ng/mL, which indicates that the detection method has good detection sensitivity and accuracy.
Evaluation of method specificity: in order to evaluate the specificity of the immunodetection method of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZP008, a plurality of strains of fungi such as fusarium oxysporum, aspergillus niger, aspergillus ochraceus and fusarium moniliforme which have certain homology with the aspergillus flavus are selected for research, the results are shown in table 1 by detecting cell disruption solutions of fungus cultures, and the method has no obvious cross reaction on the proteins of the fungi which have homology with the aspergillus flavus, thereby showing that the established aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZP008 immune rapid detection method has good specificity.
TABLE 1 Aflatoxin toxigenic bacteria virulence indicator molecule immunity rapid detection method specificity determination result
Figure BDA0003249393210000081
Method repeatability evaluation: in order to evaluate the repeatability of the established aflatoxin toxigenicity indicator molecule AFT-YJFZP008 immune rapid detection method, 4 toxigenic strains of aflatoxin, namely positive 1, positive 2, positive 3 and positive 4, and 1 non-toxigenic strain of aflatoxin, namely negative 5 are randomly selected for research, and the data variation between plates and plates of a measurement result is analyzed. The results of the above studies are shown in Table 2, and the calculated coefficient of variation in the plate is 0.6% -5.7%, and the coefficient of variation between plates is 1.2% -9.1%, both of which are below 10%, so the above method has good repeatability.
TABLE 2 reproducible determination results of fast detection method of virulence indicator AFT-YJFZP008 immunity
Figure BDA0003249393210000091
The method comprises the following steps of: in order to evaluate the detection accuracy of the method and examine the practicability of the method, the peanut and corn samples are taken as examples for research and evaluation, aflatoxin toxigenicity-producing indicator molecule AFT-YJFZP008 is added into the corn and peanut samples, the recovery rate of the detection result is closer to 100 percent, and the more accurate and practical the method is. The research result is shown in a table 3, and the result shows that the established detection method is used for detecting the virulence indicator AFT-YJFZP008 in peanuts and corns, the recovery rate reaches 83% -106%, and the method is high in accuracy, can be applied to detection of actual samples, and has good practicability.
TABLE 3 Aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection method additive recovery test results
Figure BDA0003249393210000092
Figure BDA0003249393210000101
Example 5 screening of Aflatoxin Green prevention and control Material plant essential oil
1) Preparing the plant essential oil to be screened: can be prepared by conventional plant essential oil preparation method, or obtained by commercial purchase. This example illustrates the use of the present invention with 5 kinds of green material plant essential oils, namely, cinnamon Ceylon essential oil (Cinnamunu verum), Ocimum basilicum essential oil (Ocimum basilicum), Cymbopogon nardus essential oil (Cymbopogon nardus), Mentha essential oil (Mentha arvensis), and Eucalyptus globulus essential oil (Eucalyptus globulus), which are commercially available, but not limited to these plant essential oils.
2) Preparing aflatoxin toxigenic bacteria: taking Aspergillus flavus distribution, virulence and infection research in typical peanut producing areas in China-Master thesis of China academy of agriculture sciences, Zhang xing, page 33, published Aflatoxin toxigenic strain HuNdx-7 as an example, selecting a small amount of bacterial liquid of the HuNdx-7 strain, inoculating the bacterial liquid on DG18 solid culture medium for activation, carrying out inversion culture under the constant temperature dark condition at 28 ℃, taking 0.1 percent of Tween water to wash spores after yellow green spores grow on DG18 culture medium, collecting the spores to prepare spore suspension, counting under a high-power microscope, calculating the concentration of the spores in the spore suspension, taking the spore suspension to transfer to 200ml of Sabouraud liquid culture medium until the final concentration of the spores is 5 multiplied by 105cfu/ml。
3) Prevention and control test: and (3) respectively culturing the 5 plant essential oils and the aflatoxin toxigenic strain spore suspension together in a Sasa liquid culture medium containing No. 6 peanut powder of Zhonghua, wherein the volume concentration of the plant essential oil is not higher than 200 mu L/mL (20 mu L/mL is taken in the embodiment, the culture is carried out at the temperature of 28 ℃ and 200rpm for 1 day, each treatment is repeated, and the Sasa culture medium is used for replacing the strain to be screened and cultured for a certain time to obtain a control group.
4) The content determination of the indicating molecule AFT-YJFZP008 and the selection of green prevention and control materials: the method established in the example 4 is adopted to determine the content of the indicator molecule AFT-YJFZP008 of the test treatment group and the control group, and the inhibition rate of the strain to be screened on the AFT-YJFZP008 is calculated by the following method:
AFT-YJFZP008 inhibition rate (control AFT-YJFZP008 content-treatment AFT-YJFZP008 content)/control AFT-YJFZP008 content x 100%
The research results are shown in Table 5, and the results show that the inhibition rates of AFT-YJFZP008 are obviously different among different strains, and the higher the inhibition rate of AFT-YJFZP008 is, the better the effect of simultaneously inhibiting the synthesis of aflatoxin sources and the growth of toxigenic bacteria is, so that the green prevention and control material for aflatoxin with better effect is deduced and screened, and the green prevention and control material can be used for further preparation development and development utilization.
TABLE 5 inhibition effect of 12 green prevention and control materials to be screened on AFT-YJFZP008
Figure BDA0003249393210000102
Figure BDA0003249393210000111
5) And (3) verification and confirmation: in order to further verify the reliability of the screening result of the one-step method, a conventional multi-step method, namely a bacteriostatic test and a toxin production inhibition test, is adopted.
Co-culturing the plant essential oil obtained in the step 1) and the final spore suspension obtained in the step 2) in a Sabouraud's liquid culture medium at 28 ℃ and 200rpm for 1 day, wherein 3 times of treatment are set, and culturing the plant essential oil and the final spore suspension for a certain time by using the Sabouraud's liquid culture medium instead of the strain to be screened to obtain a control group. The aflatoxin levels of a treatment group and a control group are measured by adopting a high performance liquid chromatography-mass spectrometry combined method in GB 5009.22-2016 national food safety standard food for measuring B and G families of aflatoxins, and the inhibition rate of the strain to be screened on the aflatoxins is calculated by the following method:
the inhibition rate of aflatoxin is (content of aflatoxin in control group-content of aflatoxin in treatment group)/content of aflatoxin in control group is multiplied by 100%
The results of the aflatoxin inhibition calculation are shown in table 6.
The hyphae are cleaned and dried, the dry weight of the hyphae of a treatment group and a control group is measured, and the inhibition rate of the strains to be screened on toxigenic bacteria is calculated in the following way:
toxigenic bacteria inhibition rate (control toxigenic bacteria dry weight-treatment toxigenic bacteria dry weight)/control toxigenic bacteria dry weight × 100%
The inhibition effect on the growth of toxigenic bacteria was calculated and the results are shown in Table 6.
TABLE 6 inhibition effect of the 12 strains of green prevention and control materials to be screened on the growth of aflatoxin and toxigenic bacteria
Figure BDA0003249393210000112
According to the research results, the growth inhibition effect of the cinnamomum zeylanicum essential oil (material 01) on aflatoxin and toxigenic bacteria is obviously better than that of other strains, so that the technical scheme is correct and reliable.
Example 6 screening of Aflatoxin Bionical Green prevention and control Material
1) Preparing a bionic green prevention and control material to be screened: in the embodiment, only 3 kinds of artificially synthesized bionic green prevention and control materials are used
The preparation method of the 3 bionic green prevention and control materials of the citral derivative comprises the following steps: the synthetic route of the lemon oxime is shown as follows, and the specific steps are as follows: citral (500mg, 3.28mmol) and hydroxylamine hydrochloride (290mg, 4.17mmol) were dissolved in 5mL of water, and sodium hydrogencarbonate (345mg, 4.11mmol) was dissolved in 10mL of water to prepare a reaction system. NaHCO is slowly dissolved3Adding the aqueous solution dropwise into the reaction system, stirring at 55 deg.C for 5 hr, extracting the aqueous layer with diethyl ether for 3 times, mixing diethyl ether organic phases, and adding anhydrous Na2SO4Drying the ether organic phase overnight, filtering to remove solid, concentrating the dried ether organic phase under reduced pressure, and separating by rapid silica gel column chromatography to obtain yellow oily liquid as lemon oxime.
Figure RE-GDA0003394379480000121
Synthesizing a bionic green prevention and control material: the synthetic route is as follows: will be provided withDissolving 0.75mmol of 4-methylbenzoic acid or 4-bromobenzoic acid or 4-iodophenylbutyric acid in anhydrous CH together with TBTU and DMAP2Cl2Stirring at room temperature for 30min, adding 100mg of above lemon oxime, stirring for reaction for 6h, adding water, quenching, and reacting with NaHCO3Washing with saturated NaCl solution for three times, anhydrous Na2SO4Drying overnight, filtering, concentrating the filtrate under reduced pressure, and separating by rapid silica gel column chromatography to obtain final oily liquid which is citral bionic green prevention and control material. In the synthesis, 4-methylbenzoic acid or 4-bromobenzoate or 4-iodophenylbutyric acid is adopted as a reaction raw material, and the corresponding products, namely the bionic green prevention and control materials, sequentially comprise: 3, 7-dimethyl-2, 6-octadienealdoxime- (4-methylbenzoyl) ester-Ie, 3, 7-dimethyl-2, 6-octadien-1-aldoxime-O- (4- (4-bromophenyl) butyryl) ester-IIc, 3, 7-dimethyl-2, 6-octadien-1-aldoxime-O- (4- (4-iodophenyl) butyryl) ester-IIe.
Figure BDA0003249393210000122
Wherein, the reaction raw material RCOOH containing R is respectively 4-methylbenzoic acid, 4-bromobenzoic acid or 4-iodophenylbutyric acid, and the corresponding products are sequentially: 3, 7-dimethyl-2, 6-octadienealdoxime- (4-methylbenzoyl) ester-Ie, 3, 7-dimethyl-2, 6-octadien-1-aldoxime-O- (4- (4-bromophenyl) butyryl) ester-IIc, 3, 7-dimethyl-2, 6-octadien-1-aldoxime-O- (4- (4-iodophenyl) butyryl) ester-IIe.
2) Preparing aflatoxin toxigenic bacteria: taking Aspergillus flavus distribution, virulence and infection research in typical peanut producing areas in China-Master thesis of China academy of agriculture sciences, Zhang xing, page 33, published Aflatoxin toxigenic strain HuNdx-7 as an example, selecting a small amount of bacterial liquid of the HuNdx-7 strain, inoculating the bacterial liquid on DG18 solid culture medium for activation, carrying out inversion culture under the condition of constant temperature and darkness at 28 ℃, taking 0.1 percent of Tween water to wash spores after yellow green spores grow on DG18 culture medium, collecting the spores to prepare spore suspension, counting under a high-power microscope, calculating the concentration of the spores in the spore suspension, taking the spore suspension, and obtaining the spore suspensionThe suspension was transferred to 200ml of Sabouraud's broth to a final spore concentration of 5X 105cfu/ml。
3) Prevention and control test: respectively culturing the series of bionic green prevention and control materials to be screened, namely the bionic citral derivatives and the aflatoxin toxigenic strain spore suspension in a Sabouraud's liquid culture medium containing Luhua No. 8 peanut powder, wherein the final concentration of the bionic green prevention and control materials to be screened is not higher than 200 mu L/mL-30 mu L/mL is taken in the embodiment, the bionic green prevention and control materials are co-cultured for 1 day at the temperature of 28 ℃ and 200rpm, each treatment is set to be 3 times, and a control group is obtained by culturing the Sabouraud's culture medium instead of the strains to be screened for a certain time.
4) The content determination of the indicating molecule AFT-YJFZP008 and the selection of green prevention and control materials: the method established in the example 4 is adopted to determine the content of the indicator molecule AFT-YJFZP008 of the test treatment group and the control group, and the inhibition rate of the strain to be screened on the AFT-YJFZP008 is calculated by the following method:
the inhibition ratio is (content of control AFT-YJFZP 008-content of treatment AFT-YJFZP 008)/content of control AFT-YJFZP008 x 100%
The inhibition rate of the green prevention and control material on AFT-YJFZP008 is calculated, and the result is shown in Table 7. According to the principle that the higher the inhibition rate of AFT-YJFZP008, the better the effect of inhibiting the synthesis of aflatoxin sources and the growth of toxigenic bacteria, the better-effect green prevention and control material of aflatoxin, namely the biomimetic substance 3, 7-dimethyl-2, 6-octadiene-1-aldoxime-O- (4- (4-iodophenyl) butyryl) ester-IIe, can be easily screened out from Table 7, and can be used for further development and utilization of biocontrol agents.
TABLE 7 inhibiting effect of 3 kinds of green prevention and control material to be screened on AFT-YJFZP008
Figure BDA0003249393210000131
The Aspergillus flavus toxigenic strain HuNdx-7 in examples 5 and 6 is merely an example, and is not limited thereto, and other Aspergillus flavus toxigenic strains such as Aspergillus flavus standard toxigenic strain 3.4408 may be used.
By combining the above examples, the technical scheme of the invention can be used for screening out the aflatoxin green prevention and control material which can inhibit the synthesis of aflatoxin sources and the growth of toxigenic bacteria simultaneously in one step, is easy to operate, strong in practicability and easy to popularize and apply, and has important significance for developing aflatoxin microorganism prevention and control technology and guaranteeing food safety.
< 110 > institute of oil crops of Chinese academy of agricultural sciences
Method for efficiently screening aflatoxin green prevention and control material in one step with less than 120 and application thereof
＜160＞ 1
＜210＞ 1
＜211＞ 18499
＜212＞ PRT
< 213 > Aspergillus flavus
＜400＞ 1
ALAALASERL EPRALASERP HESERGLYPH ELYSALAPHE ASPSERSERP HEPRLEPRLY 60
SALAPHEPRA SNALAPRASP LYSALAGLYV ALILEPRGLS ERLEHISGLN ASPTHRVALG 120
LYTHRPHEGL YLYSALAILE HISASPGLVA LSERPRVALG LYASPTHRAS PALALELEGL 180
ARGALAILEA SNASPTYRIL EASPSERGLN LEASPLYSAL ALELEPHEGL YALAALAGLY 240
SERALAGLAS PPRVALVALV ALLYSALALE THRASNGLYA LAGLYALAIL ELYSALALEV 300
ALSERHISAS PGLYTHRPHE VALALAASPA LALYSALAAS NASNTYRCYS SERASNGLNV 360
ALGLGLYPRT YRSERLETYR SERGLYARGA LAPRVALVAL GLNTYRALAL EASNARGALA 420
SERMETVALT RPGLGLALAG LNGLNVALSE RGLYLYSALA SERPRSERTY RLETHRALAT 480
HRPRARGALA VALGLGLNSE RLEASPALAI LEARGALAVA LGLYGLNALA THRGLARGAL 540
AVALILETHR ASPILEVALA SNGLNGLNAR GALAVALSER PRSERPHEGL ASPVALTRPS 600
ERGLNPRARG ALATYRGLNG LYTYRPHEHI SSERASNASP ASPLELEASN ARGCYSASPV 660
ALALATHRTH RASPVALTYR TYRSERGLYL YSCYSVALTH RALAPRSERG LYPRCYSGLY 720
GLNLYSCYST YRALALEVAL ASNHISGLPH ESERARGASP ALAASPALAC YSASNGLYGL 780
YGLYILEGLT YRASPSERPR ALAASPTHRP RLEGLPHELY SASPALAGLY SERPRLYSPR 840
VALVALGLNI LEGLYHISGL GLYASPVALG LYVALALAGL ILEGLNASNM ETARGASPAL 900
AGLYTYRGLT HRSERILETH RASPTYRTRP GLYARGASPA LAVALTYRAL ALEASPALAI 960
LETYRGLYIL EASPALAARG ASPGLARGAL ASERLEASPV ALGLPRSERL EPRTRPPRAS 1020
NASPGLYILE PHEARGTRPA RGASPPHEPH EASNHISVAL THRILELYSA SPPHEGLYTR 1080
PASPSERALA PHEASPGLNL YSGLYASNSE RLEGLYLECY SLEPRTHRAS PPHELYSASP 1140
PHEPRCYSAS PVALGLNARG PRPRASPLEA LAALAASNAS PALALYSASP PHETHRASPI 1200
LETHRALAGL YSERSERILE GLYCYSASPG LYVALASNPR GLNTHRGLYL YSASPGLYAL 1260
AGLYGLNMET PHEILEPRLE ASNPRASNAL ATYRSERPRA SNTHRLEASN LYSASPGLYA 1320
SPLEVALTHR GLNGLNASNG LLEGLNGLYL YSASPLEASP GLNASPILEG LLYSLYSASP 1380
LEASNASPGL YGLYSERSER VALGLYVALG LNASNARGLY SASPLEASNP RASNGLYSER 1440
GLNPHEILET HRPRGLYGLY LYSASPLEPR TRPTYRGLNC YSASNGLNGL ILEHISTHRL 1500
EVALSERGLY LELEARGARG ASPASNILEL EPRGLASNLE ASPASPGLYL EPRSERGLNP 1560
HEVALTYRGL LYSASPASNT HRCYSASNAL APRILEPRVA LSERPHEPRV ALALAPRTHR 1620
ASPTHRLYSA SPPRPHELYS ALAILEILET HRLESERALA ARGLEASPTH RPHEALATHR 1680
ILEASNTHRL EPHELYSASP PRPRILEASN METGLYPRIL EPRALATHRT HRASPLETHR 1740
ASNMETASPA RGARGASPPR TYRMETPHEH ISGLNALAAS NLEARGASPG LNCYSASNTY 1800
RSERLEGLNT YRTHRILEGL YASNLYSASP GLNGLLYSAR GGLNARGASP GLNILEILEG 1860
LCYSARGASP THRLEVALIL EPRPRGLYSE RARGASPTHR SERLECYSPR METALAPRPR 1920
ASNSERPHEM ETSERTHRLE PRMETTHRAL AASPPHEARG ASPTHRTHRG LYPHEILEGL 1980
THRASPPRLE LYSASPVALH ISGLYPHEAL ATHRARGASP VALVALGLAL APHEARGASP 2040
TYRALACYSP RTRPASNGLY GLYGLGLVAL SERLELYSGL ALAALASERA LAALALEALA 2100
ALAGLYTYRL YSGLALAGLY LEVALPRPHE GLNVALSERP RTHRTHRLYS GLGLTYRASP 2160
GLGLYLEARG METVALASNL YSGLYLEALA LYSGLGLYAS NGLSERVALG LNVALPRARG 2220
ASNHISALAL ESERSERASP ARGGLHISHI SGLLEALAIL EALASERLYS GLILEASNGL 2280
NILEGLNARG GLILESERPH EASNGLNALA TRPLEARGGL LESERALATH RVALMETASP 2340
HISLELESER GLNARGGLLE VALLEVALLE GLYARGGLPR GLYALAGLGL YVALCYSGLT 2400
HRTHRPRGLY VALLYSGLPR GLYILECYSG LTHRTHRPRG LYVALLYSGL ARGGLLEASP 2460
SERARGGLSE RGLPHEPHEI LEARGPHEAL ALESERTHRT RPALAARGPH EALAASNGLN 2520
METPRASNGL YCYSGLNASP LEILESERTH RCYSLYSPHE ALASERASPA SPALACYSGL 2580
GLLYSPHEGL GLILEALAPR TYRVALASNG LYLYSARGPH EGLGLYTYRL EPRASPALAA 2640
RGPHEGLASN SERASNVALL YSPHEGLASN SERASNVALL YSSERSERVA LVALARGPHE 2700
GLYLYSPRVA LGLYALAVAL GLYSERALAA LATHRALALE LYSPHEGLYT RPTRPSERAL 2760
AASPGLYALA TRPPRGLYAL ALEASPASPP HEVALVALTR PVALGLNLYS LYSPHEHISA 2820
SPSERSERAS NASPSERGLY ASNARGPHEH ISVALLETHR ALAGLNLESE RPHEPRARGP 2880
HELEASPGLA LALETHRTYR PRPRPRLYSP HEASNSERLE ALAASPARGP HEGLNTYRPR 2940
GLYASPLEPH EASPGLNGLY THRTHRILEA RGPHESERSE RCYSSERGLY THRARGPHES 3000
ERTHRVALAL AGLYSERARG PHESERVALA LAGLILELEP RGLYALALYS PHEVALGLYG 3060
LYALASERTH RASPALAPHE ALAASPPRLY SPHEVALTHR ASPASNGLYA SPSERLYSGL 3120
YALAASPARG GLYARGGLYA SPALAGLYSE RPRVALPHES ERPRASPSER LYSGLYPHEP 3180
HETHRALAPR GLYARGGLYG LYGLYGLYGL YTHRPHEGLY VALVALMETG LSERTHRHIS 3240
ARGGLYGLYS ERGLYALALE GLYLEALAPH ESERGLALAL YSGLYILEAS PVALALALYS 3300
PRTHRGLYAR GGLYILEGLN ILEASNASPP RSERILEASN ASPASPSERV ALMETILETY 3360
RALAPRALAV ALARGGLYLE ARGASNSERG LYVALHISGL YTHRPHESER SERARGPRGL 3420
GLNGLGLILE GLNLYSGLYL ETYRALAGLY HISARGGLYP RLEASNGLGL YGLYLETYRA 3480
LAGLARGGLY GLNTHRPRLE PRILELEVAL ALAASPGLYA RGGLYSERAS PCYSSERTHR 3540
THRALAGLYG LYCYSCYSGL YGLYTHRGLY CYSGLNPRAS NGLTHRLEVA LPHEGLYSER 3600
SERASPLEAL AARGGLYVAL ASPPHETHRG LASPPRLELE GLNGLYARGG LYVALGLYSE 3660
RASPALATRP THRVALSERG LSERGLYARG GLYVALLEAR GPRVALSERT HRGLYSERAR 3720
GGLYVALTYR ASPILEARGG LYTYRLYSPR SERALASERS ERGLYSERLY SGLYTYRPRT 3780
HRSERGLNGL NASNTRPVAL GLYTHRLELE LEPRARGHIS ALAGLYGLNC YSGLYGLTYR 3840
HISGLASNLY SHISPHEGLN LEILEASNTH RALAALATYR TRPLYSHISP HETHRSERLE 3900
GLGLLYSHIS PHEVALASPT HRPHEGLYLE HISGLYHISL YSHISGLYGL YPRASNPHEG 3960
LGLNLEPRIL EASNGLNPRA RGHISLEPHE GLYLEHISAR GHISTHRASP TYRSERSERG 4020
LNGLSERTHR SERTYRLYSH ISVALASPGL YPHEGLYILE HISTHRPHEA RGHISVALTY 4080
RASPALAVAL GLNASPLYSI LEALAPHEAL ASERTYRLEG LGLTYRALAA RGILEALAPR 4140
GLNPHEGLYA SPLELYSILE ALASERLEAS NASPSERTYR GLTHRLELYS LYSARGILEP 4200
HEARGLELEG LYTHRPRASP GLASPSERTR PPRGLYVALT HRSERPHEPR ASPTYRLYSI 4260
LELEPHEASP SERASNASNV ALALATHRGL YVALGLNVAL SERTHRGLYG LYTHRPHEGL 4320
YTHRARGILE ASNPRHISGL LESERILEAR GASPPRASPP HETYRASNGL ILETYRVALT 4380
HRGLSERLYS ARGILEGLNA LAPHEVALIL ETYRPRGLAS NPHEASPLYS ILESERALAT 4440
YRVALGLGLY SERSERARGI LESERHISHI SALAGLNTHR LELEGLGLYL EGLYTHRHIS 4500
ARGTYRLEGL SERCYSTHRG LARGILESER TYRLYSGLPR GLYILECYSG LTHRTHRPRG 4560
LYVALLYSIL EVALPRGLGL TYRVALPRIL ETHRLYSILE VALGLNVALG LYASPLEARG 4620
ILEVALTHRP RASPGLYLYS ILETYRASPS ERILETYRVA LARGLYSALA LYSGLSERSE 4680
RSERGLSERS ERASPSERSE RGLSERGLSE RGLSERGLSE RGLASPGLLY SLYSPHETHR 4740
ASPTHRPRVA LLETYRGLYP RLYSLYSMET GLASPASPLE ARGLYSPRLE GLYTHRGLYT 4800
HRASPLETRP PRLYSLYSTH RLELEPHETY RALASERSER HISGLALAIL ESERPHEASP 4860
SERCYSARGL YSTHRSERSE RSERTHRALA THRSERTHRS ERTHRSERTH RGLYALAALA 4920
ALALEPRTHR ALAALAPHEG LYALAVALGL GLYGLYLEME TLEGLYVALV ALLEGLYVAL 4980
LEGLYLELEA SPARGPRPRV ALILEPRLEP RPRSERASPS ERASPVALTH RALAPHEARG 5040
LEGLGLLEAS NGLNARGLEG LGLNTYRARG METGLNLESE RGLYLELESE RGLNASNGLY 5100
GLNGLYSERL YSLEPHESER ILEPRALAAS PALAGLYASP ASPTYRLYSP RLYSLEPHES 5160
ERTYRLEASP THRGLNLEAS NARGLEPHET YRASNSERLE THRPRALAGL GLNGLNPHEV 5220
ALVALASPAL AILEARGLEL YSASPLEVAL LESERLELEA SNLESERSER PHEASPALAS 5280
ERGLYTYRIL EASPARGLEA SNTHRGLYAL AVALILEPRV ALLEVALARG LEPRASPILE 5340
CYSASNTHRC YSPHELYSLE SERGLNLEGL SERGLYLYSL ESERSERILE ALALEPRARG 5400
LETHRASPLE GLILEASNAR GLETHRGLYA SNLEGLYGLY GLASPTYRGL NASPLYSMET 5460
PRMETPRILE LEVALALAAS PGLYARGASN ALAGLYILEG LNTHRSERAR GASNALAHIS 5520
GLYGLNGLIL ELELEARGAS NALALEGLNT HRMETTYRAS PTHRGLNASP LYSASNASPP 5580
RVALALAVAL PHEASPGLYS ERVALILEPR LYSASNPHEA SPASNASPGL NHISARGASN 5640
PHEGLNGLLE PHEGLYILEL YSASNGLYAS PGLNSERPRP RSERALALEG LYPRLEPRSE 5700
RVALILEGLA RGASNHISAS NVALLESERA LAILEPRGLN GLPRTYRARG ASNHISTHRA 5760
LAGLYILEGL ALAARGASNL EGLTHRGLAR GGLNLEARGA SNLEGLVALL ESERLETHRL 5820
YSASNLEVAL GLYTHRSERG LYPHETHRSE RALAARGASN ASNVALILEI LEGLNLEASN 5880
ARGASNPRAS PLESERSERT HRSERASPTH RTHRASPVAL ILEARGASNS ERILELEGLG 5940
LYPRASPVAL LYSASNSERV ALVALGLYIL ELYSPRTHRV ALGLYLETHR SERARGASNV 6000
ALGLYLEVAL SERVALSERL EASPGLYLYS ASNVALLEAS PTYRGLYALA ARGASNVALV 6060
ALLEASPTHR THRALALESE RALAASNTHR LYSASNTRPH ISARGLESER PHETHRTYRA 6120
SNCYSTHRPR SERALAASNT YRGLNTYRIL EPRALATYRL YSASNTYRPH EALAGLTHRG 6180
LGLNVALMET PHEGLNPRGL YHISILEVAL ARGASNTYRI LEVALVALAS PALAASPSER 6240
SERPRLEGLN ILEVALILEA SPGLYPHEAR GPRPRMETGL ASPILEVALS ERPRTHRLEG 6300
LASPLEILEH ISLEALALEA RGARGPRPRG LNPRASPGLY ALATHRCYSI LELYSGLYAS 6360
NGLCYSGLAL APHECYSVAL ASPGLYVALC YSLYSPRGLN GLYASPPRLY SPRGLNSERI 6420
LEASPTHRIL EVALGLYTHR ASNLEHISME TASPILELES ERASPLEALA ALAALALEAL 6480
AGLYSERILE GLYVALALAP RSERSERASN LEASPPRTHR ARGLYSPRVA LALAASPALA 6540
ALAVALVALA SNALACYSGL SERPHEPRLE SERPHEASPT HRASPVALSE RARGARGPRT 6600
RPVALGLYGL YGLNILEVAL ASNSERILEP RALASERVAL GLLYSILEAL AVALLEGLGL 6660
NVALARGGLN ASPGLYHISP HESERVALPR SERTYRALAG LYHISVALAL ATHRMETTHR 6720
SERVALSERL EARGARGGLN ASPLEPHEGL ALAILEGLAL AGLYARGGLN ILEGLNTHRS 6780
ERARGGLNLE PRPRSERLEP RTYRTHRPHE TYRTHRSERT YRTHRSERGL ASPSERTYRL 6840
YSLYSGLNLE SERGLASPGL YVALASPVAL VALVALVALA LAGLARGARG ASPGLGLHIS 6900
GLNLEASPAS PASPPHEGLG LYILEASPAS PGLMETASPG LSERGLNSER ARGARGPRAS 6960
PLESERTHRP HEPHEALATH RLESERGLIL ESERPRASPG LALAARGARG PRPHEILEIL 7020
EALAARGARG PRGLYASPAR GSERHISSER ARGSERALAA LAALALESER THRSERGLLY 7080
SSERALAALA ALALESERTH RSERGLLYSA SPTRPLEGLN VALARGSERA LAALAASPGL 7140
YLEALASERA LAILETHRSE RLYSSERALA GLYTYRTHRP RLELYSSERA LAMETTHRLE 7200
PRARGSERAL ATHRVALMET THRTYRTRPL YSTHRGLGLG LYALAPRLEP RGLYCYSPRA 7260
RGTHRTHRLE GLNARGSERA SPASPALAIL ETHRARGTHR ASNPRASPAL ALELYSALAS 7320
ERALAVALPR GLYALASERA LALYSSERGL YASNTYRSER TYRGLYVALA RGSERGLYSE 7380
RSERPHESER SERILEGLYS ERALAILESE RMETSERLYS SERGLYSERV ALALALEGLY 7440
LYSSERHISL ESERVALVAL ASPGLYGLYG LASPGLYGLN ASNILEPRLE HISPRLEILE 7500
GLNPRGLARG SERILEALAV ALLYSALAPR SERPRTHRGL PRGLYSERPR ALASERPRGL 7560
YGLYSERGLN PRARGSERIL EILESERARG METLEVALTH RASPPRLYSS ERLEGLYASP 7620
VALCYSMETA SPLEGLNTHR ILETHRTHRS ERSERASPPR ASPPRLYSAR GSERASNILE 7680
THRGLILELE PRALAGLYTH RPRLEPRGLY THRALAALAT HRALAARGGL NASNPRASNP 7740
RALAALAALA ALASERTHRG LYGLYGLNAS PGLYGLYPRA SNASPALAVA LPRARGSERP 7800
RALAALAVAL GLNGLYILET YRGLYASNAR GSERSERGLL YSPRSERILE THRILEASPG 7860
LYASNASNIL EASNLYSSER SERGLYALAV ALTHRGLYGL NSERTHRARG SERSERGLYT 7920
HRGLYTHRSE RTHRGLYALA ALAALATHRG LYTHRGLTHR ASNALAALAS ERVALALALY 7980
SLEGLNMETG LYVALSERAL AALAGLYILE ALAGLYLEAL ALEGLYILET RPALALESER 8040
SERHISPRIL EGLVALPRVA LLYSSERTHR ASPTHRSERI LEASNVALAR GSERVALTHR 8100
SERGLYPHEV ALASPGLYIL ELYSSERVAL THRSERGLYP HEVALASPGL YILELYSASP 8160
GLYLEARGTH RALALEALAA SPTYRALALE CYSALAGLAL ATHRASNMET CYSARGTHRA 8220
LASERASNPH EASPGLNPRH ISSERASPGL SERALALEGL NHISLEARGT HRALAVALPR 8280
ILEASNGLYP RASPSERPRG LYTHRPRGLG LYVALLYSTH RASPTYRSER VALCYSGLYG 8340
LTHRTHRILE PHELYSTHRG LYTYRVALAS NTYRASNVAL ASPTHRTHRA SNLEARGTHR 8400
ILEPHEGLYT RPASPILEAL AGLGLYGLNL YSTHRILESE RASNVALVAL ASPASNGLLE 8460
ALAARGTHRI LEVALSERPR ASPGLYPHEA SNTRPASPTY RGLYSERTHR ARGTHRLEGL 8520
YILEASPILE ALAARGTHRL ESERTHRASN GLGLGLYTYR GLTHRSERAL AVALARGTHR 8580
METLEVALGL YMETASPVAL THRHISPRSE RPRGLYSERS ERALAASNAL APRSERVALA 8640
LAGLYMETVA LALASERVAL ASPSERTHRL ESERGLNTRP PRALAGLILE ARGVALGLNA 8700
RGTHRASNTH RGLNVALPRA SPALACYSTH RGLNCYSPHE GLNLYSTHRG LNGLYPRHIS 8760
SERTHRPHEA SPARGTHRSE RGLYSERGLY SERSERSERP RGLPRARGTH RTHRASPVAL 8820
GLYTHRPHEG LYGLNLYSTH RTHRGLYALA PHEASPGLSE RGLYPRPRLE SERGLNLYST 8880
HRTHRASNGL YILEVALSER THRASNGLSE RGLYARGTHR THRSERGLNT RPASNVALLE 8940
ASPLELYSTH RTHRTHRLEA SPGLNGLYHI STYRGLNSER ARGTHRTHRT YRASNVALVA 9000
LALAGLNTHR LYSTHRVALA SNVALASNAS NLELYSVALA LALEVALTYR GLYASPARGV 9060
ALALATHRIL EGLYSERALA THRPHEALAA RGVALCYSAS NLEILEGLYL EMETGLYLEA 9120
RGVALASPPH ETYRASNASN LELYSVALGL LEGLNSERLY SVALGLARGT HRGLYTYRAL 9180
AALAPHEARG VALGLSERAL ASERALAASP LEILESERTH RILETHRLYS VALPHEASPA 9240
LAGLYHISTH RVALPRALAP HEGLNPRGLT HRMETPHEAR GVALPHEGLA LAGLYHISGL 9300
VALPRALATY RGLNPRGLTH RALATYRGLI LEPHEHISAR GVALGLYGLG LGLTHRPRAL 9360
ALEVALHISA SPLEASNTHR ALAMETARGV ALGLYPHELE ALASERVALG LTHRPRALAS 9420
ERILEGLALA ALASERGLLE SERLYSVALG LYGLYTHRLE ALATYRVALS ERVALGLILE 9480
GLLYSVALLY SVALGLYTHR ILEILETHRG LYASPPRLEA SPPRPRVALL ELYSVALILE 9540
PRLEGLNGLY CYSASPALAA SPGLTYRGLY ARGVALLELE HISPRLELET HRALAALAAL 9600
ALELEGLYAL ASERALAARG ALAGLNSERV ALVALGLYTH RPRPHEGLYP HEALASERGL 9660
YTHRTHRGLY GLYGLYASNA LAALAPRALA ALAPRLYSVA LASNGLYVAL GLTYRGLYGL 9720
THRARGVALG LNLEASPGLG LYLELYSARG VALSERILET RPTHRGLSER TYRGLYGLYA 9780
RGVALSERAS NASPLEALAA RGVALSERGL NILESERGLY ASNARGPRLE ASPALALEAS 9840
PGLNGLYTHR ARGVALSERT YRTHRGLTYR ASPSERTYRT YRASPHISTY RASNLYSVAL 9900
THRASNSERP RSERASNLEV ALTRPTYRSE RILESERTHR ARGVALVALA LAVALASPTH 9960
RALASERASN LYSVALVALA SNTYRTYRSE RASPASPPRT HRGLYMETSE RASPSERGLY 10020
GLASPALAPH EASPMETARG LYSVALTYRA LATHRPRASP GLNASPILEG LHISGLYARG 10080
TRPASNGLTH RILETYRVAL ILEILETHRS ERPHESERAS PTHRLETHRI LEGLNPRTYR 10140
ASPTRPASNG LPHEARGLYS TRPASNPHEI LEMETASNSE RARGTRPARG HISTYRTYRL 10200
EARGTYRALA GLYGLTYRGL PHEGLNALAA SPLEPHELYS TYRCYSALAS ERALAGLNGL 10260
ASPASNALAT HRLEGLNALA LELEARGTYR ASPLEASNLE GLASNLYSTY RGLYPRSERP 10320
HETHRALAPH EPHEGLNGLG LNASNGLLYS TYRLYSGLPR GLYALAGLGL YVALCYSGLT 10380
HRTHRPRGLY VALLYSTYRL EALASERTHR GLNMETGLPR THRASPALAA RGTYRLEASP 10440
GLNGLNILET HRALAGLTHR LYSTYRLEAS PTHRLEPRGL ILELYSTYRL ETHRASNSER 10500
GLNALALEAL AASPLEPRTY RPHEALAGLL YSTYRLEVAL ASPGLNLEAS NPRGLGLYLY 10560
STYRGLNGLY ALASERGLNC YSPRPHEARG TYRGLNPRHI STHRVALTHR THRVALSERA 10620
LAGLYALASE RASPPRARGG LYSERPRGLG LYGLYGLYAR GTYRVALASP ALAGLYGLYP 10680
HEGLPRSERI LELYSTYRVA LTHRSERASN ALAVALSERV ALGLYVALTH RHISPHEALA 10740
GLYSERARGA LAALAALALE ALAGLLEVAL TRPSERGLYA SNARGALAAL AALAPRLYSS 10800
ERALAALALE ASPALALEGL NGLNSERILE TYRLEGLNPR LYSALAALAT HRTYRCYSPR 10860
GLASNILEGL LYSALAGLAS PTYRLELEAS NPRSERPRLY SALAGLHISC YSPHEASPTY 10920
RASPLESERT YRLYSPRALA ASPLYSALAG LASNGLNALA VALALAVALG LYARGALAGL 10980
YALAVALALA ALAVALVALT YRASNASNGL LYSALAGLYL YSPRTHRLEG LYPHELEASN 11040
PRLELETYRS ERGLYALALE LYSALAGLYS ERSERPRTHR ASPILEILES ERGLYILESE 11100
RASPLYSALA ILEHISASPG LVALSERPRV ALGLYASPTH RASPALALEL EGLARGALAI 11160
LEMETGLYAL AGLGLALAAL ALYSALALES ERGLMETILE LEGLNSERGL LYSALALEVA 11220
LGLGLYSERT HRPHEALALY SALALETYRS ERSERALAAL ATHRGLYTHR TYRALASERS 11280
ERTHRTHRVA LTYRLYSALA ASNGLGLNPR THRTRPVALT YRARGALAAS NPHEGLVALG 11340
LTHRPRARGA LAASNASNTY RCYSSERASN GLNVALGLGL YPRTYRSERL ETYRSERGLY 11400
ARGALAPRVA LVALGLNTYR ALALEASNAR GALAGLNASN ASPPRASNAL APHEGLYVAL 11460
VALALAALAA RGALASERAL AILEGLNLEA SPGLYILEIL ETYRARGALA SERMETVALT 11520
RPGLGLALAG LNGLNVALSE RGLYLYSALA SERASNSERL EGLNTYRVAL ASNVALGLNV 11580
ALLYSALATH RGLYASPVAL LEPHEASNTH RLYSALAVAL GLYGLNALAT HRGLARGALA 11640
VALHISGLAS PLEASPVALA LAALAILEAS PALAALAGLV ALARGALAVA LLELELEASP 11700
GLALAASPVA LPHEMETGLG LARGALAVAL SERPRSERPH EGLASPVALT RPSERGLNPR 11760
ARGCYSGLNS ERVALPHEAS NPRASNILEP RLYSASPALA TYRSERPRHI SGLILETYRS 11820
ERARGASPPH ETHRASPILE THRALAGLYS ERSERILEGL YCYSASPGLY VALASNPRGL 11880
NTHRGLYLYS ASPGLYLEGL GLYSERPHEL YSASPLYSAS PPRGLLYSAS PPRLYSALAI 11940
LEGLLEPRAR GASPGLNILE ILEGLCYSAR GASPSERGLY LEVALMETLY SASPSERPRL 12000
ETYRPRTYRA RGASPVALHI SGLYPHEALA THRARGASPV ALLYSSERME TLYSASPVAL 12060
VALVALVALG LYGLYGLYAL ASERGLYALA TYRALAALAV ALARGASPTY RGLNVALGLM 12120
ETVALASNLY SGLALAGLYL EVALPRPHEG LNVALSERPR THRTHRLYSG LGLPRSERPH 12180
EGLNPRASPA SPVALTHRLE LELESERGLN ASPPRGLYHI STRPGLLYSG LGLYILESER 12240
ILEHISTHRC YSASPGLNAR GGLHISHISG LLEALAILEA LASERLYSGL ILEPRVALGL 12300
YTYRSERALA ALAASPILEA SPTHRASNAR GGLLEASPTH RGLNHISILE HISPRPRASP 12360
SERTYRPHEV ALSERPRLET HRARGGLPRG LYILECYSGL THRTHRPRGL YVALLYSGLP 12420
RSERASNASP PRASNPRPRG LTHRTYRSER LYSGLSERLE GLASPILEAR GLYSGLTYRL 12480
EVALALAASN GLYVALGLNA LAGLNALALE VALPRLYSPH EGLPRPRALA VALTYRASNA 12540
SPGLLELYSP HEGLYALATH RGLYASPGLT YRARGPHEGL YLYSPRVALG LYALAVALGL 12600
YSERALAALA THRALALELY SPHELEASPG LALALETHRT YRPRPRPRLY SPHEASNVAL 12660
ASPGLTHRAL APHETHRGLY ALATRPGLYA RGPHEARGGL NASPLEILES ERGLILELYS 12720
PRCYSCYSGL GLLYSPHETH RALAVALPHE THRPRSERIL EVALGLARGP HETHRASPTH 12780
RPRVALLETY RGLYPRLYSP HEVALTHRAS PASNGLYASP SERLYSPHEV ALTHRASNME 12840
TGLNALAALA LELELYSGLY PHEPRASPVA LALAALAHIS SERLETHRPR ARGGLYGLYS 12900
ERILELEPRM ETGLNGLVAL ALALETHRTH RARGGLYILE ASPVALALAL YSPRTHRGLY 12960
ARGGLYILEM ETLEASPTHR GLYARGGLYL YSGLSERCYS LYSGLYLETY RALAGLYHIS 13020
ARGGLYMETV ALPHESERIL EASPALAGLN GLYGLLYSGL YPRALAARGA RGARGGLYGL 13080
NLEGLYPHET RPGLYASNLY SGLYSERILE VALGLYPRAR GTRPLYSLEP RPHEMETGLY 13140
PRPHELEGLN SERVALASNP RLYSGLYTHR VALPHEPRSE RGLTHRGLGL YGLSERMETA 13200
LASERARGGL YVALASPPHE THRGLASPPR LELEGLNGLY ARGGLYVALL YSILESERGL 13260
YTRPASPVAL GLTHRLEGLY ASPGLILETH RHISVALGLY GLLYSPHETH RLYSGLYTYR 13320
LYSPRSERAL ASERSERGLY SERLYSHISA LAGLYGLNCY SGLYGLTYRH ISGLASNLYS 13380
HISPHETHRS ERLEGLGLLY SHISPHEVAL ASPTHRPHEG LYLEHISGLY HISLYSHISG 13440
LYGLYPRASN PHEGLGLNLE PRILEASNGL NPRARGHISG LYILEPRGLY GLYGLYILEA 13500
LATHRGLYAL AGLGLYILEL YSHISMETPH EGLYLEVALA LASERGLASP ALAGLYARGH 13560
ISPRVALGLV ALALAGLGLG LALASERLYS PRLYSHISVA LASPGLYPHE GLYILEHIST 13620
HRPHEARGHI SVALGLNLEL EGLNLEASNM ETGLTYRASP ASPASPILEL ECYSARGLYS 13680
SERLYSHISV ALTYRASPAL AVALGLNASP LYSILEALAP HEALASERTY RLEGLGLTYR 13740
ALAARGILEA SPALATHRTH RASNPRGLYM ETARGILEAS PTYRILEGLY GLYGLYASPL 13800
EPHEARGILE GLILEGLASN SERILEARGI LEGLASNGLN SERASPALAA SPGLYTYRSE 13860
RSERCYSSER THRLELYSIL EPHEGLGLNL EGLGLYMETS ERLESERLYS ILEPHESERT 13920
YRLYSMETAS NSERTHRLEA RGTYRLEPRP HEARGILEGL YLEHISPHEA RGTHRARGIL 13980
EHISLETHRV ALPRGLASPL EARGILEGLN ASPGLYSERG LNVALLYSIL EGLNGLYILE 14040
SERASNPRSE RGLYALALES ERSERGLYGL YLEGLYGLPR LYSILEGLNS ERLYSLEARG 14100
GLYLEVALGL NARGILEARG ASPALAMETA RGGLNARGIL EARGLEHISL EGLARGTHRG 14160
LYGLNLEGLY VALGLYSERA SPGLYASNPR VALVALALAG LYARGILESE RALATYRVAL 14220
GLGLYSERSE RARGILEVAL PRGLGLTYRV ALPRILETHR LYSILETYRS ERPHEPHEVA 14280
LGLYGLYALA VALPRGLASN LEARGILETY RVALTHRGLY GLSERTYRAL AGLYARGLYS 14340
ASPILEARGH ISGLYHISLY SLYSGLHISA SPLYSSERLY SPHETHRASP THRPRVALLE 14400
TYRGLYPRLY SLYSGLYASP ALAPRTHRIL EASPTHRSER ASNTYRPHEL EPHEGLYLYS 14460
LYSMETGLAS PASPLEARGL YSTYRTHRVA LPRSERTHRC YSGLYVALLY SLEGLMETTY 14520
RGLNGLYGLY ILEGLLESER ALALELEGLN METILEGLNA SPALAILEAR GLEPHESERT 14580
YRLEASPTHR GLNLEASNAR GLEGLYILET HRTYRTHRTH RTYRSERLYS LELYSASPLE 14640
VALLESERLE LEASNALALE GLNGLYGLYA RGLEASNTHR GLYALAVALI LEPRVALLEV 14700
ALARGLEASN VALILEASPP HEPRLYSLEG LNVALARGAL AALAALAARG ARGLESERAL 14760
AGLYSERARG LESERGLLEG LTRPILEARG LESERGLNLE GLSERGLYLY SLESERSERI 14820
LEALALEPRA RGLETHRASP LEGLILEASN ARGLEVALAL AHISSERVAL ALATHRTYRA 14880
LAARGLEVAL CYSPHEPHEP RTHRLYSLEV ALGLNASNAS PPHEASNTHR LELEARGMET 14940
ALAPRMETSE RGLGLASPLE ALATRPPHEA RGSERTHRPH EHISPRILEP RLYSMETGLY 15000
SERLESERAS PVALARGMET LYSSERILEG LGLLYSGLYG LGLYMETTHR ASNASPTYRI 15060
LESERALALE THRLYSASNA LAPHEILETH RASNTYRPRS ERGLGLNARG ASNALAGLYI 15120
LEGLNTHRSE RARGASNPHE SERARGPRLY SASNHISGLY THRSERTHRV ALALAPRGLN 15180
VALGLNALAS ERVALTYRAR GASNHISASN VALLESERAL AILEPRGLNG LPRTYRARGA 15240
SNILEASNME TLELETYRGL YTHRASPASP CYSSERGLYL YSASNLEASP GLLETRPILE 15300
VALGLYHISG LYALAVALAL AARGASNLEV ALGLYTHRSE RGLYPHETHR SERALAARGA 15360
SNMETHISAS PVALILEGLY ASNASPGLYT HRVALPRSER GLPHEARGAS NASNVALILE 15420
ILEGLNLEAS NARGASNSER METTHRASPC YSCYSILEGL THRTYRLEME TLYSSERGLA 15480
RGASNTHRLE ALAPHEPHES ERGLYASNGL VALILEASNA SPGLYPRSER SERLYSASNT 15540
HRPRVALPRS ERCYSPHEHI SPHEPHEILE TYRLYSGLYC YSTRPMETPH ELETYRARGA 15600
SNTYRPHEAL AGLTHRGLGL NVALMETPHE GLNPRGLYHI SILEVALARG PRASPGLYTH 15660
RGLYPHEARG LYSPRLETRP ARGHISTYRP HEGLNASNTH RGLNGLYILE ILEPHEVALV 15720
ALASPSERAS NASPARGPRV ALVALGLNVA LLEMETPRGL GLYMETASPS ERASPGLSER 15780
GLNALAILEL EASNASNILE GLYALAASPG LYGLNSERAL AGLNGLYALA SERPRGLYVA 15840
LVALILEALA SERPRSERLY SGLNASPLEP HEGLALAILE GLALAGLYAR GGLNTHRTYR 15900
ALASERCYST RPGLYGLYVA LGLYGLNGLY GLCYSARGGL YSERSERASN CYSLYSARGC 15960
YSTRPSERGL YVALPHETYR SERASNTRPI LEGLNGLLEL EARGARGSER METGLYLEGL 16020
SERARGSERA LAALAASPGL YLEALASERA LAILETHRSE RLYSSERALA ILESERGLNT 16080
YRGLYASPSE RPHEALALYS SERALAMETT HRLEPRARGS ERALAVALGL NSERASPVAL 16140
TRPARGSERA SPGLYGLNCY SSERASPLEL ELYSSERASP LYSLEASNVA LILEASPPHE 16200
PRLYSSERAS PTYRASPALA PHEILEARGS ERASPTYRGL NGLCYSALAA SPALAPRGLY 16260
GLNLYSSERA SPTYRSERAL ALEGLNSERG LNGLYLEILE LESERLEARG SERGLMETLE 16320
ALAGLGLNAS PLYSSERGLS ERASNPRGLY VALMETSERT HRARGSERGL YALAASPTHR 16380
HISLYSSERI LEVALILEAR GSERILETYR ALAILEASNS ERGLYARGSE RLEPRLEILE 16440
VALGLYASNS ERASPGLNGL GLYLYSSERG LNSERASPPH EGLSERGLPH ESERTHRALA 16500
LYSSERSERG LYALAVALTH RGLYGLNSER THRARGSERS ERSERALATY RGLSERLETH 16560
RSERALAVAL LYSSERTHRA SPTHRSERIL EASNVALARG SERVALVALG LASNASNASN 16620
ASPGLYLETH RALAALATYR ARGTHRALAL EPHEASPSER HISGLTYRAR GTHRALASER 16680
ASNPHEASPG LNPRHISSER ASPGLSERAL ALEGLNHISL EARGTHRCYS HISARGCYSC 16740
YSTHRTHRPH EALAPRASPA LATHRGLCYS GLASNCYSLY SHISTHRARG THRASPTYRS 16800
ERVALCYSGL YGLTHRTHRI LEPHELYSTH RGLYGLTHRT HRGLNILEHI SALAARGTHR 16860
GLYPRSERIL EGLNASPARG THRILESERA SNVALVALAS PASNGLLEAL AARGTHRLYS 16920
SERLEPRARG THRLEPRPRL EGLNTYRARG ASPLEASPLE LEPRLEHISG LNASNLEILE 16980
LYSTHRLEVA LSERTHRGLY ARGTHRPRAL AALAHISARG ALAARGTHRS ERGLYSERGL 17040
YSERSERSER PRGLPRARGT HRTHRASPVA LGLYTHRPHE GLYGLNLYST HRTHRGLMET 17100
THRGLNARGT HRTHRSERAS NPRGLTHRAR GTHRVALGLY SERSERCYSP RTYRCYSASP 17160
SERGLNALAP RGLNVALARG THRVALASNG LYGLYPHEGL NILEALAARG THRVALASNV 17220
ALASNASNLE LYSTHRVALT YRALAPHEAS PVALSERGLA SPGLYSERTY RLELYSTHRT 17280
YRGLVALVAL GLYASNVALT YRLYSVALAL ALEVALTYRG LYASPARGVA LALAPRASNS 17340
ERGLYALATY RLEASNGLAL AASPPHEARG VALALASERL ELEGLNARGV ALALATHRIL 17400
EGLYSERALA THRPHEALAA RGVALCYSAS NLEILEGLYL EMETGLYLEA RGVALASPAS 17460
NVALVALALA SERPHELYSV ALGLTYRSER ASPALAALAL YSVALPHEGL ALAGLYHISG 17520
LVALPRALAT YRGLNPRGLT HRALATYRGL ILEPHEHISA RGVALGLYSE RILEGLPHET 17580
HRALALEPRG LNLEGLNSER LEASPPHETH RLYSVALILE PRGLILEASP METPRSERHI 17640
SSERSERSER GLYTRPLYSV ALLEASPARG ASPPRASNHI SALALYSVAL LEPHELEGLY 17700
ARGVALLEIL EALAASPMET CYSARGARGV ALLEPRGLNV ALILEGLALA THRASNARGV 17760
ALGLNASNGL YALAVALTHR TRPGLSERAS PPRASNARGV ALGLNASNGL YALAVALTHR 17820
TRPGLSERAS PPRASNARGL YSVALSERAS NASPLEALAA RGVALTHRAL AMETARGTYR 17880
TRPTRPLECY SGLILEALAT YRCYSPHEAL ASERVALGLY GLYLYSVALV ALTHRASPSE 17940
RPHEARGVAL TYRSERVALA SPASNSERLY STRPASPASN LEASPSERAL AALALEASNT 18000
HRLYSTRPPH EALAGLASPP RSERARGTYR CYSALASERA LAGLNGLASP ASNALATHRL 18060
EGLNALALEL EARGTYRCYS GLYVALGLYV ALASNILELE TYRGLARGTY RGLALAALAI 18120
LEGLNGLYVA LALAALATHR ASPLYSTYRP HETYRGLYAS PASNTYRALA THRLEARGTY 18180
RGLYALATYR SERVALCYSS ERPRLYSTYR GLYGLTHRGL LYSSERGLYL EGLSERILEA 18240
LAALAALAAR GTYRILEALA ARGPRASPIL EMETLYSTYR LEASPGLNGL NILETHRALA 18300
GLTHRLYSTY RLEVALASPG LNLEASNPRG LGLYLYSTYR GLNPHEPRGL NTHRPRSERA 18360
RGTYRARGHI SLEPRPRGLT HRVALTHRGL YILELEGLYA RGALATHRPH ETRPTRPILE 18420
ASNSERILEL ELYSTYRTHR ALAGLGLYTY RGLALAALAT HRLYSTYRVA LASPALAGLY 18480
GLYPHEGLPR SERILELYS 18499

Claims (9)

1. The method for efficiently screening the aflatoxin green prevention and control material in one step is characterized by comprising the following steps of: the content of an indicator molecule AFT-YJFZP008 of a test treatment group and a control group of the aflatoxin green prevention and control material is measured, the inhibition rate of the green prevention and control material to the indicator molecule AFT-YJFZP008 is calculated, the aflatoxin green prevention and control material is efficiently screened according to the inhibition rate result, and the amino acid sequence of the indicator molecule AFT-YJFZP008 is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein: the aflatoxin green prevention and control material test treatment group and the control group can be realized by the following steps: mixing peanut kernel powder, aspergillus flavus toxigenic bacteria spores and a series of candidate green prevention and control materials, and culturing for a certain time in a culture medium to obtain a test treatment group, wherein the culture medium is used for replacing the candidate green prevention and control materials, and culturing for a certain time to obtain a control group.
3. The method of claim 2, wherein: the peanut varieties are not limited to medium flower No. 6 and Luhua No. 8.
4. The method of claim 2, wherein: the culture medium is liquid Sabouraud's culture medium, or other culture medium with similar effect.
5. The method of claim 1, wherein: the content of the indicator molecule AFT-YJFZP008 can be realized by but is not limited to the following steps:
a, after the treatment group and the control group are cultured for a certain time, fully homogenizing the culture to prepare a solution to be detected, adding the solution to an enzyme-labeled plate hole with the bottom coated with a nano antibody or a monoclonal antibody of an indicator molecule AFT-YJFZP008, reacting, and washing the plate;
b, adding an indicator molecule AFT-YJFZP008 polyclonal antibody for reaction, and washing the plate;
c, adding a horseradish peroxidase labeled antibody which has a binding reaction with the polyclonal antibody of the indicator molecule AFT-YJFZP008, reacting, and washing the plate;
d, adding a color development solution for reaction; adding a stop solution, reading by an enzyme-linked immunosorbent assay and calculating the content of AFT-YJFZP008 in the solution to be detected;
replacing the solution to be detected with a pure solution of indicator molecules AFT-YJFZP008 with a series of concentrations and serving as a standard substance, making a standard curve, and calculating the content of the indicator molecules AFT-YJFZP008 according to the reading of an enzyme-labeling instrument;
the inhibition rate of the green prevention and control material to an indicator molecule AFT-YJFZP008 is calculated by the following method: the inhibition rate is (content of control AFT-YJFZP 008-content of test treatment AFT-YJFZP 008)/content of control AFT-YJFZP008 x 100%.
6. The method of claim 4, wherein: the hole bottom is coated with an ELISA plate of a nano antibody or a monoclonal antibody of an indicator molecule AFT-YJFZP008, and the preparation method comprises the following steps: dissolving the nano antibody or monoclonal antibody of AFT-YJFZP008 in ELISA coating buffer solution to form 0.2-8.0 mug/mL coating solution, adding into an ELISA plate, standing overnight at 4 ℃ or standing for not less than 2h at 37 ℃, removing the coating solution from the ELISA plate, and washing the ELISA plate with ELISA conventional washing solution; then adding ELISA conventional blocking solution, standing at room temperature or 37 deg.C, blocking for not less than 1h, discarding the blocking solution, and washing the ELISA plate with ELISA conventional washing solution.
7. The method of claim 4, wherein: the ELISA color developing solution refers to ELISA conventional hydrogen peroxide and TMB color developing solution.
8. The method of claim 4, wherein: the stop solution is 2mol/L sulfuric acid aqueous solution of ELISA conventional chromogenic stop solution.
9. Use of the method of any one of claims 1 to 8 in screening for aflatoxin green control materials.
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