CN113640510B - Agricultural product aflatoxin molecule early warning method - Google Patents

Agricultural product aflatoxin molecule early warning method Download PDF

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CN113640510B
CN113640510B CN202110609135.8A CN202110609135A CN113640510B CN 113640510 B CN113640510 B CN 113640510B CN 202110609135 A CN202110609135 A CN 202110609135A CN 113640510 B CN113640510 B CN 113640510B
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aflatoxin
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yjfz01
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张奇
唐晓倩
李培武
白艺珍
姜俊
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The invention relates to an agricultural product aflatoxin molecule early warning method. Determining the concentration of aflatoxin early warning molecules in an agricultural product sample: when the concentration of the detected aflatoxin early-warning molecules is less than 0.2 mug/g, the aflatoxin pollution level is below 0.2 mug/g, and the risk is low; when the concentration of the detected aflatoxin early-warning molecules is more than 1.2 mug/g, the aflatoxin pollution level is more than 10 mug/kg, and the risk is high; when the concentration of the detected aflatoxin early-warning molecule is 0.2-1.2 mug/g, the aflatoxin early-warning molecule is AFT-YJFZ01 peptide, and the amino acid sequence is shown as SEQ ID NO. 1. The method is used for early warning the risk of agricultural products polluted by aflatoxin, and is high in speed, strong in timeliness and practicality and easy to popularize and apply.

Description

Agricultural product aflatoxin molecule early warning method
Technical Field
The invention relates to an agricultural product aflatoxin molecule early warning method.
Background
Aflatoxin has strong toxicity and great harm, is the pollutant with the largest variety of polluted foods, generally presents a pollution aggravating trend in recent years, and seriously threatens the food safety and the health of people. Aflatoxin is the most toxic mycotoxin in nature, wherein aflatoxin B1 is a class I carcinogen identified by International cancer research organization (International Agency for Research on Cancer, IARC), and has caused excessive poisoning events of human and animal populations, and becomes one of the main causes of high incidence of liver cancer cases. Statistics of data retrieved according to the last 5 years Web of Science: the aflatoxin pollutes food and raw materials more than 110 kinds, and the high-concentration pollutants are first. However, no molecular early warning research example before the pollution of the microbial toxins such as aflatoxin exists at home and abroad, and urgent needs of early warning in advance are difficult to meet.
Aflatoxins are mainly produced by toxic fungi such as aspergillus flavus. Taking peanuts as an example, the peanuts are provided with toxin-producing fungus strains of aflatoxin in the field, and after the peanut is harvested, the strains enter packaging bags, transport vehicles, warehouses, processing lines and the like along with the peanuts, and once the conditions are proper, a large amount of aflatoxin can be produced, so that the consumption safety and the life health safety of people of the peanuts and products thereof are threatened. The agricultural research bureau of the United states department of agriculture, the International semi-arid research bureau of the United nations grain and agriculture organization (FAO) and other internationally known research institutions list the toxic fungi of aflatoxins as soil-borne pathogens of crops such as peanuts for research, prevention and control.
Studies have shown that the virulence of different strains of aflatoxin are very different from strains of non-virulent fungi to high-virulence strains. Taking Aspergillus flavus strains separated from peanuts as examples, a considerable part of Aspergillus flavus strains lack toxigenic genes or toxigenic key regulatory genes, and do not have the capability of producing aflatoxin; less than about 20% of the Aspergillus flavus strains are virulent strains. How does the early warning risk assess in advance of aflatoxin contamination? This has been a worldwide problem in the art.
According to the difficult problem, the inventor groups discover the toxicity-producing fungus toxicity-producing indicator molecules of the aflatoxin through more than ten years of systematic researches, and the aflatoxin-producing indicator molecules have the early warning function of aflatoxin pollution in advance for the first time, so that the aflatoxin-producing indicator molecules can be used as aflatoxin early warning molecules. The research discovers that obvious positive correlation exists between the occurrence level of the early warning molecule and the aflatoxin pollution level, so that by utilizing the correlation, the agricultural product aflatoxin molecular early warning method and the application thereof are further invented, the early warning method can be used for early warning of the early occurrence risk of agricultural product aflatoxin, and scientific basis is provided for the prevention and control of the source of agricultural product aflatoxin and the early prevention and control of the early agricultural product aflatoxin.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an agricultural product aflatoxin molecular early warning method and application thereof, which are used for early warning the risk of agricultural products polluted by aflatoxin, and have the advantages of high speed, strong timeliness and practicability and easy popularization and application.
In order to solve the technical problems, the invention adopts the following technical scheme:
the early warning method for the aflatoxin molecules of the agricultural products comprises the steps of measuring the concentration of the aflatoxin early warning molecules in the agricultural product samples: when the concentration of the detected aflatoxin early-warning molecules is less than 0.2 mug/g, the aflatoxin pollution level is below 0.2 mug/g', which is a low risk; when the concentration of the detected aflatoxin early-warning molecules is more than 1.2 mug/g, the aflatoxin pollution level is more than 10 mug/kg, and the risk is high; when the concentration of the detected aflatoxin early-warning molecule is 0.2-1.2 mug/g, the aflatoxin early-warning molecule is AFT-YJFZ01 peptide, and the amino acid sequence is shown as SEQ ID NO. 1.
In the scheme, the quantitative detection of the concentration of the aflatoxin early-warning molecule can be realized by adopting the amino acid sequence of the aflatoxin virulence early-warning molecule AFT-YJFZ01 or a part of the amino acid sequence, preparing an antibody corresponding to the protein through a conventional antibody preparation flow, realizing the quantitative detection of the early-warning molecule protein, and also realizing the quantitative detection of the aflatoxin early-warning molecule protein in one-to-one correspondence through other detection technical means, so that the purpose is realized, wherein the part of the amino acid sequence refers to a part of the whole sequence having one-to-one correspondence with the aflatoxin early-warning molecule protein.
Specifically, the AFT-YJFZ01 detection method for the aflatoxin early-warning molecule AFT-YJFZ01 is used for measuring the concentration of AFT-YJFZ01 in an agricultural product sample, and the AFT-YJFZ01 concentration in the agricultural product sample can be quantitatively detected by adopting the aflatoxin early-warning molecule AFT-YJFZ01 immune rapid detection method, and the method comprises the following steps:
preparing an agricultural product sample to be detected, namely adding the agricultural product sample to be detected into an enzyme-labeled plate hole of which the bottom is coated with a nano antibody or a monoclonal antibody of aflatoxin early-warning molecule AFT-YJFZ01, reacting, and washing the plate;
adding aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody for reaction, and washing the plate;
adding a horseradish peroxidase labeled antibody which is combined with an aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody, reacting, and washing a plate;
adding a color development liquid for reaction; and adding a stop solution, and reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by using an enzyme-labeled instrument.
The method comprises the following specific steps: 100-200 mu L of each hole is added into an enzyme-labeled plate hole of a nano antibody or a monoclonal antibody of which the bottom is coated with aflatoxin early-warning molecules AFT-YJFZ01, the reaction is carried out at room temperature or 37 ℃ for not less than 1h, then the reacted liquid is discarded, the enzyme-labeled plate is washed, then the AFT-YJFZ01 rabbit-derived polyclonal antibody is added, then 100-200 mu L of each hole is added, the reaction is carried out at room temperature or 37 ℃ for not less than 1h, then the liquid is discarded, then 100-200 mu L of horseradish peroxidase-labeled goat anti-rabbit antibody is added into each hole, the reaction is carried out at room temperature or 37 ℃ for not less than 1h, then the liquid is discarded, the enzyme-labeled plate is washed, then ELISA chromogenic liquid and stop liquid are added in sequence, and finally the concentration of the AFT-YJFZ01 in a sample to be detected is read and calculated through the enzyme-labeled instrument. The AFT-YJFZ01 concentration can also be measured by adopting an aflatoxin early-warning molecule AFT-YJFZ01 detection method with other equivalent effects.
According to the scheme, the AFT-YJFZ01 pure product solution (100-200 mu L per hole) serving as the standard substance with a series of concentrations is used for replacing the liquid to be measured, and is used for manufacturing a standard curve, and the concentration of AFT-YJFZ01 in the sample to be measured is calculated.
According to the scheme, the method for preparing the agricultural product sample to be detected liquid to be detected comprises the following steps: weighing an agricultural product sample to be measured, crushing, transferring to a sample diluent, homogenizing by a conventional method, standing, and taking the supernatant to prepare a sample to be measured.
The sample diluent can be 0.01mol/L phosphate buffer solution containing 0.1% sorbitol and soft sugar, and the preparation method comprises the following steps: sorbitol and soft sugar 0.5g, naCl 4.0g, na 2 HPO 4 ·12H 2 O 1.45g、KCl 0.1g、KH 2 PO 4 0.1g, deionized water was added to a volume of 500mL.
The hole bottom is coated with a nano antibody or a monoclonal antibody of aflatoxin early-warning molecule AFT-YJFZ01, and the preparation method comprises the following steps: dissolving the nanometer antibody or monoclonal antibody of AFT-YJFZ01 in ELISA coating buffer solution to form coating solution of 0.2-8.0 mug/mL, adding the coating solution into an ELISA plate (100-200 mug/hole), standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2 hours, removing the coating solution in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; then adding ELISA routine blocking solution (200-300 mu L is added to each hole), standing at room temperature or 37 ℃ for blocking for at least 1h, discarding the blocking solution, and washing the ELISA plate with ELISA routine washing solution.
According to the scheme, the polyclonal antibody of AFT-YJFZ01 is different from the nanometer antibody or the animal source of the monoclonal antibody of AFT-YJFZ01, and the nanometer antibody or the monoclonal antibody and the rabbit polyclonal antibody can be prepared by directly using the aflatoxin-producing strain virulence early-warning molecule as an antigen, specifically:
the aflatoxin early warning molecule AFT-YJFZ01 nano antibody or monoclonal antibody can be obtained by the following method: AFT-YJFZ01 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then a known conventional nano antibody or murine monoclonal antibody preparation technical scheme is utilized to develop and obtain the alpaca or Balb/c mice;
the aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody can be obtained by the following method: the AFT-YJFZ01 is used as an immune antigen, a conventional mode is adopted to immunize test rabbits such as New Zealand white rabbits, and a known conventional polyclonal antibody preparation technical scheme is utilized to develop and obtain a rabbit-derived polyclonal antibody of aflatoxin early-warning molecule AFT-YJFZ 01;
the horseradish peroxidase-labeled antibody which is subjected to a combination reaction with the polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic early warning molecule AFT-YJFZ01 is a horseradish peroxidase-labeled goat anti-rabbit antibody, and can be directly purchased.
The ELISA coating buffer solution refers to a conventional carbonate buffer solution, and the preparation method comprises the following steps: weighing NaHCO 3 1.465g、Na 2 CO 3 0.795g, deionized water is added to fix the volume to 500mL.
The ELISA chromogenic liquid refers to conventional hydrogen peroxide and TMB chromogenic liquid for ELISA.
The stop solution is a conventional chromogenic stop solution 2mol/L sulfuric acid aqueous solution for ELISA, and the preparation method comprises the following steps: 44mL of concentrated sulfuric acid is added into 300mL of deionized water, stirred until cooling is achieved, and finally the volume is fixed to 400 mL.
The invention combines national and foreign aflatoxin limit regulations of European Union, the United states, china and the like, and sets the aflatoxin concentration to be low risk when 1.0 mug/kg and high risk when more than 10 mug/kg, and further combines research to find that: when the aflatoxin concentration is below 1.0 mug/kg, the AFT-YJFZ01 concentration is less than 0.2 mug/g; when the aflatoxin content is more than 10 mug/kg, the AFT-YJFZ01 concentration is more than 1.2 mug/g, and the agricultural product aflatoxin molecular early warning method is determined: determining the concentration of aflatoxin early-warning molecules in an agricultural product sample, wherein when the determined concentration of the aflatoxin early-warning molecules is less than 0.2 mug/g, the aflatoxin pollution level is below 0.2 mug/g, and the risk is low; when the concentration of the detected aflatoxin early-warning molecules is more than 1.2 mug/g, the aflatoxin pollution level is more than 10 mug/kg, and the risk is high; when the concentration of the detected aflatoxin early-warning molecules is 0.2-1.2 mug/g, the aflatoxin early-warning molecules are medium risks.
Further, the results were verified using 293 agricultural product samples: when the concentration of AFT-YJFZ01 is less than 0.2 mug/g, the aflatoxin pollution level of 86% sample is below 1.0 mug/kg, which is low risk; when the concentration of AFT-YJFZ01 is measured to be more than 1.2 mug/g, the aflatoxin pollution level of a 76% sample is above 10 mug/kg, and the risk is high. The following is indicated: the accuracy of predicting the low risk of aflatoxin in agricultural products by the method is more than 80%, and the accuracy of predicting the high risk can be more than 75%.
In conclusion, the early warning of the aflatoxin of the agricultural product is realized by detecting the concentration of the aflatoxin early warning molecule in the agricultural product and evaluating the pre-pollution risk of the aflatoxin of the agricultural product according to the obvious correlation between the concentration of the aflatoxin early warning molecule and the aflatoxin pollution level, so that a scientific basis is provided for the prevention and control of the source and the early control of the aflatoxin of the agricultural product.
The invention has the beneficial effects that:
1. the method has the advantages of high early warning speed of the aflatoxin of the agricultural products, accuracy and easiness in operation, and 2. Wide applicable agricultural product range, strong practicability and easiness in popularization and application, and 3. Has important significance in promoting the high-quality development of the agricultural products and guaranteeing the food safety.
Drawings
FIG. 1 is a standard curve of an aflatoxin early-warning molecule AFT-YJFZ01 immune rapid detection method.
Detailed Description
Example 1 preparation of aflatoxin Pre-alarm molecule AFT-YJFZ01 which can function as Standard substance
The preparation of the culture medium was carried out as follows: 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 were prepared to obtain a Chlamydomonas medium. Randomly selecting 10 strains of published open literature (national institute of peanut production area Aspergillus flavus distribution, virulence and infection) namely national institute of agriculture, namely national institute of Chinese agricultural, namely, shuoshi institute of science, author Zhang Xing, page 33, namely published toxigenic fungus strains HLJ-1, heNZY-2, huBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-108-19, jcnt-1, huNdx-7, HBHA-8-17 and the like, respectively inoculating the 10 strains into the Chlamydia medium, culturing for 5 days at 28 ℃ at 200rpm/min, fully homogenizing and crushing cells by a conventional method, and purifying to obtain aflatoxin early-warning molecules AFT-YJFZ01 by using a conventional protein purification system, protein electrophoresis, immunoaffinity and other methods. Test results show that AFT-YJFZ01 can be prepared in the strain culture of the toxigenic fungi, under the same culture conditions, the amount of AFT-YJFZ01 prepared by HBHA-8-17 is the largest, and the amount of AFT-YJFZ01 prepared by HLJ-1 is the smallest.
The immunoaffinity method is to prepare an immunoaffinity column by using a nanometer antibody or a monoclonal antibody of aflatoxin early warning molecule AFT-YJFZ01 through a conventional method, and then to enrich and purify from aflatoxin toxigenic fungi cell disruption liquid by using the immunoaffinity method, and dissolve with deionized water to obtain the product. Specifically, the aflatoxin toxigenic fungi cell disruption solution can be diluted by using a sample solution, filtered by using filter paper, continuously added into the immunoaffinity column, and after the aflatoxin toxigenic fungi cell disruption solution is basically drained, the immunoaffinity column is washed by using a conventional eluent, and finally the column is eluted by using a glycine buffer solution with the pH of 2.2 or a 70% methanol aqueous solution, the solution is timely removed by a conventional ultrafiltration centrifugation method after the eluent is collected, and then the protein remained in the ultrafiltration centrifuge tube is dissolved by using sterile water from the ultrafiltration centrifuge tube, so that the aflatoxin early-warning molecule AFT-YJFZ01 aqueous solution can be obtained.
The initial acquisition of aflatoxin toxigenic bacteria toxigenic early warning molecule AFT-YJFZ01 is carried out by the following mining method:
(1) Taking an aspergillus flavus strong virulence strain, and culturing to obtain a 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 a test animal with the aspergillus flavus antigen to obtain a nano antibody library or a monoclonal antibody library;
(3) Obtaining protein combined solutions of aspergillus flavus strains with different virulence, detecting the proteins of the aspergillus flavus strains with different virulence 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 positively correlated with the aspergillus flavus strain virulence, namely an aspergillus flavus strain virulence early-warning molecular antibody, and a protein corresponding to the aspergillus flavus strain virulence early-warning molecular antibody, namely the discovered aspergillus flavus strain virulence early-warning molecule.
In the scheme, the aspergillus flavus strong virulence strain in the step (1) is separated and identified from the natural world by a conventional method or is obtained by artificial transformation, and the virulence is identified to be not less than 10 mug/kg by a NY/T2311-2013 standard method.
And (3) the aspergillus flavus strains with different virulence in the step (3) are not less than 3 strains, and the virulence is at least 3 layers higher, middle and lower as the result of the identification by the NY/T2311-2013 standard method.
The culture medium adopted in the culture of the aspergillus flavus strong virulence strain is a Chlamydia medium or other nutrients for the 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 disruption instrument method.
The amount of the carbodiimide added to the combined extracellular secreted protein mixture and intracellular protein mixture is 0.005-0.1 g per 1.0 mL.
The coupling reaction is carried out at 15-37 ℃ for 2-6 h and at 4-10 ℃ overnight.
The immunization is a conventional immunization mode, and Aspergillus flavus antigens are inoculated. The test animal refers to a white mouse or alpaca or other test animals with similar effects.
According to the scheme, the antibody preparation process refers to a conventional nanobody preparation process or a conventional hybridoma monoclonal antibody preparation process based on cell fusion.
According to the scheme, the detection of the proteins of the aspergillus flavus strains with different virulence is realized by adopting a conventional Western Blot technical process, namely, the proteins of the aspergillus flavus strains with different virulence 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 scheme, the direct method refers to coupling the antibodies in the antibody library with a signal material by a conventional method, and then performing an immune binding reaction with the corresponding proteins transferred onto the nitrocellulose membrane.
According to the scheme, the indirect method is that the antibodies in the antibody library are subjected to immune binding reaction with the corresponding proteins transferred onto the nitrocellulose membrane, and then the second antibodies and the conjugate of the signal material are subjected to immune binding reaction with the antibodies bound onto the nitrocellulose membrane.
The signal material in the detection is horseradish peroxidase, colloidal gold, 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 aflatoxin early-warning molecule AFT-YJFZ01 antibody can also be prepared by using all peptide fragments or part of peptide fragments of the aflatoxin early-warning molecule AFT-YJFZ01 after the whole sequence of the aflatoxin early-warning molecule AFT-YJFZ01 is known through a conventional antibody preparation technical process.
Example 2 preparation of nanobody of aflatoxin Pre-alarm molecule AFT-YJFZ01
AFT-YJFZ01 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then the preparation technical scheme of known conventional nano antibodies or mouse monoclonal antibodies is utilized to develop and obtain the alpaca or Balb/c mice.
Dissolving AFT-YJFZ01 obtained by the preparation method in conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1mg/mL, mixing and emulsifying with Freund's complete adjuvant in an equal volume, immunizing alpaca by subcutaneous or intradermal multipoint injection at back, and enhancing immunity for 1 time every 2-4 weeks, wherein Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during enhancing immunity. The immune effect is monitored by adopting a conventional ELISA flow until serum titer of alpaca is not increased any more, then, the operations of venous blood collection, total RNA extraction, cDNA synthesis, VHH gene amplification, VHH gene fragment recovery, connection of the VHH gene and a double enzyme digestion pCANTAB 5E (his) carrier, electric conversion of a connection product, construction of a nanobody gene library, rescue of the nanobody gene library and the like of the alpaca are finished by referring to a method of patent document CN103866401A, and finally, the rescued nanobody gene library is obtained.
Fixing AFT-YJFZ01 obtained by the preparation on solid-phase carriers such as 96-well ELISA plates according to gradients of 8 mug/well, 2 mug/well, 0.5 mug/well and 0.1 mug/well, panning the saved nanobody gene library for 2-4 times by a method of a patent document CN103866401A, identifying antibodies generated by each phage clone by using AFT-YJFZ01 and indirect non-competitive ELISA, identifying phage corresponding to positive results as phage positive clones, preparing the nanobody by the positive clones through a conventional method of nanobody preparation, namely the nanobody of AFT-YJFZ01, for further application research work, and preferably characterizing the nanobody with strong specificity and high affinity by ELISA method.
Example 3 preparation of monoclonal antibody of Aft-YJFZ01
AFT-YJFZ01 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then the preparation technical scheme of known conventional nano antibodies or mouse monoclonal antibodies is utilized to develop and obtain the alpaca or Balb/c mice.
The AFT-YJFZ01 obtained by the preparation method is dissolved in a conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1mg/mL, and is mixed and emulsified with Freund's complete adjuvant in an equal volume, BALB/c mice are subjected to boost immunization 1 time every 2-4 weeks through subcutaneous or intradermal multipoint injection, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during boost immunization. And (3) monitoring the immune effect by adopting a conventional ELISA flow, after the serum titer of the BALB/c mice is no longer increased, then separating immune mouse spleen cells, fusing the spleen cells with mouse myeloma cells SP2/0, completing the selective culture operation of a semisolid culture medium on hybridoma cells by using a method of reference patent document CN103849604A, and after a needle point white spot grows on the semisolid culture medium, respectively picking the white spots into 96-hole culture plates with the built-in hybridoma conventional culture medium, thereby obtaining a monoclonal hybridoma resource library.
The monoclonal antibody obtained by the culture supernatant of the monoclonal hybridoma is obtained by the method of the reference CN103849604A, AFT-YJFZ01 obtained by the preparation is fixed on a solid-phase carrier such as a 96-well ELISA plate according to the gradient 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, positive clones are picked up, and the AFT-YJFZ01 monoclonal antibody is obtained and used for further application research work, and the AFT-YJFZ01 monoclonal antibody with the characteristics of strong specificity and high affinity is preferably detected.
Example 4 preparation of Rabbit-derived polyclonal antibody of Aft-YJFZ01
AFT-YJFZ01 is used as an immune antigen, test rabbits such as New Zealand white rabbits are immunized by a conventional mode, and a known conventional rabbit polyclonal antibody preparation technical scheme is utilized to develop the immune antigen.
The AFT-YJFZ01 prepared by the method is directly used as an antigen, the solution with the concentration not lower than 0.1mg/mL is mixed and emulsified with Freund's complete adjuvant in an equal volume, new Zealand white rabbits are subjected to subcutaneous or intradermal multipoint injection at the back, then the immunization is enhanced for 1 time every 2-4 weeks, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during the enhancement. And monitoring the immune effect by adopting a conventional ELISA flow, and preparing and obtaining serum of the immune animal by a conventional method after the serum titer of the immune animal is not increased, namely the rabbit-derived polyclonal antibody of aflatoxin early-warning molecule AFT-YJFZ01.
Example 5 establishment of an immune Rapid detection method for Aft-YJFZ01, an Aflatoxin early warning molecule
Basic operation procedure of AFT-YJFZ01 immune rapid detection method, namely double-antibody sandwich indirect non-competitive ELISA method: coating a nanometer antibody of AFT-YJFZ01 in the ELISA plate, and washing the plate; adding a sealing liquid for sealing and washing the plate; adding AFT-YJFZ01 or a liquid to be tested for reaction, and washing the plate; adding AFT-YJFZ01 rabbit-source polyclonal antibody for reaction, and washing the plate; adding horseradish peroxidase labeled goat anti-rabbit antibody for reaction, and washing the plate; adding a color development liquid for reaction; adding stop solution, enzyme-labeled instrument toxin and calculating the result. The following work is accomplished using this basic procedure.
Determination of optimal concentration of antibody: a plurality of parallel experiments are simultaneously carried out by adopting a chessboard titration method, the nanometer antibodies with different concentrations are used for coating, in addition, the rabbit polyclonal antibodies are set to be different concentrations, and finally, the optimal working concentrations of the nanometer antibodies and the rabbit polyclonal antibodies are determined according to the result, namely, the concentrations of the two antibodies corresponding to the point with the OD450nm value of approximately 1.0 are selected under the principle of saving the antibody consumption, ELISA result researches show that the other concentrations of the nanometer antibodies and the rabbit polyclonal antibodies can be detected, the optimal coating concentration of the nanometer antibodies is 2.0 mug/mL, and the optimal concentration of the rabbit polyclonal antibodies is 3.0 mug/mL.
Determination of optimal antibody coating conditions: coating is the first step in ELISA method research, and the quality of the coating effect has a very critical effect on the ELISA result. In order to determine the influence of different coating conditions on the detection result, three different conditions of coating at 4 ℃ overnight, constant temperature coating at 37 ℃ for 2h and constant temperature coating at 37 ℃ for 1h are selected to be coated in the hole, and the detection result shows that the three coating modes can be used for coating, and the coating at 4 ℃ is the optimal coating condition.
Determination of optimal blocking agent: after the antibody is coated, in order to avoid interference of unoccupied sites of the ELISA plate hole on subsequent steps of ELISA, inert proteins, namely blocking agents, need to be used for occupying the sites, and improper blocking agents can be combined with the secondary antibody in a non-specific way, so that false positive conditions are caused. The study adopts three different blocking agents of 3% BSA/PBST, 3% skimmed milk powder/PBST and 5% skimmed milk powder/PBST for blocking, and the study results show that the blocking agents can achieve the purpose of blocking in different degrees, and the blocking effect of 5% skimmed milk powder/PBST is optimal.
Determination of the closing time: the study is carried out by respectively setting three different sealing durations of sealing at 37 ℃ for 1h, 2h and 3h, and the detection result shows that the value of OD450nm value of a positive hole/OD 450nm value of a negative hole is maximum under the setting of sealing at 37 ℃ for 2h, the OD450nm value of a positive sample is more than 1, and the sealing at 37 ℃ for 2h is the optimal sealing time.
Determination of the reaction time of the rabbit polyclonal antibody: the study adopts three reaction durations of 30min, 50min and 1h of constant temperature reaction at 37 ℃ to carry out reaction, and the detection result shows that: the optimal reaction time of the rabbit polyclonal antibody is obtained by reacting for 50min at the constant temperature of 37 ℃.
ELISA standard curve of aflatoxin early-warning molecule AFT-YJFZ01 is drawn: AFT-YJFZ01 molecules are respectively diluted to 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30 and 300ng/mL, 200 mu L of each hole is used for entering the hole, and ELISA standard curves of AFT-YJFZ01 are drawn under the optimal conditions, and the results are shown in figure 1. The correlation coefficient of the double-antibody sandwich ELISA method established under the optimal condition reaches 0.9980, and the detection limit of AFT-YJFZ01 molecules reaches 0.1ng/mL, which shows that the detection method has good detection sensitivity and accuracy.
Method specificity evaluation: in order to evaluate the specificity of the immune detection method of the aflatoxin early-warning molecule AFT-YJFZ01, several strains of fungi including fusarium oxysporum, aspergillus niger, aspergillus ochraceus, fusarium moniliforme and the like which have certain homology with the aflatoxin are researched and selected, and the cell disruption liquid of the fungi cultures is detected, and the results are shown in a table 1, and the method has no obvious cross reaction on the protein of the fungi with the homology with the aflatoxin, so that the established immune rapid detection method of the aflatoxin early-warning molecule AFT-YJFZ01 has good specificity.
TABLE 1 specificity determination results of aflatoxin early warning molecular immunorapid detection method
Figure GDA0003292171960000101
Method repeatability evaluation: in order to evaluate the repeatability of the established aflatoxin early-warning molecule AFT-YJFZ01 immune rapid detection method, randomly taking the non-toxic fungus strains of 4 aflatoxins, namely positive 1, positive 2, positive 3, positive 4 and 1 aflatoxins, namely negative 5, and analyzing the data variation in and among plates of the measurement results. The results of the above study are shown in tables 2 and 3, and the calculated intra-plate variation coefficient is 0.5% -3.5%, and the calculated inter-plate variation coefficient is 0.9% -5.7%, which are below 7%, so that the method has good repeatability.
TABLE 2 in-plate repeatability determination results of early warning molecule AFT-YJFZ01 immune rapid detection method
Figure GDA0003292171960000111
TABLE 3 determination results of the repeat test between plates of early warning molecule AFT-YJFZ01 immune rapid detection method
Figure GDA0003292171960000112
Evaluation of accuracy of the method: in order to evaluate the detection accuracy of the method and also to examine the practicability of the method, peanut and corn samples are selected as examples for research and evaluation, aflatoxin early warning molecule AFT-YJFZ01 is added to the corn and peanut samples, and the more the recovery rate of the detection result is close to 100%, the more accurate and practical the method is explained. The research results are shown in Table 4, and the results show that the established aflatoxin early warning molecular immunity rapid detection method is used for detecting early warning molecules AFT-YJFZ01 in peanuts and corns, the recovery rate of the early warning molecules AFT-YJFZ01 reaches 82.5% -109.5%, and the method is high in accuracy, can be applied to actual sample detection, and has good practicability.
TABLE 4 addition recovery test results of aflatoxin Pre-alarm molecular immunity Rapid detection method
Figure GDA0003292171960000113
Figure GDA0003292171960000121
Example 6 correlation of Aflatoxin Pre-alarm molecule concentration in agricultural products with Aflatoxin contamination level of agricultural products
The agricultural product samples such as peanut, corn, rice, wheat, walnut, pistachio and the like are randomly selected to be 293 parts, the concentration of aflatoxin early-warning molecule AFT-YJFZ01 in the samples is measured by adopting the method of the embodiment 5, and the aflatoxin level is measured by adopting a high performance liquid chromatography-mass spectrometry method in the determination of aflatoxin B and G in national food safety standards of GB 5009.22-2016. The measurement result shows that: when the concentration of AFT-YJFZ01 is less than 0.2 mug/g, the aflatoxin pollution level of 86% sample is below 1.0 mug/kg, which is low risk; when the concentration of AFT-YJFZ01 is higher than 1.2 mug/g, the aflatoxin pollution level of a 76% sample is higher than 10 mug/kg, and the risk is high; the rest of the AFT-YJFZ01 concentration is measured to be the stroke risk at 0.2-1.2 mug/g.
Further adopting a conventional Chlamydia culture medium method to randomly culture 6 aflatoxin-producing strains from different producing areas, adopting the early warning molecule detection method and the aflatoxin detection method to track and monitor the growth regulation of AFT-YJFZ01 and aflatoxin in the culture process, finding that AFT-YJFZ01 can be detected after 16 hours of culture, and the aflatoxin is almost zero at the moment, wherein the result shows that the early warning molecule AFT-YJFZ01 is produced before aflatoxin is produced.
Therefore, the agricultural product aflatoxin molecule early warning method is established based on the aflatoxin early warning molecule AFT-YJFZ 01: the method is characterized in that when the concentration of AFT-YJFZ01 in the agricultural products is measured to be less than 0.2 mug/g, the risk is low, and the measured agricultural products can be safely eaten or stored in the aspect of guidance production; when the measured AFT-YJFZ01 concentration is more than 1.2 mug/g, the risk is high, and on the aspect of guiding production, prevention and control and even reduction measures are required to be adopted to prevent and control aflatoxin pollution hazard; when the concentration of AFT-YJFZ01 is measured to be 0.2 mu g/g-1.2 mu g/g, the method is a medium risk, and the method is used for guiding production, so that the measured agricultural products need to be continuously tracked and monitored. The accuracy of the method for predicting the low risk of aflatoxin in agricultural products exceeds 80 percent, and the accuracy of predicting the high risk exceeds 75 percent.
Example 7 application of the method for early warning aflatoxin molecules in agricultural products
By adopting the molecular early warning method, agricultural products such as peanuts from 10 county-level peanut producing areas, corns from 3 county-level producing areas, rice from 3 county-level producing areas, wheat from 3 county-level producing areas, 1 walnut, 1 pistachio and the like are predicted. The prediction result shows that: 1) The AFT-YJFZ01 concentration of peanuts in 1 county-level production area is more than 1.63 mug/g, the peanut belongs to high risk, and effective aflatoxin prevention and control or reduction measures need to be adopted for products. 2) AFT-YJFZ01 concentration of 2 county-level producing area peanuts, 1 county-level producing area corn and 1 producing area walnut is in the range of 0.27 mu g/g-0.83 mu g/g, and is sequentially 0.58 mu g/g, 0.30 mu g/g, 0.83 mu g/g and 0.27 mu g/g, which belongs to medium risk, and pollution risk needs to be continuously tracked and monitored.
As demonstrated by the above specific examples in combination,
the technical scheme of the invention can be used for early warning the risk of aflatoxin in agricultural products and guiding production in time, 1. The early warning speed of the aflatoxin of the agricultural products is high, namely, the aflatoxin can be finished within half a day, and the method is accurate and easy to operate, 2. The range of the agricultural products which can be applied is wide, the practicability is high, the popularization and the application are easy, and 3. The method has important significance in promoting the high-quality development of the agricultural products and guaranteeing the food safety.
< 110 > institute of oil crop and oil crop of national academy of agricultural sciences
Aflatoxin molecular early warning method for agricultural products less than 120
＜160＞ 1
＜210＞ 1
＜211＞ 33172
＜212＞ PRT
< 213 > Aspergillus flavus
＜400＞ 1
AIGVEEPEAD PTYYHNAIGV EEPEADPTYY HNNKAIGVEE PEADPTYYHN NKTTASMVWE 60
EAQQVSGKAS VETPASIEAA SELSKAVSPS FEDVWSQPRD GAGQMFIPLN PNAYSPNTLN 120
KDGAGQMFIP LNPNAYSPNT LNKGSPKDGV YVDKSVTSGF VDGIKDGLRD VGGPIEDQNS 180
LQVGDRDVHG FATRFEQLPI NQPRFGKPVG AVGSAAFGKP VGAVGSAATA LKGDNEIPQA 240
ATAHDSAWDF FSQQPSGDNE IPQAATAHDS AWDFFSQQPS SLHGPNFEQL PINQPRGPTL 300
LEDFIFRGVD FTEDPLLQGR GVGAHGVFTS YHGGPNFEQL PINQPRHVDG FGIHTFRLAS 360
VETPASIEAA SELSKLFSYL DTQLNRLFYN SLTPAEQQFV VDAIRNAGIQ TSRNAGIQTS 420
RDGVYVDKNS LTPAEQQFVV DAIRNYFAET EQVMFQPGHN YFAETEQVMF QPGHIVRPEE 480
YVPITKPLQI VIDGFRPNAY SPNTLNKPVG AVGSAATALK QDLFEAIEAG RQLSEDGVDV 540
VVVAERSEDG VDVVVVAERS LTPAEQQFVV DAIRSMVWEE AQQVSGKSPS FEDVWSQPRS 600
VETPASIEAA SELSKSVTSG FVDGIKDGLR TTDVGTFGQK VDFTEDPLLQ GRVETPASIE 660
AASELSKVGF LASVETPASI EAASELSKYP EWELGVQIMD EEDQLKFDHE RVPERFGFDL 720
FDPTKFDLFD PTKFGKPVGA VGSAATNPDF MRQDLFEAIE AGRFVTDNGD SKLVKFVTDN 780
GDSKSVTSGF VDGIKGFDLF DPTKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKIVPEEY 840
VPITKDGVYV DKSVTSGFVD GIKFENSNVK SSVVRVGGPI EDQNSLQVGD RGDNEIPQAA 900
TAHDSVTSGF VDGIKDTTDV GTFGQKLKGV GAHGVFTGDN EIPQAATAHF VVDAIRTLLE 960
DFIFRFTEDP LLQGRPDLIH AVKPRVDGFG IHTFRGVGAH GVFYLDTQLN RNVIIQLNRK 1020
PVGAVGSAAT ALKDGVYVDK NNVIIQLNRI VPEEYVPITK LGKTPAEQQF VVDAIRFENS 1080
NVKEQVMFQP GHIVRGVDFT EDPLLQGRAI GVEEPEADPT YYHNAIGVEE PEADPTYYHN 1140
ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKASMVWEEA QQVSGKASMV 1200
WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKASMVWEEA 1260
QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS 1320
GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS 1380
MVWEEAQQVS GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE 1440
EAQQVSGKAS MVWEEAQQVS GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ 1500
VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKAVSPSFED VWSQPRAVSP SFEDVWSQPR 1560
AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED VWSQPRAVSP 1620
SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED 1680
VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ 1740
PRAVSPSFED VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV 1800
SPSFEDVWSQ PRAVSPSFED VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF 1860
EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED VWSQPRDGAG QMFIPLNPNA YSPNTLNKDG 1920
AGQMFIPLNP NAYSPNTLNK DGAGQMFIPL NPNAYSPNTL NKDGAGQMFI PLNPNAYSPN 1980
TLNKDGAGQM FIPLNPNAYS PNTLNKDGAG QMFIPLNPNA YSPNTLNKDG AGQMFIPLNP 2040
NAYSPNTLNK GSPKDGAGQM FIPLNPNAYS PNTLNKGSPK DGVYVDKDGV YVDKDGVYVD 2100
KSVTSGFVDG IKDGVYVDKS VTSGFVDGIK FENSNVKSSV VRFGFDLFDP TKFGFDLFDP 2160
TKFGKPVGAV GSAATALKFG KPVGAVGSAA TALKFGKPVG AVGSAATALK FGKPVGAVGS 2220
AATALKFGKP VGAVGSAATA LKFGKPVGAV GSAATALKFG KPVGAVGSAA TALKFGKPVG 2280
AVGSAATALK FGKPVGAVGS AATALKFGKP VGAVGSAATA LKFGKPVGAV GSAATALKFG 2340
KPVGAVGSAA TALKFGKPVG AVGSAATALK FVTDNGDSKF VTDNGDSKLV KFVTDNGDSK 2400
LVKFVTDNGD SKLVKGPTLL EDFIFRGVDF TEDPLLQGRG VDFTEDPLLQ GRGVDFTEDP 2460
LLQGRGVDFT EDPLLQGRGV DFTEDPLLQG RGVDFTEDPL LQGRHGGPNF EQLPINQPRH 2520
GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2580
GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2640
GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2700
GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2760
GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2820
GGPNFEQLPI NQPRHGGPNF EQLPINQPRH VDGFGIHTFR HVDGFGIHTF RHVDGFGIHT 2880
FRHVDGFGIH TFRHVDGFGI HTFRHVDGFG IHTFRHVDGF GIHTFRHVDG FGIHTFRHVD 2940
GFGIHTFRHV DGFGIHTFRH VDGFGIHTFR HVDGFGIHTF RHVDGFGIHT FRHVDGFGIH 3000
TFRHVDGFGI HTFRHVDGFG IHTFRHVDGF GIHTFRHVDG FGIHTFRHVD GFGIHTFRHV 3060
DGFGIHTFRI VPEEYVPITK IVPEEYVPIT KIVPEEYVPI TKLFSYLDTQ LNRLFSYLDT 3120
QLNRLFSYLD TQLNRLFSYL DTQLNRLFYN SLTPAEQQFV VDAIRLFYNS LTPAEQQFVV 3180
DAIRNAGIQT SRNAGIQTSR DGVYVDKNNV IIQLNRNYFA ETEQVMFQPG HNYFAETEQV 3240
MFQPGHIVRN YFAETEQVMF QPGHIVRNYF AETEQVMFQP GHIVRNYFAE TEQVMFQPGH 3300
IVRNYFAETE QVMFQPGHIV RNYFAETEQV MFQPGHIVRN YFAETEQVMF QPGHIVRNYF 3360
AETEQVMFQP GHIVRNYFAE TEQVMFQPGH IVRNYFAETE QVMFQPGHIV RNYFAETEQV 3420
MFQPGHIVRN YFAETEQVMF QPGHIVRNYF AETEQVMFQP GHIVRNYFAE TEQVMFQPGH 3480
IVRNYFAETE QVMFQPGHIV RQDLFEAIEA GRQDLFEAIE AGRQLSEDGV DVVVVAERQL 3540
SEDGVDVVVV AERQLSEDGV DVVVVAERQL SEDGVDVVVV AERSLQGKAS MVWEEAQQVS 3600
GKSLQGKASM VWEEAQQVSG KSPSFEDVWS QPRSVTSGFV DGIKSVTSGF VDGIKDGLRS 3660
VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS 3720
VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS 3780
VTSGFVDGIK DGLRTTDVGT FGQKTTDVGT FGQKTTDVGT FGQKTTDVGT FGQKTTDVGT 3840
FGQKLKTTDV GTFGQKLKVG FLASVETPAS IEAASELSKV GFLASVETPA SIEAASELSK 3900
VGFLASVETP ASIEAASELS KVGFLASVET PASIEAASEL SKVGFLASVE TPASIEAASE 3960
LSKVGFLASV ETPASIEAAS ELSKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKVPVHNN 4020
NRDGAGQMFI PLNPNAYSPN TLNKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKYPEWEL 4080
GVQIMDEEDQ LKYPEWELGV QIMDEEDQLK ALFNRDIATG KANNYCSNQV EGPYSLYSGR 4140
DIATGKVSIA KDYACPWNGG EEVSLKDYAC PWNGGEEVSL KVEYSDAAKE GDPEMYGNNE 4200
TVNKVCAKAN NYCSNQVEGP YSLYSGREPG ICETTPGVKE QTASVVNGTA VIKGFSATGD 4260
YPRGGPGSSS MIGLMQENGP CRGYLEDIAY VLDSGIKGYY DISHFDPDIG LMQENGPCRL 4320
AAEGDPEMYG NNETVNKNAP LSIWMNGGPG SSSMIGLMQE NGPCRNQVEG PYSLYSGRNY 4380
CSNQVEGPYS LYSGRPGGCK DQIIECRQVE GPYSLYSGRQ YGNFSFTRSN QVEGPYSLYS 4440
GRTNASYVGG LVRTVYDMAM EAWSKTVYDM AMEAWSKPGG TVYDMAMEAW SKPGGCKVAL 4500
VYGDRDYACP WNGGEEVSLK VFEAGHEVPA YQPETAYEIF VFEAGHEVPA YQPETAYEIF 4560
HRVSIWTESY GGRYCSNQVE GPYSLYSGRY GPSFTAFFQE QNEKVALVYG DRVEYSDAAK 4620
FRISYKEPGI CETTPGVKFT AFFQEQNEKS IWTESYGGRN YIVVDADSSF WFFESRISYK 4680
EPGIHDDRVS IWTESYGGRY GPSFTAFFQE QNEKVALVYG DRDYAANNYC SNQVEGPYSL 4740
YSGRANNYCS NQVEGPYSLY SGRANNYCSN QVEGPYSLYS GRANNYCSNQ VEGPYSLYSG 4800
RANNYCSNQV EGPYSLYSGR ANNYCSNQVE GPYSLYSGRA NNYCSNQVEG PYSLYSGRAN 4860
NYCSNQVEGP YSLYSGRANN YCSNQVEGPY SLYSGRANNY CSNQVEGPYS LYSGRANNYC 4920
SNQVEGPYSL YSGRANNYCS NQVEGPYSLY SGRANNYCSN QVEGPYSLYS GRANNYCSNQ 4980
VEGPYSLYSG RANNYCSNQV EGPYSLYSGR ANNYCSNQVE GPYSLYSGRA NNYCSNQVEG 5040
PYSLYSGRAN NYCSNQVEGP YSLYSGRANN YCSNQVEGPY SLYSGRDIAT GKVSIAKDYA 5100
CPWNGGEEVS LKDYACPWNG GEEVSLKGDP EMYGNNETVN KVCAKANNYC SNQVEGPYSL 5160
YSGRGGPGSS SMIGLMQENG PCRIGLMQEN GPCRIGLMQE NGPCRISYKE PGICETTPGV 5220
KISYKEPGIC ETTPGVKISY KEPGICETTP GVKISYKEPG ICETTPGVKI SYKEPGICET 5280
TPGVKISYKE PGICETTPGV KISYKEPGIC ETTPGVKNAP LSIWMNGGPG SSSMIGLMQE 5340
NGPCRNAPLS IWMNGGPGSS SMIGLMQENG PCRNAPLSIW MNGGPGSSSM IGLMQENGPC 5400
RNAPLSIWMN GGPGSSSMIG LMQENGPCRN APLSIWMNGG PGSSSMIGLM QENGPCRNAP 5460
LSIWMNGGPG SSSMIGLMQE NGPCRNAPLS IWMNGGPGSS SMIGLMQENG PCRNAPLSIW 5520
MNGGPGSSSM IGLMQENGPC RNAPLSIWMN GGPGSSSMIG LMQENGPCRN APLSIWMNGG 5580
PGSSSMIGLM QENGPCRNAP LSIWMNGGPG SSSMIGLMQE NGPCRPGGCK DQIIECRTVY 5640
DMAMEAWSKT VYDMAMEAWS KTVYDMAMEA WSKTVYDMAM EAWSKTVYDM AMEAWSKTVY 5700
DMAMEAWSKT VYDMAMEAWS KTVYDMAMEA WSKPGGTVYD MAMEAWSKPG GTVYDMAMEA 5760
WSKPGGCKTV YDMAMEAWSK PGGCKTVYDM AMEAWSKPGG CKTVYDMAME AWSKPGGCKT 5820
VYDMAMEAWS KPGGCKTVYD MAMEAWSKPG GCKTVYDMAM EAWSKPGGCK TVYDMAMEAW 5880
SKPGGCKVAL VYGDRDYACP WNGGEEVSLK VALVYGDRDY ACPWNGGEEV SLKVALVYGD 5940
RDYACPWNGG EEVSLKVALV YGDRDYACPW NGGEEVSLKV ALVYGDRDYA CPWNGGEEVS 6000
LKVALVYGDR DYACPWNGGE EVSLKVALVY GDRDYACPWN GGEEVSLKVA LVYGDRDYAC 6060
PWNGGEEVSL KVALVYGDRD YACPWNGGEE VSLKVALVYG DRDYACPWNG GEEVSLKVFE 6120
AGHEVPAYQP ETAYEIFHRV FEAGHEVPAY QPETAYEIFH RVFEAGHEVP AYQPETAYEI 6180
FHRVFEAGHE VPAYQPETAY EIFHRVFEAG HEVPAYQPET AYEIFHRVFE AGHEVPAYQP 6240
ETAYEIFHRV FEAGHEVPAY QPETAYEIFH RVFEAGHEVP AYQPETAYEI FHRVFEAGHE 6300
VPAYQPETAY EIFHRVSIWT ESYGGRVSIW TESYGGRVSI WTESYGGRVS IWTESYGGRV 6360
SIWTESYGGR VSIWTESYGG RVSIWTESYG GRVSIWTESY GGRVSIWTES YGGRVSIWTE 6420
SYGGRVSIWT ESYGGRVSIW TESYGGRYGP SFTAFFQEQN EKYGPSFTAF FQEQNEKYGP 6480
SFTAFFQEQN EKAAWLFEDS QAKADEINQI FDAISYMKAD VPSGSTNITH GRAFPCFDEP 6540
ALKAGMIADA GALASSGYQS TSGLLSLLKA VEQSLDAIRD GHILQQFKFA AGETSAIHPN 6600
IRGFDNEAEF IVWNEIVARI VDVLLDEKIV DVLLDEKNSG ASRLNADHSA IYRLTFTGIL 6660
NDNMAGFYRN GGEKEYNVVY DRNQDIYMPL GGLRNVGFPV VTVAEDAASS SIKSSHPIEV 6720
PVKSSHPIEV PVKRTGDVRP EEDTTLYPVM LGLRTHEIGW EFSEKTKQGL DENTMLTERT 6780
LGLALSDEVK VYATPDQDIE HGRYLASTQM EPTDARYLGE DVFIQGVRQG LLTVEDRGSV 6840
FSIVLKAAQE MFQRQGLDEN TMLTERTDVE SWLKAGMIAD AGALASSGYQ STSGLLSLLK 6900
FAAGETSAIH PNIRLTFTGI LNDNMAGFYR NGGEKEYNVV YDRNGGEKEY NVVYDRNGGE 6960
KEYNVVYDRN GGEKEYNVVY DRNQDIYMPL GGLRVYATPD QDIEHGRYLA STQMEPTDAR 7020
AWYENGITNC VGDNTRCCDS GVEQLVSFSD VSDFKDADAC NGGGIEYDSP ADTPLEFKDN 7080
TCNAPIPVSF PVAPTDTKDP YMFHQANLRD QCNYSLQYTI GNKFAANGNY GSETTAAVIN 7140
NFNGRFTTSA SDGFDGMQVN PRGNGVIEAA AGKGNVAGNI LVIAKITTAD MDGISSWLPT 7200
INGKIYYNCD TPACTVQEWI DTSAGSGDFS NLLATEKKPL GTGTDLWPKL DDLFVWWTTP 7260
ANRLVDWPIV TITHQEMSAN MNAGSSYFVE VGHNMNAGSS YFVEVGHNGN GVIEAAAGKQ 7320
AWVETMVQEF VRSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 7380
VNPRVIANGN VAGNILVIAK YFTSNGIIPP AITGLHNGDA LRQITGVTLS AKSASDGFDG 7440
MQVNPRDPYM FHQANSDVSD FKEAGLKGII PPAITGLHNG DALRYFTSNG IIPPAITGLH 7500
NEISFNQAWL RLVDWPIVTI THQEMSANFL DRKPLGTGTD LANGNVAGNI LVIAKHQEMS 7560
ANFLDRYNRD QCNYSLQYTI GNKAWYENGI TNCVGDNTRA WYENGITNCV GDNTRAWYEN 7620
GITNCVGDNT RAWYENGITN CVGDNTRAWY ENGITNCVGD NTRAWYENGI TNCVGDNTRA 7680
WYENGITNCV GDNTRAWYEN GITNCVGDNT RDADACNGGG IEYDSPADTP LEFKDADACN 7740
GGGIEYDSPA DTPLEFKDAD ACNGGGIEYD SPADTPLEFK DNTCNAPIPV SFPVAPTDTK 7800
DNTCNAPIPV SFPVAPTDTK DPYMFHQAND PYMFHQANLR DPYMFHQANL RDPYMFHQAN 7860
LRDPYMFHQA NLRDQCNYSL QYTIGNKEIS FNQAWLREIS FNQAWLREIS FNQAWLREIS 7920
FNQAWLREIS FNQAWLREIS FNQAWLREIS FNQAWLRFAA NGNYGSETTA AVINNFNGRF 7980
AANGNYGSET TAAVINNFNG RFAANGNYGS ETTAAVINNF NGRFAANGNY GSETTAAVIN 8040
NFNGRFAANG NYGSETTAAV INNFNGRFAA NGNYGSETTA AVINNFNGRF AANGNYGSET 8100
TAAVINNFNG RFAANGNYGS ETTAAVINNF NGRFAANGNY GSETTAAVIN NFNGRITTAD 8160
MDGISSWLPT INGKITTADM DGISSWLPTI NGKITTADMD GISSWLPTIN GKITTADMDG 8220
ISSWLPTING KITTADMDGI SSWLPTINGK KPLGTGTDLW PKKPLGTGTD LWPKKPLGTG 8280
TDLWPKKPLG TGTDLWPKKP LGTGTDLWPK KPLGTGTDLW PKKPLGTGTD LWPKKPLGTG 8340
TDLWPKKPLG TGTDLWPKKP LGTGTDLWPK KPLGTGTDLW PKKPLGTGTD LWPKLDDLFV 8400
WWTTPANRLD DLFVWWTTPA NRLDDLFVWW TTPANRLDDL FVWWTTPANR LVDWPIVTIT 8460
HQEMSANLVD WPIVTITHQE MSANFLDRLV DWPIVTITHQ EMSANFLDRM NAGSSYFVEV 8520
GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA 8580
AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS 8640
YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN 8700
GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKTPLLNQ QNSMWPYFTT SASDGFDGMQ 8760
VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8820
VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8880
VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8940
VNPRVIANGN VAGNILVIAK VIANGNVAGN ILVIAKYFTS NGIIPPAITG LHNGDALRAS 9000
HTVDKNGIWS SEVKATDNYI ANAAAAVAKD LLQDIVTWDD KEAGIYLIAR GPLNEGGLYA 9060
ERGTNYVALS LWALESDGAK HTDYSSQEST SYKLGSFELS YTTPVLTGYG NVESPEQPKL 9120
SGQDASAITW KLTGNLGGED YQDKLTGNLG GEDYQDKVRN SAYNYWVPEL PTEGTSPGFS 9180
TSKPGIGFYT AQFDLDLPKQ GFHQPQPPSE SWESGSPLEG LSKSPGSFFV VRSSYDDSAW 9240
VSADLPKTSY DYGSPITETR YPDADYMQYV MDQARNGIWS SEVKVLVLYG GPKASPSYLT 9300
ATPRYLDTLP EIKTLHLEQS PSTPYAQLYV NGYQYGKKAD IVVPFPWGGP GFEKAQLYVN 9360
GYQYGKAQLY VNGYQYGKAT DNYIANAAAA VAKLSGQDAS AITWKLTGNL GGEDYQDKVR 9420
NGIWSSEVKQ GFHQPQPPSE SWESGSPLEG LSKSSYDDSA WVSADLPKYP DADYMQYVMD 9480
QARYPDADYM QYVMDQARAL TNGAGAIKEA IADVLEHLGE NDEDIAVYAP NPFYKGFAPL 9540
EYLGSNFENG ELPKGFDNAG FVMGTSSSLF NQFILRGKMP MPILVADGRH FQLINTAAYW 9600
KIPNVAIAVS GGGYRLNGTD IPNFLKLNLS SFDASGYIDR LPDICNTCFK MPMPILVADG 9660
RNSILEGPDV KSAAALSTSE KDWLQVRSSS LFNQFILRTF LNLGLNKTNT KLPDICNTCF 9720
KTSLTDYWGR SSFDASGYID RSTSEKDWLQ VRGTDIPNFL KLNLGLNKGF DNAGFVMGTS 9780
SSLFNQFEYL GSNFENGELP KMPILVADGR DLYDAVKINT AAYWKSIALG DDFKKALTNG 9840
AGAIKALTNG AGAIKALTNG AGAIKALTNG AGAIKGKMPM PILVADGRGK MPMPILVADG 9900
RGKMPMPILV ADGRGKMPMP ILVADGRGKM PMPILVADGR GKMPMPILVA DGRHFQLINT 9960
AAYWKHFQLI NTAAYWKLPD ICNTCFKLPD ICNTCFKMPI LVADGRMPMP ILVADGRMPM 10020
PILVADGRMP MPILVADGRM PMPILVADGR MPMPILVADG RMPMPILVAD GRMPMPILVA 10080
DGRMPMPILV ADGRMPMPIL VADGRNSILE GPDVKNSILE GPDVKNSILE GPDVKSAAAL 10140
STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS 10200
AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL 10260
QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE 10320
KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL 10380
STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS 10440
AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL 10500
QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL STSEKDWLQV RTNTKLPDIC 10560
NTCFKTNTKL PDICNTCFKT NTKLPDICNT CFKAASLPAS FSGFKALVSH DGTFVADAKE 10620
NSLVWHQQVL GWLNKFNAVF SGTLKGDAGS PVFSPDSKGD AGSPVFSPDS KKIAYWQMAD 10680
ESYEADHRIQ AFVIYPENFD KLFSIPADAG DDYKPKLNPE GLISAPRLPV SEGLSLFNIL 10740
QERMINWIQG SDLGRNAESP YPPFGGASDY DLSPDGKRSE AIPNPSGDVA VFSQSQYSFK 10800
SEAIPNPSGD VAVFSQSQYS FKTAVPINGP DSPGTPEGVK TAVPINGPDS PGTPEGVKGD 10860
AGSPVFSPDS KTLATANKID PELKTLYVYT VGSEETIPSL AADWDRTTSQ WNVLDLKVVT 10920
TDSGDVRWIQ GSDLGRALVS HDGTFVADAK ALVSHDGTFV ADAKGDAGSP VFSPDSKKIA 10980
YWQMADESYE ADHRIAYWQM ADESYEADHR IAYWQMADES YEADHRLFSI PADAGDDYKP 11040
KLFSIPADAG DDYKPKLNPE GLISAPRRSE AIPNPSGDVA VFSQSQYSFK RSEAIPNPSG 11100
DVAVFSQSQY SFKTAVPING PDSPGTPEGV KTAVPINGPD SPGTPEGVKT AVPINGPDSP 11160
GTPEGVKGDA GSPVFSPDSK TTSQWNVLDL KTTSQWNVLD LKAASNFDGD TLVLGYDSGN 11220
GNPETSFMTL MRAGSTIAVT DVQITGGAVG IKASGTCSGP IQSAPTSYWL ADQDHSGDAR 11280
DAGSPKPVVQ IGHEGDVGVA EIQNMRFSVA EILPGAKGDG STDDSASLNA ILANNAANCK 11340
GTCSGPIQSA PTSYWLADQD HSGDARIVGE AWAVITGAGD AFKNSQILIQ NLSHDNSNAI 11400
AVDSKNSQIL IQNLSHDNSN AIAVDSKDNI KNVVLDTTAL SANTKSAPTS YWLADQDHSG 11460
DARVGTIITG DPLDPPVLKV TNSPSNLVWY SISTRYPAEV FLPGGTYQLG KSLGSLVLLD 11520
SSSINSGPVV RDTLVIPPGS RAQPTYAEYS NDQIVNVKSG TCSGPIQSAP TSYWLADQDH 11580
SGDARAGSTI AVTDVQITGG AVGIKAQPTY AEYSNDQIVN VKAQPTYAEY SNDQIVNVKD 11640
AGSPKPVVQI GHEGDVGVAE IQNMRFSVAE ILPGAKNSQI LIQNLSHDNS NAIAVDSKNS 11700
QILIQNLSHD NSNAIAVDSK NSQILIQNLS HDNSNAIAVD SKNSQILIQN LSHDNSNAIA 11760
VDSKDNIKNS QILIQNLSHD NSNAIAVDSK DNIKNSQILI QNLSHDNSNA IAVDSKDNIK 11820
NVVLDTTALS ANTKSAPTSY WLADQDHSGD ARVTNSPSNL VWYSISTRVT NSPSNLVWYS 11880
ISTRAGALLL GKAGVIPESL HQDTVGTFGK DRLETTAGSW ALLGSVVPRG IMDETYYQAL 11940
EFCQRGKTPE GGYAQFLTNK GPLHGIPFIV KIHQTQPYLN AILQVNPDAF KLETTAGSWA 12000
LLGSVVPRNS VVGIKPTVGL TSRTIVSPDG FNWDYGSTRT PEGGYAQFLT NKTTREEGID 12060
AALKVDFYNN LKDYLSEVEN TKAALSEWAD MRALGTETDG SVINPAQREE GIDAALKDAV 12120
YALDAIYGID ARAALSEWAD MRAALSEWAD MRAGVIPESL HQDTVGTFGK AGVIPESLHQ 12180
DTVGTFGKAG VIPESLHQDT VGTFGKAGVI PESLHQDTVG TFGKGIMDET YYQALEFCQR 12240
IHQTQPYLNA ILQVNPDAFK NSVVGIKPTV GLTSRNSVVG IKPTVGLTSR NSVVGIKPTV 12300
GLTSRTIVSP DGFNWDYGST RTTREEGIDA ALKAVWPGDM GVAVPAAFVS TGDLESVKAY 12360
QGYFHSNDDL LNRDGSASYV MPDKDRAVWP GDMGVAVPAA FVSTGDLESV KDSAFPGLWE 12420
ENIYAPSSRG GSGALGLAFS EAKGVYYVDD TATITVSGGG SHRIWYSGAY TLQTNAVPVN 12480
TGRNALQTMY DTQDKNALQT MYDTQDKTTG AFDESGPPLS QKSPDGYTLQ FSVPPGTKSS 12540
EKPSITIDGN NINKTTGAFD ESGPPLSQKI APQFGDLKAL ELIRRFQASW DKSPDGYTLQ 12600
FSVPPGTKIS SFEIQGHFKA VWPGDMGVAV PAAFVSTGDL ESVKAYQGYF HSNDDLLNRA 12660
YQGYFHSNDD LLNRAYQGYF HSNDDLLNRA YQGYFHSNDD LLNRDGSASY VMPDKDGSAS 12720
YVMPDKDRAV WPGDMGVAVP AAFVSTGDLE SVKGVYYVDD TATITVSGGG SHRGVYYVDD 12780
TATITVSGGG SHRISSFEIQ GHFKNALQTM YDTQDKNALQ TMYDTQDKNA LQTMYDTQDK 12840
NALQTMYDTQ DKTTGAFDES GPPLSQKNAL QTMYDTQDKT TGAFDESGPP LSQKNALQTM 12900
YDTQDKTTGA FDESGPPLSQ KNALQTMYDT QDKTTGAFDE SGPPLSQKSP DGYTLQFSVP 12960
PGTKSPDGYT LQFSVPPGTK SSEKPSITID GNNINKSSEK PSITIDGNNI NKSSEKPSIT 13020
IDGNNINKTT GAFDESGPPL SQKDNILPEN LDDGLPSQFV YEKFGPHEYN GDQYTSIIRL 13080
VIDLSGNGGG YILQGYDTFR NVGLVSVSLD GKPSSDPMQG IGGIKQLFPS IVQDGYTRSD 13140
KYAGEYEFQA DLFKSFEPST PAEFQAVLEK YAGEYEFQAD LFKPFAASTP GFDGYFSGSA 13200
RAASTPGFDG YFSGSARYDL NLENKAFNLA HDGHFRHFTS LEEKFFPDLL TKSIAIGGRP 13260
SSDPMQGIGG IKFGPHEYNG DQYTSIIRFG PHEYNGDQYT SIIRQLFPSI VQDGYTRQLF 13320
PSIVQDGYTR QLFPSIVQDG YTRSDKYAGE YEFQADLFKE PGAEGVCETT PGVKFANQMP 13380
NGCQDLISTC KGCQDLISTC KQLPKNPTGV KSAGYTPLKV NGVEYGETRS YSGYVDTSPE 13440
SHTFTALADY ALCAEATNMC RTIFGWDIAE GQKTIFGWDI AEGQKKVYEA GHEVPYYQPI 13500
ASLYKEPGAE GVCETTPGVK LSGLPSLDSR SYSGYVDTSP ESHTFFHNPE TAPITLWLNK 13560
IWPSYKVYEA GHEVPYYQPI ASVNGVEYGE TRTALADYAL CAEATNMCAE GVCETTPGVK 13620
FANQMPNGCQ DLISTCKFAN QMPNGCQDLI STCKFANQMP NGCQDLISTC KFANQMPNGC 13680
QDLISTCKFA NQMPNGCQDL ISTCKFANQM PNGCQDLIST CKKIWPSYKK IWPSYKSAGY 13740
TPLKVNGVEY GETRSAGYTP LKVNGVEYGE TRSAGYTPLK VNGVEYGETR SAGYTPLKVN 13800
GVEYGETRSA GYTPLKVNGV EYGETRSAGY TPLKVNGVEY GETRSAGYTP LKVNGVEYGE 13860
TRSAGYTPLK VNGVEYGETR SAGYTPLKVN GVEYGETRSA GYTPLKVNGV EYGETRSAGY 13920
TPLKVNGVEY GETRTALADY ALCAEATNMC RTALADYALC AEATNMCRTA LADYALCAEA 13980
TNMCRTALAD YALCAEATNM CRTALADYAL CAEATNMCRT ALADYALCAE ATNMCRTALA 14040
DYALCAEATN MCRTALADYA LCAEATNMCR TALADYALCA EATNMCRTIF GWDIAEGQKT 14100
IFGWDIAEGQ KTIFGWDIAE GQKTIFGWDI AEGQKTIFGW DIAEGQKTIF GWDIAEGQKT 14160
IFGWDIAEGQ KTIFGWDIAE GQKTIFGWDI AEGQKTIFGW DIAEGQKTIF GWDIAEGQKT 14220
IFGWDIAEGQ KTIFGWDIAE GQKKVNGVEY GETRVNGVEY GETRVNGVEY GETRYKEPGA 14280
EGVCETTPGV KYKEPGAEGV CETTPGVKYK EPGAEGVCET TPGVKYKEPG AEGVCETTPG 14340
VKYKEPGAEG VCETTPGVKA SDFWANELVT WWNKEAEPSQ EYVSYSHGVF LRFSYEEGEK 14400
FLNKGAKDDV FIKGGSILPM QEVALTTRKA TGDVLFNTKN AHGQEILLRN HNVLSAIPQE 14460
PYRQYQLSTV GLPAMQQYNT LGFHQCRSSE AEPSQEYVSY SHGVFLRTLG GSVDLTFYSG 14520
PTQAEVTKWA SVIDATKSEA EPSQEYVSYS HGVFLRAEPS QEYVSYSHGV FLRAEPSQEY 14580
VSYSHGVFLR AEPSQEYVSY SHGVFLRAEP SQEYVSYSHG VFLRASDFWA NELVTWWNKE 14640
AEPSQEYVSY SHGVFLREAE PSQEYVSYSH GVFLREAEPS QEYVSYSHGV FLRFSYEEGE 14700
KFLNKGGSIL PMQEVALTTR GGSILPMQEV ALTTRNAHGQ EILLRNAHGQ EILLRNHNVL 14760
SAIPQEPYRN HNVLSAIPQE PYRNHNVLSA IPQEPYRNHN VLSAIPQEPY RNHNVLSAIP 14820
QEPYRSEAEP SQEYVSYSHG VFLRWASVID ATKWASVIDA TKDPSINDDS VMIYAPAVRF 14880
LDEALTYPPP KGIQINDPSI NDDSVMIYAP AVRIASAMLD EEDEKYFNVK NGDQSPPSAL 14940
GPLPSVIERP SINDDSVMIY APAVRTDYSV CGETTIFKVY LTGESYAGQY IPYIASAMLD 15000
EEDEKVYLTG ESYAGQYIPY IASAMLDEED EKYFNVKIAS AMLDEEDEKT DYSVCGETTI 15060
FKNGDQSPPS ALGPLPSVIE RVYLTGESYA GQYIPYTLIA GAGLLGTAHT ERETTIFKNG 15120
DQSPPSALGP LPSVIERTDV QKALHVPRDD SVMIYAPAVR FLDEALTYPP PKGIQINDPS 15180
INDDSVMIYA PAVRGIQIND PSINDDSVMI YAPAVRGIQI NDPSINDDSV MIYAPAVRGI 15240
QINDPSINDD SVMIYAPAVR IASAMLDEED EKIASAMLDE EDEKYFNVKI ASAMLDEEDE 15300
KYFNVKIASA MLDEEDEKYF NVKIASAMLD EEDEKYFNVK IASAMLDEED EKYFNVKIAS 15360
AMLDEEDEKY FNVKIASAML DEEDEKYFNV KNGDQSPPSA LGPLPSVIER TDYSVCGETT 15420
IFKNGDQSPP SALGPLPSVI ERTDYSVCGE TTIFKNGDQS PPSALGPLPS VIERVYLTGE 15480
SYAGQYIPYI ASAMLDEEDE KYFNVKAHIL PPNGRDLNPN GSQFITPGGK DLNPNGSQFI 15540
TPGGKNDPVA VFDGSVIPKE AGLVPFQVSP TTKFHVLTAQ LSFPRFRDLN PNGSQFITPG 15600
GKLDRPPVIP LPPSDSDVTA FRNDPVAVFD GSVIPKPVAV FDGSVIPKTI SNVVDNELAR 15660
TTNGIVSTNE SGRDPVAVFD GSVIPKFALS TWARILPATS QVSTKAHILP PNGRAHILPP 15720
NGRAHILPPN GRDLNPNGSQ FITPGGKFAL STWARFALST WARFHVLTAQ LSFPRFHVLT 15780
AQLSFPRFRD LNPNGSQFIT PGGKFRDLNP NGSQFITPGG KFRDLNPNGS QFITPGGKND 15840
PVAVFDGSVI PKTISNVVDN ELARTISNVV DNELARTISN VVDNELARTT NGIVSTNESG 15900
RDWSDSYYQG PAFKFGLGAD NTLAFEVVTA DGQLVTASRG VGSDAWTVSE SGRITNEYVP 15960
QLEAVTPGSG CYQNEGNFRN VLENNPTGMA SVLRSKWDPN NFFYVLKTTA LTDLGIAYKV 16020
SAGVMGYQIL NAAHAKVSYT EYDSYYDHYN KYMGPLPYGN LAVATYQYGG RWDPNNFFYV 16080
LKDWSDSYYQ GPAFKGVGSD AWTVSESGRG VGSDAWTVSE SGRGVGSDAW TVSESGRGVG 16140
SDAWTVSESG RITNEYVPQL EAVTPGSGCY QNEGNFRNVL ENNPTGMASV LRNVLENNPT 16200
GMASVLRVSA GVMGYQILNA AHAKVSAGVM GYQILNAAHA KVSAGVMGYQ ILNAAHAKVS 16260
AGVMGYQILN AAHAKVSAGV MGYQILNAAH AKVSAGVMGY QILNAAHAKV SAGVMGYQIL 16320
NAAHAKVSYT EYDSYYDHYN KYMGPLPYGN LAVATYQYGG RALMNGAGAI KDAGYETSIT 16380
DYWGRDFFNH VTIKDGNWTT CVGCAILSRG FVPLEYVGSK HVYDAVQDKP TVYGFVPLEY 16440
VGSKTNTQVP DACTQCFQKQ ADMPMPLLVA DGRTAFSDIL AKSIALTDTF KILDSATYYK 16500
ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK 16560
ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK DAGYETSITD YWGRDAGYET SITDYWGRDA 16620
GYETSITDYW GRDAGYETSI TDYWGRDAGY ETSITDYWGR DFFNHVTIKD FFNHVTIKDF 16680
FNHVTIKDFF NHVTIKGFDN AGFVMGTSSS LFNQFMPLLV ADGRMPLLVA DGRMPMPLLV 16740
ADGRMPMPLL VADGRMPMPL LVADGRMPMP LLVADGRMPM PLLVADGRMP MPLLVADGRM 16800
PMPLLVADGR QADMPMPLLV ADGRQADMPM PLLVADGRQA DMPMPLLVAD GRQADMPMPL 16860
LVADGRTSIT DYWGRAHDDT VNYLYEELKK ATAFAVATYA NDLSSIPKGG DPNNVVALGG 16920
HTDSVEAGPG INDDGSGIIS NLVIAKGPYS AIVGISLEDG QKNLGCSEAD YPSDVEGKQP 16980
QVHLWSNADQ TLKTMTYSPS VEVTADVAVV KTTYNVVAQT KVGDEEIEAK AHDDTVNYLY 17040
EELKKAHDDT VNYLYEELKK AHDDTVNYLY EELKKQPQVH LWSNADQTLK TMTYSPSVEV 17100
TADVAVVKAG QFPISANDGA TSTKAGVLSW SYTWSPADKE AASAALAAGY KFDGVTWDEE 17160
NWLLKFDPSA AIYPWTSGRF VGGASTDAFA DPKLLPEEGI YITPNLPPQI PYVKPSAAIY 17220
PWTSGRVVLT LTGIEPSTIY TAEEENQVRA YVASDSELEY VTWTVDNRGD WEVTSILSID 17280
QERTLIPADK IPTGKTLSTN EEGYETSAVR VSQTNPTVTL SLLNIASKYP ILFTPYGGPG 17340
AQEVTKDGTD GWLDNLLSMK TLSTNEEGYE TSAVRKFYDS MYTERTLIPA DKIPTGKATS 17400
GGTSAAAPVF AGLVGMLNDA RDFTDITAGS SIGCDGVNPQ TGKGFPDVAA HSLTPRPDVA 17460
AHSLTPRPNS ALPQVLSNSY GDEEQTVPEY YAKSALPQVL SNSYGDEEQT VPEYYAKSYG 17520
DEEQTVPEYY AKVCNLIGLM GLRLKDLVLS LAWYQESAVS KATSGGTSAA APVFAGLVGM 17580
LNDARATSGG TSAAAPVFAG LVGMLNDARA WYQESAVSKA WYQESAVSKD FTDITAGSSI 17640
GCDGVNPQTG KGFPDVAAHS LTPRGFPDVA AHSLTPRGFP DVAAHSLTPR SALPQVLSNS 17700
YGDEEQTVPE YYAKVCNLIG LMGLRDAEEE PYDWSNEGRG ISDGIDWQAG YSAVQKMDDA 17760
EQYEATSRNI YIQSATLDGK PYSKSLNYIP VEDFDYKTLE YSYDDFTIAQ MARTMINPQD 17820
YTGENPLWKD NGIFVNSRSI NGYPLPGGAF VRMDDAEQYE ATSRTLEYSY DDFTIAQMAR 17880
DIDFGENGDG IKDYVPNTQI PVTVAANTFP GGQEGFIDFV KFQESPEGAD FWGARTKVTI 17940
SPELSIRVFQ TAFGPAGTML TYEPIVRADA IVAAIRLAED HINWVEIRVT ISPELSIRHE 18000
LGVDEIWRDI DFGENGDGIK DIDFGENGDG IKFQESPEGA DFWGARLAED HINWVEIRAG 18060
VKPSNYVGDI FGTLGGTPDF GPGRCDVATT DVYYSGKSKY TAEGYEAATK TASNFDQPHS 18120
DESALQHLRY CASAQEDNAT LQALLRYTAE GYEAATKYVD AGGFEPSIKL QALLRSKYTA 18180
EGYEAATKTA SNFDQPHSDE SALQHLRGHL TAMTGDGVND APSLKTAALV QGASDSGHFK 18240
TGTLTANQLS IRAYGIVVAT AKTGDGVNDA PSLKMLTGDA LAIAKLAIEH EVDAHGKIEN 18300
MLSHLSKDKS ETETETEIEI SNKIKEIQEA GDVRLSDELE DDNAPIGFET TKSVEIAQLH 18360
SEVEALVEKY TVTAGELTSD FKEVEVEKEW TKTETKTEIE IERIKEIQEA GDVRASSDDS 18420
NYGWEDSKGI QDAGVIATAK IGAQSTVLLK NFGEIGDASE YVYPEGLERY TPPNFSSWTR 18480
VNEFVDVQRA VDIVSQMTLT EKDSPNWDVD SDALPAIPEG AKEIPVGYSA ADIDTNRGVD 18540
YQPGGSSNLA DPIADAEGCK RNTLAFFSGN EVINDGPSSK YGLVEIDDGK VKTLADFDAL 18600
KYGLVEIDDG KVKDLMQAMA DFGPKFPGGN NLEGDTLDGR TGEVYASAVI VSKYPSNLDA 18660
WIPVDGSALS LKYVEVGNED NLNDGLDSYK FQAFYDAIKE QTYTGSFYVK ASLGHPEPWT 18720
VKDLMQAMAD FGPKELPSGP YFVSLYTGEV FKGISPEAHQ SLTTFTRLYP DDNLAFIQAG 18780
ISDEKQLLLA GGGWDGKSLF VSVYSVGTTD YRLYYTPTAE KPLAGLRGIS PEAHQSLTTF 18840
TRQLLLAGGG WDGKDSLSEA IAYAKGGGGG TFGVVMESTH RIANECQNQE LFWALRSQGG 18900
TAVIEEFPSW YEFYQKYVVP NAVTVGNAHF AATRSSGQGT LSLWTRAVTV GNAHFAATRG 18960
GGGGTFGVVM ESTHRGGGGG TFGVVMESTH RGGGGGTFGV VMESTHRIAN ECQNQELFWA 19020
LRSSGQGTLS LWTRYVVPNA VTVGNAHFAA TRYVVPNAVT VGNAHFAATR YVVPNAVTVG 19080
NAHFAATRAS SVSGVITLSD GRTTADSDGN FSFENVRVQD ETWELSDGSY ITKYDWSDFI 19140
NSAKYEEFEV PAGTLVKIWQ IGTLDRAYTQ YTETSVYGML KDGDLVTQQN ELQGKHEGWI 19200
DEAAVQEAKL HYGAYSIKSN LFNSYSENQV LLPASVYGSY KETYGSAAGW DKLADALAAS 19260
SLPEAWWGEN YEPLLNIKGG GGGTFGVVIE STHRIANECQ NQDLFWALRV EPQLSFVAAV 19320
IKYVTSNAVS VGVTHFAGSR AVFETAEGRG GGGGTFGVVI ESTHRYVTSN AVSVGVTHFA 19380
GSRDAEVAPP NDPVDPMAPD SSTKFEGYLP DARGSTGNVL VDVSHVLPSF RVGISWLSTE 19440
KLSITATGGD GNGDSQIYVQ KGAVNWEDGY RDAEVAPPND PVDPMAPDSS TKDSVKEDDY 19500
EDLFNYICAK KFTDTPVLYG PKMLDDAGIY LITDLSSPSE SINRSDGQCS DLLKQQLSFV 19560
MNQWYEKYGA YSVCSPKTLP AIESKKFTDT PVLYGPKMLD DAGIYLITDL SSPSESINRS 19620
DGQCSDLLKA DAAGSHGEAL NEVQAKAKAD AAGSHGEALN EVQAKLEAAE QALSEARVGA 19680
LESQLSTEQD AIKEAAESAG TTHSQQLQEL RDALEAAEAA AKIQEQLKGQ TPLPILVADG 19740
RNNILEGPDV KSHLSVVDGG EDGQNIPLHP LIQPERTIDY WTELVDTVKT STTLPEVCSK 19800
AMLNGAGALK AMLNGAGALK AMLNGAGALK AMLNGAGALK AMLNGAGALK GQTPLPILVA 19860
DGRINLGLNK NNILEGPDVK SHLSVVDGGE DGQNIPLHPL IQPERTFINL GLNKTSLTDY 19920
WGRFNVDETA FTGAWGRIGS LAITDVSLPF FKIQGISNPS GALSSGGLGE PKVQNGAVTW 19980
ESDPNRATTV YGESIKSIYA INSGRELDTQ HIHPPDSYFV SPLTRGFTEI DELWNGVTAE 20040
TNAAQDLRQA QVAHDFWQKF YHQVVELNRK DAELTDAGVK LNTGAVIPVL VRELDTQHIH 20100
PPDSYFVSPL TRLNTGAVIP VLVRLNTGAV IPVLVRQAQV AHDFWQKQAQ VAHDFWQKAI 20160
NDYIDSQLDK KGVQISTNIP KSSPWIMLGG SYPGMRYQSL EYQQSLCYRL FSLALKISIP 20220
IDHEDPSMGT YQNRAINDYI DSQLDKKCSS HDDCSDELAC TDGVCACTAD SAVTCSWEGH 20280
CAGAKLNLQY QASGDAKKSL VDFSAARGDN PSILGLRSAV TCSWEGHCAG AKIGTTIDDI 20340
KCTADSAVTC SWEGHCAGAK CTADSAVTCS WEGHCAGAKC TADSAVTCSW EGHCAGAKCT 20400
ADSAVTCSWE GHCAGAKAFP DVAAQGMNFA VYDKELYNIG DYQADANSGS KIAFASYLEE 20460
YARQGLQDIT LGASIGCTGR YADLENFENY LAPWAKAFPD VAAQGMNFAV YDKAFPDVAA 20520
QGMNFAVYDK ELYNIGDYQA DANSGSKYAD LENFENYLAP WAKYADLENF ENYLAPWAKA 20580
GSSPTDIISG ISDKTDALDS AIKKVEQAID DIIAKSAADG LASAITSKSG DDISTTDALA 20640
LPEPVQALTK SPTDIISGIS DKTDALDSAI KAQNDPNAFG VVAARLGACP PGKETALLGD 20700
KPNAFGVVAA RQNDPNAFGV VAARAATYCP ENIEKIENQS DADGYSSCST LKLTGLTTLT 20760
TLSFAALTKS DKLNVIDFPK VGSIEFTALP QLQSLDFTKL NVIDFPKTVN GGFQIARGVA 20820
AWLFERLSFG SIDLENANIN RSLSFIPGVL YDGSPIGKQE STFAAVERSC FEIGKFVDPL 20880
IGSNNGGNVF AGASLPYGMA KGWTQGGSNA DVVLTDAYVK VGISYISTDR AGNWQNLYKA 20940
QHPFLTIVDP EAQSRSVFSQ NESVAAGLKT TAVLFDEGKE ICLAALGRAL VIVSDSIRTD 21000
TIHGVGQNSF YKASHPIEVP VKASHPIEVP VKIMSILLGG AIPDDLKPLV LFDSVTKTAT 21060
ESEPSLSDIE KTGYVNYNVD TTNLRKHNPL VLFDSVTKTS PFPYDSKKHN PLVLFDSVTK 21120
KHNPLVLFDS VTKKHNPLVL FDSVTKTGYV NYNVDTTNLR APTPPDFSLG YIQSKIEQDG 21180
SESLLTNEYA PLKGYAFIWN MPAQGRTSGW GGNPGGYRYQ LASYLRDYLD EYLVFPPAGV 21240
QPQKIYVTGE SYAGRVDHLP DVPFDVGEMY SGLVPIDKDD KNFQELFGIK KTPLDDFRAD 21300
DVLEVNPLAD PEVVSYFRLD ADAITAQYFG NDAPWYRSDY ASFLYGPYRL VFAPQEEKAT 21360
WDGVDADKIR FQYPGDLFDQ GTTIRFTGDA ATVNSIAARP DSDEIYFGGQ FEKNLEVLSL 21420
TKHVAAYSFG SKAWTALGGG VNGPVHKNLA LLDGKDLTDY LMKSYELPDG QVITIGNERV 21480
APEEHPVLLT EAPINPKQEY DESGPSIVHR QEYDESGPSI VHRVAPEEHP VLLTEAPINP 21540
KVAPEEHPVL LTEAPINPKV APEEHPVLLT EAPINPKAEL PEGYPESSAN PAFRDIQYLE 21600
NYQGQGYSGP AVKFVTVTEE TDPDLFWALR LSLGDSGACK DAEVEPANWG VEGRLGGAQL 21660
FTSRTVSLVE DGSNTPATLS AGTFARSLVD IYRTVSLVED GSNTPATLSA GTFARAYVEM 21720
MQCTDEKEPL VRLEKDLPGT TLSSKYYGYG GGNPLGPAQG IGFANELIAR LEKDLPGTTL 21780
SSKLSEAGHS VLLIEKTPIE SDATSYLNDR GGPMGTYLVS ASERVILSAG TFGTPKAVIT 21840
DIVNQQRLIN QVELSEDKTI ARGGSNNFGI VTRLLLSDAM WYTRERPSAQ LLSGKFDTLG 21900
TSGPTAKLAN AYTWEGGRAV ADRIPLAIHD EVSPVGDTDA LLERAPVVQY ALNRDGYMGY 21960
HGVPQIPIYA YKAIHDEVSP VGDTDALLER AIHDEVSPVG DTDALLERAI HDEVSPVGDT 22020
DALLERDGYM GYHGVPQIPI YAYKLAEESA ALGVKVNPII LTGDMWRVSD NAGLGDWVPN 22080
PDRFPDGLTP LVEDVTKALG GTSTINGMAY TRSQLSDYAA ATVKAEDVQI DVWQKALGGT 22140
STINGMAYTR EIGFTVQEDN TDGKIDFGSA PNIVNAIAED RNSPVWPDGI QQTYEYPNRA 22200
STNMITSNSG AGLVPQDENR IGGSAGTELQ SDKITGEVWP VLMAYGQKSA ISQYGDSFAK 22260
SQSDFESEFS TAKVNAGIGI GPDDLVSFIK GNAMDHFSSI MERYGPTYAA YFLDQNEKAI 22320
TETQYEEAKD LTLDLPDIGL QYAGTVKTVA ALINWRNIWD GTTAQNVKKE DLETSSYSFS 22380
GDGKVDFASL QSAATQSTTY SNAPAVKTCS LVFLFPKKED LETSSYSFSG DGKFDGILGL 22440
GFDTISVNKY GSGSLSGFVS QDTLKWYSVY DLGNGAVGLA KDAYSPHEIY SRIFEQLEGM 22500
SLSKTYEVVG NVYKDQVLKD VALIKAVPGT AQQAAAIKDA GEFDVERGTV VTNDQCGGAS 22560
SVRLHLVTAE EADIKIIYDM PDGSSCKGID VAKPTGRVNG VWTVTHSPFE GLSALKSAMT 22620
LPRVNGVWTV THSPFEGLSA LKVPTVLMSP WVGKSIDQFF NDAKNVAPDD PDHSITGGNQ 22680
QVYSTYHPNA KESTLHLVLR IQDKEGIPPD QQRIQDKEGI PPDQQRIQDK EGIPPDQQRD 22740
VSLVANHIDT VGKTAATVNT WTGGWSDSKA IGTYQNSGLS QYTVRYSEGV HFPARTVANW 22800
LVREVAGDVD NAVNPAWRLG AGVQGFEAYE AANAQGLRLD GGVIEDFAQK NPDLSSTSDT 22860
TDVIRGPDEP YSGQYDEERQ FISVTNPTGA EPVPKFLSEL LEDEYFTKFV NIWLENTDYE 22920
SAANDPHLSK LLEYDIASGT PVYRNNNQGL EALTISPDGK VYAISDQDDT GPWIRDAYQN 22980
DFAARVDPST AVDYNHYSDA ADRSGDVQTL QFAWALQHLS ERTGNQTGQL GTYFNKNINM 23040
LLYGTDDCSG KGMVFSIDAQ GEKNINMLLY GTDDCSGKNP NPDQAFLQVR FWVATGDSSK 23100
AALDALQQSI YLQPKFSDGN GLFYQYERVA VAGYDDTTGG VGPLLAQKAI AIALQSSHRQ 23160
TGSVDGYAYT DANKNDLITY LKSVVENNND GLTAAYRVAP NSGAYLNEAD FRSLPLIVGN 23220
SDQEGKANEQ PTWVYRSDYQ ECADAPGQKF GATGDEYREP SNDPNPPETY SKVALLFSER 23280
LADGSIPTRS ISSDEDSAET EQSDSSDPKV AIIDGLADPW RTMGVGYATN DDSTIRDVPI 23340
MQELNTNTIR DVDPIVIKVE TGVIKPGMVV TFAPANVTTE VKIVVIGHVD SGKDDADLQE 23400
LGAKSSAFAF RKDPSDAIPS IPSIPISYKE AIPFLKEAAE IDSHWERDDY MPFIEVPRDD 23460
YMPFIEVPRA LQGVDELVEK DALHPQFDNF YQEQPKASYG AGVTIQDRDL SVFFTRINDL 23520
LPVYVELLQK AALTNMVNAA KGEITPEQYE KIINEPTAAA IAYGLDKQLT SEEIWQAEEK 23580
LDKPAGDYTI RDYSNNWESG ALKLAPNQMT GSLDATYLKL APNQMTGSLD ATYLKCASDG 23640
SAETCSWEGH CKCASDGSAE TCSWEGHCKC ASDGSAETCS WEGHCKCASD GSAETCSWEG 23700
HCKCASDGSA ETCSWEGHCK LAAISVEPGK ASELGCSSGD LDCLCKVGEC AQMCISNMNA 23760
KGTLLSSNQG SQAADVKNDD DFLNYGISSK APSVITYDEA TKYEAAGITV HKDDYLPFID 23820
MPSEVTQIDA KIPMALDDWP TLSNMIFSGK DNFDTSSVTH RGYQINFLSN SAKDNIQGIT 23880
KPAIRDNIQG ITKPAIGPLG LSPKLQLWDT AGQERAEDYL LNPSPKNFGI GQDIQPKIDA 23940
TTNPGMRQLG LAMLGNKLVI DGLKEVTEIP ATADASRVNV DYTEVPRLAV NMVPFPRYCD 24000
LILGEWRDQI KDVLRIDYIG GGDLFRGSSP NVLYKSANWT PPEGIVRAAS TGSMAEQYTK 24060
AATGTYASST TVYKAENQAV AVGRALVEGS TFAKALYSSA ATGTYASSTT VYKATGTYAS 24120
STTVYKATTV YGESIKAYAD GYVQIVQTYA ASTGSMAEQY TKDLTWSYAA LLTANNRFNV 24180
DETAFTGAWG RGQSAQGASP GVVIASPSKI GADGQSAQGA SPGVVIASPS KIGSLAITDV 24240
SLPFFKIQGI SNPSGALSSG GLGEPKKYTV PSTCGVKPSG ALSSGGLGEP KQAILNNIGA 24300
DGQSAQGASP GVVIASPSKS DPDYFYTWTR SIYAINSGRT GTYASSTTVY KTVGSSCPYC 24360
DSQAPQVRVQ NGAVTWESDP NRVQNGAVTW ESDPNRKPDY FYTWTRYTVP STCGVKSSAA 24420
TGTYASSTTV YKGAVTWESD PNRIVGSISQ LGSWNPSSAT ALSAVQSDVW RDINTVLGSI 24480
HTFDPQNIGA DGQSAQGASP GVVIASPSKA ASTGSMAEQY TKAENQAVAV GRAENQAVAV 24540
GRALVEGSTF AKALVEGSTF AKALYSSAAT GTYASSTTVY KAYADGYVQI VQTYAASTGS 24600
MAEQYTKDLT WSYAALLTAN NRDLTWSYAA LLTANNRIQG ISNPSGALSS GGLGEPKIQG 24660
ISNPSGALSS GGLGEPKNIG ADGQSAQGAS PGVVIASPSK PDYFYTWTRQ AILNNIGADG 24720
QSAQGASPGV VIASPSKQAI LNNIGADGQS AQGASPGVVI ASPSKQAILN NIGADGQSAQ 24780
GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS KQAILNNIGA DGQSAQGASP 24840
GVVIASPSKQ AILNNIGADG QSAQGASPGV VIASPSKQAI LNNIGADGQS AQGASPGVVI 24900
ASPSKQAILN NIGADGQSAQ GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS 24960
KQAILNNIGA DGQSAQGASP GVVIASPSKQ AILNNIGADG QSAQGASPGV VIASPSKQAI 25020
LNNIGADGQS AQGASPGVVI ASPSKQAILN NIGADGQSAQ GASPGVVIAS PSKSAVQSDV 25080
WRSAVQSDVW RSAVQSDVWR SAVQSDVWRS AVQSDVWRSD PDYFYTWTRS DPDYFYTWTR 25140
SDPDYFYTWT RSDPDYFYTW TRSDPDYFYT WTRSDPDYFY TWTRSIYAIN SGRSIYAINS 25200
GRSIYAINSG RTVGSSCPYC DSQAPQVRTV GSSCPYCDSQ APQVRVQNGA VTWESDPNRV 25260
QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN RVQNGAVTWE SDPNRVQNGA 25320
VTWESDPNRV QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN RVQNGAVTWE 25380
SDPNRVQNGA VTWESDPNRV QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN 25440
RKVQNGAVTW ESDPNRKVQN GAVTWESDPN RKVQNGAVTW ESDPNRKASM VWEEAQQVSG 25500
KAVSPSFEDV WSQPRDGAGQ MFIPLNPNAY SPNTLNKDVG GPIEDQNSLQ VGDRFGFDLF 25560
DPTKGPTLLE DFIFRHGGPN FEQLPINQPR HVDGFGIHLF SYLDTQLNRL FYNSLTPAEQ 25620
QFVVDAIRNN VIIQLNRQDL FEAIEAGRSL TPAEQQFVVD AIRSVTSGFV DGIKVGFLAS 25680
VETPASIEAA SELSKTTDVG TFGQKDVHGF ATRIVPEEYV PITKFVTDNG DSKASMVWEE 25740
AQQVSGKASM VWEEAQQVSG KASMVWEEAQ QVSGKASMVW EEAQQVSGKA SMVWEEAQQV 25800
SGKASMVWEE AQQVSGKAVS PSFEDVWSQP RAVSPSFEDV WSQPRAVSPS FEDVWSQPRA 25860
VSPSFEDVWS QPRAVSPSFE DVWSQPRDGA GQMFIPLNPN AYSPNTLNKD GAGQMFIPLN 25920
PNAYSPNTLN KHGGPNFEQL PINQPRHGGP NFEQLPINQP RHGGPNFEQL PINQPRHGGP 25980
NFEQLPINQP RLFSYLDTQL NRNNVIIQLN RNNVIIQLNR NNVIIQLNRS VTSGFVDGIK 26040
SVTSGFVDGI KAAALAELVW SGNRAELVWS GNRASNSLQY VNVQVKDAYS PHEIYSREYL 26100
VANGVQAQAL VPKGIMLDTG RGVQAQALVP KHIVGATAPL WGEQVDDINV SHIVGATAPL 26160
WGEQVDDINV SSMIFEQLEG MSLSKVIPEI DMPSHSSSGW KYNVMANPDA NTPNFNYGGN 26220
GGSWCAPYKT YEVVGNVYKD IEADLQHAET VWGALHAFLV MWEDIALSAD NAHDVPKAAA 26280
LAELVWSGNR AAALAELVWS GNRAAALAEL VWSGNRAAAL AELVWSGNRA SNSLQYVNVQ 26340
VKASNSLQYV NVQVKASNSL QYVNVQVKAS NSLQYVNVQV KDAYSPHEIY SRDAYSPHEI 26400
YSRDAYSPHE IYSRDAYSPH EIYSRDAYSP HEIYSRDAYS PHEIYSRDAY SPHEIYSREY 26460
LVANGVQAQA LVPKEYLVAN GVQAQALVPK EYLVANGVQA QALVPKGIML DTGRIFEQLE 26520
GMSLSKIFEQ LEGMSLSKIF EQLEGMSLSK IFEQLEGMSL SKTYEVVGNV YKYNVMANPD 26580
ANTPNFNYGG NGGSWCAPYK YNVMANPDAN TPNFNYGGNG GSWCAPYKYN VMANPDANTP 26640
NFNYGGNGGS WCAPYKYNVM ANPDANTPNF NYGGNGGSWC APYKATSGGT SAAAPVFAGL 26700
VGMLNDARAW YQESAVSKDF TDITAGSSIG CDGVNPQTGK GFPDVAAHSL TPRPDVAAHS 26760
LTPRPNSALP QVLSNSYGDE EQTVPEYYAK SALPQVLSNS YGDEEQTVPE YYAKSNSYGD 26820
EEQTVPEYYA KSYGDEEQTV PEYYAKYLDQ QITAETKAWY QESAVSKAWY QESAVSKAWY 26880
QESAVSKAWY QESAVSKGFP DVAAHSLTPR GFPDVAAHSL TPRGFPDVAA HSLTPRGFPD 26940
VAAHSLTPRG FPDVAAHSLT PRPDVAAHSL TPRSALPQVL SNSYGDEEQT VPEYYAKYLD 27000
QQITAETKYL DQQITAETKA NEQPTWVYRF FCPTDYLIDV RGTPSVLTEQ GLVKLGVPGN 27060
ELAIEIGSTG DYNARQGTPS VLTEQGLVKS LPLIVGNSDQ EGKTFQGTPS VLTEQGLVKY 27120
PVVQNPVTLA ESSCQSVFNP NIPKNPSAGP GWDQAKPTNG PLAKFQGTPS VLTEQGLVKA 27180
NEQPTWVYRA NEQPTWVYRG TPSVLTEQGL VKNPSAGPGW DQAKPTNGPL AKSLPLIVGN 27240
SDQEGKSLPL IVGNSDQEGK SLPLIVGNSD QEGKARNHGT STVAPQVQAS VYRASVDISN 27300
VDTYSSTEVA NDDSFQQVGK ATFLVWDQQR KASVDISNVD TYSSTEVAND DSFQQVGKNH 27360
GTSTVAPQVQ ASVYRSSTEV ANDDSFQQVG KTLYLTDTDA GVPMIDPRTV YAFDVSEDGS 27420
YLKVASNGYV ITGAGKARNH GTSTVAPQVQ ASVYRARNHG TSTVAPQVQA SVYRATFLVW 27480
DQQRATFLVW DQQRATFLVW DQQRKASVDI SNVDTYSSTE VANDDSFQQV GKTVYAFDVS 27540
EDGSYLKVAS NGYVITGAGK VASNGYVITG AGKIENQSDA DGYSSCSTLK LTGLTTLTTL 27600
SFAALTKSAS SLNSIGDTFK SDKLNVIDFP KSLNSIGDTF KVGSIEFTAL PQLQSLDFTK 27660
TVNGGFQIAR LNVIDFPKGQ LGFWGNKGTY SGDLQLNGVK LNVIDFPKSD KLNVIDFPKS 27720
DKLNVIDFPK TVNGGFQIAR TVNGGFQIAR AVGSDEWTVR LGITYTTYSK MTNDYISALT 27780
KSEMLAEQDK MTNDYISALT KSTITTPWKS VVENNNDGLT AAYRVAPNSG AYLNEADFRY 27840
FYGDNYATLR SGAYLNEADF RTAVGSDEWT VRLGITYTTY SKMTNDYISA LTKMTNDYIS 27900
ALTKMTNDYI SALTKSEMLA EQDKMTNDYI SALTKSEMLA EQDKMTNDYI SALTKSEMLA 27960
EQDKMTNDYI SALTKSVVEN NNDGLTAAYR SVVENNNDGL TAAYRSVVEN NNDGLTAAYR 28020
SVVENNNDGL TAAYRETTMF VLQGFGASMA RLNALQGGRL VQNDFNTLLR QDILGGMVDS 28080
YTDPKTGETT QIHARYGLEA AVPLMYESTG LGLGDMTKVS VADVKIDYIG GGDLFRLVQN 28140
DFNTLLRQDI LGGMVDSYTD PKYGLEAAVP LMYESTGLGL GDMTKALEAY KVNGKAVDFS 28200
GHDEFQGKEP SNDPNPPETY SKFGATGDEY RSDYQECADA PGQKYIARPD IMKSTLPDLS 28260
EVIKVNGKEV GQFKSVDNFH LLTVYAVDFS GHDEFQGKFG ATGDEYRYIA RPDIMKFLDE 28320
ALTYPPPKGI QINDPSINDD SVMIYAPAVR IASAMLDEED EKYFNVKNGD QSPPSALGPL 28380
PSVIERPSIN DDSVMIYAPA VRTDYSVCGE TTIFKTVDDE EGVAAQFKIA SAMLDEEDEK 28440
FLDEALTYPP PKFLDEALTY PPPKGIQIND PSINDDSVMI YAPAVRGIQI NDPSINDDSV 28500
MIYAPAVRIA SAMLDEEDEK IASAMLDEED EKYFNVKIAS AMLDEEDEKY FNVKNGDQSP 28560
PSALGPLPSV IERNGDQSPP SALGPLPSVI ERAGAVAAVV YNNEKHGIPG GGIATGAEGI 28620
KLVLGDAVPE SAAPMGLTPP TKSDKELVSS SAFQSHVKSF EGFPKRLVAH SVATYARIAD 28680
LGKEEYNHPT RAGAVAAVVY NNEKHGIPGG GIATGAEGIK HGIPGGGIAT GAEGIKLVLG 28740
DAVPESAAPM GLTPPTKLVL GDAVPESAAP MGLTPPTKLV LGDAVPESAA PMGLTPPTKS 28800
DKELVSSSAF QSHVKFTDTP VLYGPKKFTD TPVLYGPKML DDAGIYLITD LSSPSESINR 28860
QQLSFVMNQW YEKYGAYSVC SPKSDGQCSD LLKWDVDLYS RLITDLSSPS ESINRDLSSP 28920
SESINRKFTD TPVLYGPKKF TDTPVLYGPK QQLSFVMNQW YEKQQLSFVM NQWYEKCDVA 28980
TTDVYYSGKG GTPDFGPGRS KYTAEGYEAA TKVATIGSAT FARYCASAQE DNATLQALLR 29040
YTAEGYEAAT KYVDAGGFEP SIKSKYTAEG YEAATKVATI GSATFARVAT IGSATFARYV 29100
DAGGFEPSIK DADACNGGGI EYDSPADTPL EFKDNTCNAP IPVSFPVAPT DTKEISFNQA 29160
WLRFAANGNY GSETTAAVIN NFNGRITTAD MDGISSWLPT INGKVIANGN VAGNILVIAK 29220
SDVSDFKEAG LKDADACNGG GIEYDSPADT PLEFKDADAC NGGGIEYDSP ADTPLEFKDA 29280
DACNGGGIEY DSPADTPLEF KFAANGNYGS ETTAAVINNF NGRANNYCSN QVEGPYSLYS 29340
GRVSIWTESY GGRYGPSFTA FFQEQNEKTV YDMAMEAWSK ISYKEPGICE TTPGVKSAGY 29400
APLKPGGCKD QIIECRANNY CSNQVEGPYS LYSGRANNYC SNQVEGPYSL YSGRANNYCS 29460
NQVEGPYSLY SGRISYKEPG ICETTPGVKV SIWTESYGGR AIMGAEEAAK TGGAMWPYRT 29520
LIPDVVGIFA GTPKFVTNMQ AALLKALSEM ILQSEKDYYA SMLQQPKANF EVETPRGITG 29580
TSIARANFEV ETPRDYYASM LQQPKFVTNM QAALLKFVTN MQAALLKTGG AMWPYRTGGA 29640
MWPYRAEDYL LNPSPKLSDL TGDTEYAQLS QKTDDQVSLF ETTIRTDSGF AGLTNVNAAN 29700
GGGRYDNQES FLFAEVLKIT GQEIYRAEDY LLNPSPKTDD QVSLFETTIR AGFAGDDAPR 29760
QEYDESGPSI VHRSYELPDG QVITIGNERV APEEHPVLLT EAPINPKDLT DYLMKIIAPP 29820
ERSYELPDGQ VITIGNERVA PEEHPVLLTE APINPKALLF GAAGSAEDPV VVKNVGFPVV 29880
TVAEDAASSS IKVYATPDQD IEHGRAVEQS LDAIRQGLLT VEDRGPLNEG GLYAERLSGQ 29940
DASAITWKLT GNLGGEDYQD KASPSYLTAT PRALMNGAGA IKDAGYETSI TDYWGRPTVY 30000
GFVPLEYVGS KTAFSDILAK TNTQVPDACT QCFQKMPMPL LVADGRTAFS DILAKAFPDV 30060
AAQGMNFAVY DKELYNIGDY QADANSGSKI AFASYLEEYA RLETIGDTFK QGLQDITLGA 30120
SIGCTGRAFP DVAAQGMNFA VYDKELYNIG DYQADANSGS KQGLQDITLG ASIGCTGRLV 30180
EGAAAGIVVA SPSKQGVLNN IGADGKSSSA YESLTSAVKA SALIAYGNSL ISSDKSVYGI 30240
NNGRPDYFYT WTRPDYFYTW TRSNPDYFYT WTRSNPDYFY TWTRSNPDYF YTWTRSNPDY 30300
FYTWTRSNPD YFYTWTRSNP DYFYTWTRFS VAEILPGAKN VVLDTTALSA NTKVGTIITG 30360
DPLDPPVLKY PAEVFLPGGT YQLGKADKET DIGSAIEKAS AIQLDGIIYR QLSGHVGPLT 30420
SSSSKETDIG SAIEKADKET DIGSAIEKQL SGHVGPLTSS SSKQLSGHVG PLTSSSSKIY 30480
SFFVGGAVPE NLRQTSSEQN PSLEEIQAAQ ATVLPHSPVS NVKSAGEYNT FSPEWPVPLT 30540
KTFDENDTYE IGNKSIDQFF NDAKVPTVLM SPWVGKDGQE ATFHFDRVDW SPSFRAAEVI 30600
NYYTPDHVPV FNAMSIDQFF NDAKNAFITN YPSEQRYDTA TFIDKRIDAT TNPGMRQLGL 30660
AMLGNKNMHD VIGNDGTVPS EFRIDATTNP GMRIDATTNP GMRNAFITNY PSEQRNAFIT 30720
NYPSEQRQLG LAMLGNKAQI TAVNLEARGD GGGGPTFEHL EKSMDNDNTS LLVFGKMGES 30780
VDDFFARAQI TAVNLEARMG ESVDDFFARG IVSGEGEESS DPVKVDNVVA SFKYMQQLLD 30840
QTKVVWQDSV RAQNDPNAFG VVAARDPNAF GVVAARNDPN AFGVVAARAA TYCPENIEKA 30900
QNDPNAFGVV AARAQNDPNA FGVVAARTIF GWDIAEGQKS AGYTPLKVNG VEYGETRSAG 30960
YTPLKVNGVE YGETRSAGYT PLKVNGVEYG ETRGGSILPM QEVALTTRWA SVIDATKKAT 31020
GDVLFNTKHT ADGAWAKGGS ILPMQEVALT TRGGSILPMQ EVALTTRWAS VIDATKDGLE 31080
GSFKWDNLDS AALNTKVEDG NLILTMPKVE DGNLILTMPK VEDGNLILTM PKWDNLDSAA 31140
LNTKWDNLDS AALNTKSAIS QYGDSFAKSQ SDFESEFSTA KVNAGIGIGP DDLVSFIKSA 31200
ISQYGDSFAK SQSDFESEFS TAKGFNIVVA PGLDGRHVDV PLTGEDEITI LAIHDEVSPV 31260
GDTDALLERA PVVQYALNRY LVDQLNPEGK AIHDEVSPVG DTDALLERAI HDEVSPVGDT 31320
DALLERAPVV QYALNRAPVV QYALNRDAEL TDAGVKQAQV AHDFWQKLNT GAVIPVLVRL 31380
GDLSANPIER VLPQVIEATN RIYVTGQSYA GRLGDLSANP IERVLPQVIE ATNRVLPQVI 31440
EATNRNDPVA VFDGSVIPKE AGLVPFQVSP TTKTLGIDIA RGQTPLPILV ADGRTSTTLP 31500
EVCSKNNILE GPDVKGQTPL PILVADGRIN TAAYWKEAIA DVLEHLGEND EDIAVYAPNP 31560
FYKNSILEGP DVKMPMPILV ADGRMPMPIL VADGRNDDDF LNYGISSKSP VTSEYTSVRS 31620
IFEAANEKAI NDYIDSQLDK YLTNSQALAD LPYFAEKGVQ ISTNIPKGVQ ISTNIPKEII 31680
STYSIDGLRS VYQTMTDRYN TDAELYKGGS ELGFRSAADG LASAITSKSG DDISTTDALA 31740
LPEPVQALTK AGSSPTDIIS GISDKTDALD SAIKYDYENV DSDGANKYNL SNGAPAPETV 31800
TNKSMPTSGA VDLVAKCSSH DDCSDELACT DGVCACTADS AVTCSWEGHC AGAKGDNPSI 31860
LGLRCTADSA VTCSWEGHCA GAKDSPNWDV DSDALPAIPE GAKTLADFDA LKTLADFDAL 31920
KINPGPLARL YPDDNLAFIQ AGISDEKLYT GEVFKTDEGK GQEPPAAIVE VQKVAIIDGL 31980
ADPWRSVNIV NYTPSDSYTY SDNSGSWQSV KVTTGGQGAE FTLAKDQTTW SVDGNVVRVT 32040
TGGQGAEFTL AKVTTGGQGA EFTLAKAGQF PISANDGATS TKEAASAALA AGYKQTYTSC 32100
NPLKKTLNYA DALDGENYPQ TPSRVAVAGY DDTTGGVGPL LAQKINPSSG LLEPQTPLAV 32160
SPGSGPRVAV AGYDDTTGGV GPLLAQKTTG AFDESGPPLS QKNALQTMYD TQDKAALDAL 32220
QQSIYLQPKF SDGNGLFYQY ERLGAEVVTA GRIYAVADTQ ERLINQVELS EDKAVITDIV 32280
NQQRAVITDI VNQQRGENIL SAPLITYAPA GPELDEKELG FTAVGGEGKD IQYLENYQGQ 32340
GYSGPAVKIL QYAQGRDYSN NWESGALKDD YMPFIEVPRE AAEIDSHWER TIPIDNDVDY 32400
VVTGYRVYWV DSGPRTGDGV NDAPSLKDGQ EQEILARADA IVAAIRSALI AFEKNINMLL 32460
YGTDDCSGKG MVFSIDAQGE KTGTDQASVG YYKDAVYALD AIYGIDARDN ILPENLDDGL 32520
PSQFVYEKIL VENLQDQTAK ILVENLQDQT AKAEPYVTGS SAASGSNFVA DFAEAGTDGK 32580
QISYWAFTTP AVKFEPPAVY NDELKDAEEE PYDWSNEGRL SDELEDDNAP IGFETTKDQE 32640
MAVAAFRTSD DFASQMDGRC MGCDSTSIDV SRCMGCDSTS IDVSRDASGG DQITEWQDIY 32700
LPPITKGIQD AGVIATAKGV GSDAWTVSES GRGIDGDKGL VVKDLYGNIV MSGGSTLYPG 32760
IADRAGFAGD DAPRQEYDES GPSIVHRSYE LPDGQVITIG NERSYELPDG QVITIGNERL 32820
SGGVAVIKTT AVLFDEGKIG GSAGTELQSD KDLALVDPGL ELSYNTKLVG GSDFGEDEAK 32880
TLSTNEEGYE TSAVRASYGA GVTIQDRSAY VVYDLSNNEI SLANTKVPYL IGANTDEGTS 32940
FAIRLPVEAF QALASSTSET KDAGNAATND PLFPFSRAAL PGTEVLFADS VAKVTSAQYY 33000
VNPKIQAFVI YPENFDKVGA GVNVGELYAF ADKALTQYSV KTTYNVVAQT KTYANLPQAL 33060
VNSGAIKVIP LQGCDADEYG RDNIQGITKP AIRLANAYTW EGGRYQGASQ CPFRTMGVGY 33120
ATNDDSTIRC ASDGSAETCS WEGHCKCASD GSAETCSWEG HCKLILPGEL AK 33172

Claims (9)

1. An agricultural product aflatoxin molecule early warning method is characterized in that: determining the concentration of aflatoxin early warning molecules in an agricultural product sample: when the concentration of the detected aflatoxin early-warning molecules is less than 0.2 mug/g, the aflatoxin pollution level is below 1.0 mug/kg, and the risk is low; when the concentration of the detected aflatoxin early-warning molecules is more than 1.2 mug/g, the aflatoxin pollution level is more than 10 mug/kg, and the risk is high; when the concentration of the detected aflatoxin early-warning molecule is 0.2-1.2 mug/g, the aflatoxin early-warning molecule is a AFT-YJFZ01 peptide, and the amino acid sequence is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein: the method for quantitatively detecting the concentration of AFT-YJFZ01 in an agricultural product sample by adopting an aflatoxin early warning molecule AFT-YJFZ01 immune rapid detection method comprises the following steps:
preparing an agricultural product sample to be detected, namely adding the agricultural product sample to be detected into an enzyme-labeled plate hole of which the bottom is coated with a nano antibody or a monoclonal antibody of aflatoxin early-warning molecule AFT-YJFZ01, reacting, and washing the plate;
adding aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody for reaction, and washing the plate;
adding a horseradish peroxidase labeled antibody which is combined with an aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody, reacting, and washing a plate;
adding a color development liquid for reaction;
and adding a stop solution, and reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by using an enzyme-labeled instrument.
3. The method of claim 2, wherein: 100-200 mu L of each hole is added into an enzyme-labeled plate hole of a nano antibody or a monoclonal antibody of which the bottom is coated with aflatoxin early-warning molecules AFT-YJFZ01, the solution after reaction is removed after the reaction is placed at room temperature or 37 ℃ for no less than 1h, the enzyme-labeled plate is washed, then the AFT-YJFZ01 rabbit-derived polyclonal antibody is added, then 100-200 mu L of each hole is added, the solution is removed after the reaction is placed at room temperature or 37 ℃ for no less than 1h, the enzyme-labeled plate is washed, then 100-200 mu L of horseradish peroxidase-labeled goat anti-rabbit antibody is added into each hole, the solution is removed after the reaction is placed at room temperature or 37 ℃ for no less than 1h, the enzyme-labeled plate is washed, then the chromogenic solution and the stop solution are sequentially added, and finally the concentration of the AFT-YJFZ01 in a sample to be detected is read and calculated through an enzyme-labeled instrument.
4. The method of claim 2, wherein: and replacing the solution to be measured with a serial concentration of an early warning molecule AFT-YJFZ01 pure product solution serving as a standard substance, and preparing a standard curve, so as to calculate and obtain the concentration of AFT-YJFZ01 in the sample to be measured.
5. The method of claim 2, wherein: the method for preparing the agricultural product sample to be measured liquid comprises the following steps: weighing an agricultural product sample to be measured, crushing, transferring to a sample diluent, homogenizing by a conventional method, standing, and taking the supernatant to prepare a sample to be measured.
6. The method of claim 5, wherein: the sample diluent is 0.01mol/L phosphate buffer solution containing 0.1% sorbitol and soft sugar, and the preparation method comprises the following steps: sorbitol and soft sugar 0.5. 0.5g each, naCl 4.0. 4.0g, na 2 HPO 4 ·12H 2 O 1.45 g、KCl 0.1g、KH 2 PO 4 0.1 And g, adding deionized water to a volume of 500mL.
7. The method of claim 2, wherein: the hole bottom is coated with a nano antibody or a monoclonal antibody of aflatoxin early-warning molecule AFT-YJFZ01, and the preparation method comprises the following steps: dissolving the nanometer antibody or monoclonal antibody of AFT-YJFZ01 in ELISA coating buffer solution to form coating solution of 0.2-8.0 mug/mL, adding the coating solution into an ELISA plate, standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2h, removing the coating solution in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; then adding ELISA routine blocking solution, standing at room temperature or 37 ℃ to block at least 1h, discarding the blocking solution, and washing the ELISA routine washing solution.
8. The method of claim 2, wherein: the color developing solution refers to conventional hydrogen peroxide and TMB color developing solution for ELISA.
9. The method of claim 2, wherein: the stop solution is a conventional chromogenic stop solution of ELISA, namely 2mol/L sulfuric acid aqueous solution.
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