CN113607959A - Aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection kit and application - Google Patents

Aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection kit and application Download PDF

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CN113607959A
CN113607959A CN202110594331.2A CN202110594331A CN113607959A CN 113607959 A CN113607959 A CN 113607959A CN 202110594331 A CN202110594331 A CN 202110594331A CN 113607959 A CN113607959 A CN 113607959A
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yjfz01
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张奇
李培武
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 immune rapid detection kit for aflatoxin toxigenic bacteria virulence indicator molecules. The kit comprises an ELISA plate, a polyclonal antibody, a pure solution of aflatoxin toxigenic indicator molecules, a Chao's culture medium, a horse radish peroxidase labeled antibody, an ELISA developing solution, a stop solution and a sample diluent, wherein the bottom of the ELISA plate is coated with a nano antibody or a monoclonal antibody of the aflatoxin toxigenic indicator molecules; the aflatoxin toxigenic bacteria virulence indicator molecule is AFT-YJFZ01 peptide, and the amino acid sequence of the aflatoxin toxigenic bacteria virulence indicator molecule is shown in SEQ ID NO. 1. The kit can be used for quickly and quantitatively detecting the aflatoxin toxigenicity-producing indicator molecule AFT-YJFZ01 of the aflatoxin toxigenicity-producing strain, can be used for identifying and comparing the aflatoxin-producing capacity of the aflatoxin toxigenicity-producing strains of aspergillus, and is easy to operate, strong in practicability and easy to popularize and apply.

Description

Aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection kit and application
Technical Field
The invention relates to an immune rapid detection kit for aflatoxin toxigenic bacteria virulence indicator molecules and application thereof.
Background
Aflatoxin has strong toxicity and great harm, is a pollutant which pollutes most foods, generally presents a pollution aggravation 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. However, no molecular early warning research paradigm before aflatoxin and other microbial toxins are polluted exists at home and abroad so far, and the urgent need of early warning is difficult to meet.
The existing aflatoxin early warning method is mainly established based on an aflatoxin detection technology and is used for toxin pollution level evaluation or postpartum pollution degree and consumption risk evaluation, once detection and discovery are carried out, pollution often occurs, and urgent requirements of early warning in advance and guidance and prevention and control are difficult to meet. The aflatoxin detection technology is used for carrying out pollution early warning, and is a successful experience for carrying out food safety supervision in developed countries such as Europe and America. A Rapid early warning System (Rapid Alert System for Food and Feed, RASFF) of European Union is used for obtaining the aflatoxin content in Food and Feed by using limited standards and detection, and rapidly early warning the Food and Feed input into the European Union from each country. The American research institution establishes early warning models such as multivariate Rogeridi regression analysis and superposition Gaussian processing based on aflatoxin detection technology and pollution monitoring data, and is mainly used for evaluating mycotoxin pollution degree and consumption risk of agricultural products such as postpartum corns.
By integrating the research progress of nearly two decades at home and abroad, the lack of the early warning molecular detection technology of the aflatoxin at present is the fundamental reason. Aiming at the bottleneck problem, an inventor group successfully discovers an aflatoxin toxigenicity-producing indicator molecule and an early warning function thereof through more than ten years of attack and closure research, and the aflatoxin toxigenicity-producing indicator molecule is a first early warning molecule.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an immune rapid detection kit for aflatoxin toxigenic bacteria virulence-producing indicator molecules and application thereof, which are used for identifying the aflatoxin-producing capability of aspergillus strains and identifying whether aflatoxin toxigenic strains with strong virulence are present in products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like, and are easy to popularize and apply.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
providing an aflatoxin toxigenic indicator molecule immune rapid detection kit, which comprises an enzyme label plate, a polyclonal antibody, a pure solution of aflatoxin toxigenic indicator molecule AFT-YJFZ01 serving as a standard substance, a Zernike culture medium or other culture media suitable for growth of aflatoxin, an ELISA peroxidase labeled antibody, a developing solution, a stop solution and a sample diluent, wherein the hole bottom of the enzyme label plate is coated with a nano antibody or a monoclonal antibody of the aflatoxin toxigenic indicator molecule AFT-YJFZ 01;
the aflatoxin toxigenic bacteria virulence indicator molecule is AFT-YJFZ01 peptide, and the amino acid sequence of the aflatoxin toxigenic bacteria virulence indicator molecule is shown in SEQ ID NO. 1.
According to the scheme, the elisa plate with the hole bottom coated with the nano antibody or the monoclonal antibody of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 is prepared by the following method: dissolving the nano antibody or monoclonal antibody of AFT-YJFZ01 in ELISA coating buffer solution to form 0.2-8.0 mu g/mL coating solution, adding the coating solution into an ELISA plate (with 100-200 mu L/hole), standing overnight at 4 ℃ or standing for not less than 2h at 37 ℃, removing the coating solution in the ELISA plate, and washing the ELISA plate by using ELISA conventional washing solution; then adding ELISA conventional blocking solution (300 mu L per well), standing at room temperature or 37 ℃ for blocking for not less than 1h, then removing the blocking solution, and then washing the ELISA plate by using ELISA conventional washing solution.
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.
In the scheme, the polyclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 is a rabbit-derived polyclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01, and the horseradish peroxidase labeled antibody is a horseradish peroxidase labeled goat anti-rabbit antibody.
In the scheme, the polyclonal antibody of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 is different from the animal source of the nano antibody or the monoclonal antibody of the aflatoxin toxigenic indicator molecule AFT-YJFZ01, and the obtained aflatoxin toxigenic indicator molecule can be directly used as an antigen to prepare and obtain the nano antibody or the monoclonal antibody and the rabbit source polyclonal antibody;
the aflatoxin toxigenic bacteria can produce nano antibodies or monoclonal antibodies of virulence indicator molecules AFT-YJFZ01, AFT-YJFZ01 can be used as immune antigens, alpaca or Balb/c mice can be immunized in a conventional mode, and the nano antibodies or the mouse-derived monoclonal antibodies can be prepared by using the known conventional preparation technical scheme.
The rabbit polyclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 can be prepared by using AFT-YJFZ01 as an immune antigen, immunizing test rabbits such as New Zealand white rabbits in a conventional manner and the like, and then using a known conventional rabbit polyclonal antibody preparation technical scheme.
The Chaudhur culture medium is a conventional Chaudhur culture medium, can be prepared by self, and can also be obtained by directly purchasing commodities.
The horseradish peroxidase-labeled goat anti-rabbit antibody can be directly purchased from commercial products.
According to the scheme, the ELISA color developing solution refers to ELISA conventional hydrogen peroxide and TMB color developing solution.
The stop solution is ELISA conventional chromogenic stop solution: the preparation method of the 2mol/L sulfuric acid aqueous solution 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 sample diluent is 0.01mol/L phosphate buffer solution containing 0.1% of sorbitol and raffinose, and the preparation method comprises the following steps: sorbitol and sugar 0.5g, NaCL 4.0g, Na2HPO4·12H2O 1.45g、KCL 0.1g、KH2PO40.1g, deionized water is added to make the volume to 500 mL.
The use method of the aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection kit is provided:
culturing by using a culture medium suitable for growth of the toxigenic aspergillus flavus in a Chao's culture medium in the kit and preparing a strain to be identified or a liquid to be detected of a sample to be identified;
diluting a solution to be detected of a strain to be identified by using a sample diluent, adding the diluted solution to be detected of the strain to be identified or the solution to be detected of the sample to be identified into a hole of an ELISA plate in a kit by 100-200 mu L per hole, placing the hole at room temperature or 37 ℃ for reaction for not less than 1h, removing the liquid after the reaction, and washing the ELISA plate;
then adding AFT-YJFZ01 polyclonal antibody in the kit, adding 100-200 mu L of antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, and washing the ELISA plate;
then adding 200 mu L of horseradish peroxidase labeled antibody 100-,
and then adding the ELISA developing solution and the stop solution in the kit in sequence, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-linked immunosorbent assay (ELISA) reader.
According to the scheme, the method further comprises the following steps: and (3) evaluation of a detection result: according to the result of the enzyme-labeling instrument, the content of aflatoxin virulence indicator molecules AFT-YJFZ01 produced by aflatoxin virulence producing bacteria in unit volume of the aflatoxin solution to be detected is obtained, and the virulence producing capacity of aflatoxin of the strain to be identified is analyzed. If the concentration of aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 in the strain to be identified is higher, the aflatoxin production capacity of the strain to be identified, namely the virulence is stronger;
according to the result of the enzyme-labeling instrument, the content of aflatoxin toxigenic indicator AFT-YJFZ01 in unit volume of sample liquid to be identified is obtained, and whether the sample to be identified contains a strong toxigenic strain of aflatoxin or not is judged. If the aflatoxin toxigenic bacteria in the sample to be identified has high concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01, the sample to be identified contains the aflatoxin-producing virulent strain.
According to the scheme, aflatoxin toxigenic bacteria with series concentrations and functions as standard substances are used for producing pure solutions (each hole is 100-200 mu L) of virulence indicator molecules AFT-YJFZ01 to replace the liquid to be detected, the liquid to be detected is used for making a standard curve, and the concentration of AFT-YJFZ01 in the sample to be detected is obtained through calculation.
According to the scheme, the preparation of a solution to be detected of the strain to be identified comprises the following steps: culturing the strain to be identified to obtain a solution to be detected of the strain to be identified; the method specifically comprises the following steps: culturing the strain to be identified in a conventional Chaudou culture medium or other culture media suitable for the growth of the strain, wherein the culture environment temperature is 15-35 ℃, and the culture time is not less than 12 hours, then fully homogenizing the mixture of the culture medium and the culture, and diluting by 1-10 times with sterile water to obtain a solution to be detected of the strain to be identified;
preparing the sample solution to be identified: culturing and diluting a sample to be identified to obtain a solution to be detected of the sample to be identified;
the method specifically comprises the following steps: weighing a sample to be identified, transferring the sample to a sample diluent, shaking the sample to be identified to be uniform at room temperature to prepare a uniform dispersion liquid of the sample to be detected, adding 10-1000 mu L of the uniform dispersion liquid of the sample to a conventional Homex culture medium containing 6-600mL or other culture media suitable for growth of the virus-producing aspergillus flavus, placing the mixture in a shaking culture at 200 +/-50 rpm at 15-35 ℃, and sampling the mixture after culturing for 6-24h to form the liquid to be detected of the sample to be identified.
According to the scheme, the samples are products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like.
The kit can be used for identifying the capability of producing aflatoxin by the aspergillus strain, and the higher the content of aflatoxin toxigenic bacteria virulence indicator molecules in the strain to be identified is, the stronger the capability of producing aflatoxin by the strain to be identified, namely virulence productivity is; the kit can be used for identifying whether aflatoxin virulence producing strains with strong virulence exist in products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like, and if the aflatoxin virulence producing indicator molecules in a sample to be identified are high in content, the sample to be identified contains the aflatoxin virulence producing strains;
the strain virulence of the aspergillus flavus is an index for measuring the capability of the strain to produce the aflatoxin, and the stronger the strain virulence, the more the strain can produce the aflatoxin in the same time and under the same culture conditions. The invention utilizes aflatoxin toxigenic bacteria obtained by research to produce virulence indicator molecules, combines nano antibodies or monoclonal antibodies of aflatoxin toxigenic bacteria virulence indicator molecules AFT-YJFZ01 and rabbit polyclonal antibodies, constructs a sandwich immunoassay method by the antibodies, and finally assembles a kit; in the application of the kit, the characteristic that the aflatoxin toxin-producing bacteria virulence indicator molecule is used for determining the concentration and the strain virulence is in positive correlation is utilized, so that the kit is used for identifying the aflatoxin-producing capacity of aspergillus strains and identifying whether aflatoxin toxin-producing strains with strong virulence exist in farmlands, agricultural products and feeds, is practical and easy to popularize, provides key tongs and scientific basis for timely discovering aflatoxin pollution risk and early prevention and control, and has important significance for promoting high-quality development of agricultural industry and ensuring food safety.
The invention has the beneficial effects that:
1. the kit can be used for quickly and quantitatively detecting the indicator molecule AFT-YJFZ01 for the virulence production of the aflatoxin toxin-producing bacteria, 2 can be used for identifying and comparing the capability of producing the aflatoxin by the aflatoxin toxin-producing strains in the aspergillus, 3 can be used for identifying whether the aflatoxin toxin-producing strains with strong virulence exist in farmlands, agricultural products and feeds, 4 is easy to operate, strong in practicability and easy to popularize and apply, and 5 has important significance for promoting the high-quality development of the agricultural industry and guaranteeing the food safety.
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FIG. 1 is a standard curve of an aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 immune rapid detection method.
Detailed Description
Example 1 preparation of Aflatoxin toxigenicity-producing indicator molecule AFT-YJFZ01 of Aflatoxin toxigenicity-producing bacteria capable of functioning as standard substance in kit
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, virulence and infection research in typical peanut producing areas in China-Master thesis of Chinese academy of agricultural sciences, Zhang apricot, page 33-published toxigenic strains HLJ-1, HeNZY-2, HuBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-122-2, JSnnt-1, HuNdx-7, HBHA-8-17 and the like, respectively inoculating the strains into the Cnahs culture medium, culturing for 5 days at 28 ℃ and 200rpm/min, fully homogenizing by a conventional method, crushing cells, purifying by a conventional protein purification system, protein electrophoresis, immunoaffinity and the like to obtain the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ 01. The test results show that AFT-YJFZ01 can be prepared from the toxigenic strain culture, the maximum amount of AFT-YJFZ01 can be prepared from HBHA-8-17 under the same culture conditions, and the minimum amount of AFT-YJFZ01 can be prepared from HLJ-1.
The immunoaffinity method is characterized in that a sample solution is used for diluting aflatoxin toxigenic bacteria cell crushing solution, the aflatoxin toxigenic bacteria cell crushing solution is continuously added into an immunoaffinity column after being filtered by conventional filter paper, when the aflatoxin toxigenic bacteria cell crushing solution is basically drained, the column is washed by conventional eluent of the immunoaffinity column, finally, the column is eluted by glycine buffer solution with the pH of 2.2 or 70% methanol water solution, the solution is timely removed by a conventional ultrafiltration centrifugal method after the eluent is collected, and then, the protein remained in an ultrafiltration centrifugal tube is dissolved out from the ultrafiltration centrifugal tube by sterile water, so that the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 water solution can be obtained.
The discovery method for the initial acquisition of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 is as follows:
the method for discovering the aspergillus flavus strain to produce virulence indicator molecule AFT-YJFZ01 is as follows:
(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 solutions 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 the conventional WesternBlot 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.
Example 2 preparation of Nanobody of Aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01
AFT-YJFZ01 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.
Dissolving AFT-YJFZ01 obtained by the preparation in conventional PBS buffer solution or physiological saline until the concentration is not lower than 0.1mg/mL, mixing and emulsifying the solution with Freund's complete adjuvant in equal volume, immunizing alpaca by back subcutaneous or intradermal multipoint injection, then boosting the immunity for 1 time every 2-4 weeks, and replacing Freund's complete adjuvant with Freund's incomplete adjuvant during boosting. 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 pCANTAB 5E (his) vector double enzyme digestion treatment, connection product electrotransformation, construction of a nano antibody gene bank, rescue of the nano antibody gene bank and the like on the immune alpaca according to a method of patent document CN103866401A, and finally obtaining the rescued nano antibody gene bank.
Fixing AFT-YJFZ01 obtained by the preparation on solid phase carriers such as a 96-hole enzyme label plate according to gradients of 8 mu g/hole, 2 mu g/hole, 0.5 mu g/hole and 0.1 mu g/hole, carrying out elutriation on the rescued nano antibody gene library for 2-4 times according to a method of patent document CN103866401A, identifying the antibody generated by each phage clone by AFT-YJFZ01 and indirect non-competitive ELISA, wherein the phage corresponding to a positive result is a phage positive clone, and preparing a nano antibody, namely the nano antibody of AFT-YJFZ01, by a conventional method for preparing the nano antibody through the nano antibody, wherein the nano antibody is used for further application and research work, preferably characterized by the ELISA method and has strong specificity and high affinity.
Example 3 preparation of monoclonal antibody against Aflatoxin toxigenic bacteria producing virulence indicator molecule AFT-YJFZ01
AFT-YJFZ01 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.
Dissolving AFT-YJFZ01 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's complete adjuvant in an equal 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's complete adjuvant with Freund's 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 splenocytes of the immunized mouse, fusing the splenocytes with murine myeloma cells SP2/0, and selectively culturing hybridoma cells by using a semisolid culture medium by the method of reference 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-YJFZ01 obtained by the preparation is fixed on a solid phase carrier such as a 96-well enzyme label 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, and a positive clone is selected to obtain the AFT-YJFZ01 monoclonal antibody which is used for further application and research work, preferably the AFT-YJFZ01 monoclonal antibody which has the characteristics of strong specificity and high affinity.
Example 4 preparation of Rabbit-derived polyclonal antibody of Aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01
AFT-YJFZ01 is used as an immune antigen, a test rabbit such as a New Zealand white rabbit 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-YJFZ01 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. And (3) monitoring the immune effect by adopting a conventional ELISA process, and preparing the serum of the immune animal by a conventional method after the serum titer of the immune animal is not increased any more, namely the rabbit-derived polyclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ 01.
Example 5 establishment of immune Rapid detection method for Aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01
The basic operation program of the immune rapid detection method of AFT-YJFZ01, namely the double-antibody sandwich indirect non-competitive ELISA method: coating AFT-YJFZ01 nano antibody in an enzyme label plate, and washing the plate; adding sealing liquid for sealing, and washing the plate; adding AFT-YJFZ01 or a solution to be detected for reaction, and washing the plate; adding AFT-YJFZ01 rabbit source polyclonal antibody for reaction, and washing the plate; adding horse radish peroxidase labeled goat anti-rabbit antibody 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 the optimal concentration of antibody: 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-derived polyclonal antibody is set to have different concentrations, and finally, the optimal working concentrations of the nano-antibodies and the rabbit-derived polyclonal antibody are 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, and ELISA result researches show that although other concentrations of the nano-antibodies and the rabbit-derived polyclonal antibody can also realize detection, the optimal coating concentration of the nano-antibodies is 2.0 mug/mL, and the optimal concentration of the rabbit-derived polyclonal antibody is 3.0 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 the research result shows that although the three sealing agents can achieve the sealing purpose 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 numerical 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 positive sample OD450nm value 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 ℃.
And (3) drawing an ELISA standard curve of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ 01: AFT-YJFZ01 molecules were diluted to 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300ng/mL, and 200. mu.L/well, and ELISA standard curves for AFT-YJFZ01 were prepared under the optimal conditions as described above, and the results are shown in FIG. 1. The correlation coefficient of the double-antibody sandwich enzyme-linked immunosorbent assay method established under the optimal optimization condition reaches 0.9980, and the detection limit of the double-antibody sandwich enzyme-linked immunosorbent assay method on AFT-YJFZ01 molecules reaches 0.1ng/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-YJFZ01, 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-YJFJF 01 immune rapid detection method has good specificity.
TABLE 1 Aflatoxin toxigenic bacteria virulence indicator molecule immunity rapid detection method specificity determination result
Figure RE-GDA0003292174000000091
Method repeatability evaluation: in order to evaluate the repeatability of the established aflatoxin toxigenicity indicator molecule AFT-YJFZ01 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 intra-plate and inter-plate data variation of the measurement result is analyzed. The results of the above studies are shown in tables 2 and 3, and the calculated coefficient of variation in the plate is 0.5% to 3.5%, and the coefficient of variation between plates is 0.9% to 5.7%, both of which are below 7%, so that the above method has good repeatability.
TABLE 2 in-board repeatability determination results of the fast detection method of virulence indicator AFT-YJFZ01 immunity
Figure RE-GDA0003292174000000092
Figure RE-GDA0003292174000000101
TABLE 3 results of the repetitive determination between plates of the fast immunity detection method for producing virulence indicator molecules AFT-YJFZ01
Figure RE-GDA0003292174000000102
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, the aflatoxin toxigenicity indicator molecule AFT-YJFZ01 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 table 4, and the result shows that the established aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection method is used for detecting virulence indicator molecules AFT-YJFZ01 in peanuts and corns, the recovery rate 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 Aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection method additive recovery test results
Figure RE-GDA0003292174000000103
Figure RE-GDA0003292174000000111
Example 6 composition and shelf life of Aflatoxin toxigenic bacteria virulence indicator AFT-YJFZ01 immune rapid detection kit
The aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 immune rapid detection kit mainly comprises an enzyme label plate, a rabbit source polyclonal antibody, an aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 pure solution, a culture medium, a horse radish peroxidase-labeled goat anti-rabbit antibody, an ELISA developing solution, a stop solution and a sample diluent, wherein the hole bottom of the enzyme label plate is coated with a nano antibody or a monoclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01, the rabbit source polyclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01, the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 pure solution plays a role of a standard substance, and the horse radish peroxidase-labeled goat anti-rabbit antibody is combined with the ELISA developing solution, the stop solution and the sample diluent.
The preparation method of the ELISA plate with the hole bottom coated with the nano antibody or the monoclonal antibody of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 comprises the following steps: dissolving the nano antibody or the monoclonal antibody of AFT-YJFZ01 in an ELISA (enzyme-linked immunosorbent assay) coating buffer solution carbonate solution to form a coating solution of 2 mu g/mL, adding the coating solution into a 96-hole ELISA plate at a concentration of 100 and 200 mu L/hole, standing overnight at 4 ℃ or standing for not less than 2 hours at 37 ℃, removing the coating solution in the ELISA plate, and washing the ELISA plate by using an ELISA conventional washing solution; then adding 200-300 mu L of ELISA conventional blocking liquid into each hole, placing the hole at room temperature or 37 ℃ for blocking for not less than 1h, then removing the blocking liquid, and washing the ELISA plate by using the ELISA conventional washing liquid to obtain the ELISA plate with the hole bottom coated with the nano antibody or the monoclonal antibody of the aflatoxin toxigenicity indicator molecule AFT-YJFZ 01.
The nano antibody or the monoclonal antibody of the aflatoxin toxigenicity indicator molecule AFT-YJFZ01 can be prepared by the methods of example 2 and example 3.
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 rabbit-derived polyclonal antibody of the aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 can be prepared and obtained by the method of example 4.
The Chaudhur culture medium is a conventional Chaudhur culture medium, can be prepared by self, and can also be obtained by directly purchasing commodities.
The horseradish peroxidase-labeled goat anti-rabbit antibody can be directly purchased from commercial products.
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 sample diluent is 0.01mol/L phosphate buffer solution containing 0.1% of sorbitol and sugar, and the preparation method comprises the following steps: sorbitol and sugar 0.5g, NaCL 4.0g, Na2HPO4·12H2O 1.45g、KCL 0.1g、KH2PO40.1g, deionized water is added to make the volume to 500 mL.
The shelf life research result of the kit shows that the kit can be stored for at least more than 6 months at 4 ℃.
Example 7 Aflatoxin toxigenicity-producing indicator molecule AFT-YJFZ01 immune rapid detection kit for identifying and comparing aflatoxin-producing abilities of strains
Step one, preparing a solution to be detected of a strain to be identified: according to the strong and weak strain virulence, selecting 10 strains of toxin-producing strains HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, Jcnt-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 and the like which are published in the publication of China typical peanut producing area Aspergillus flavus distribution, virulence producing capability and infection research, page 33, according to the study on the Master of Chinese academy of agriculture, culturing the strains to be identified in a Czochralski culture medium in the kit at the culture environment temperature of 15-35 ℃ for not less than 12h, fully homogenizing a mixture of the culture medium and the culture, and diluting the mixture by 1-10 times with a sample diluent of the kit to obtain a solution to be detected of the strains to be identified.
Secondly, measuring the liquid to be measured of the strain to be identified: the liquid to be detected is added into the hole of the enzyme label plate of the kit by 100-, or adding 100-200 muL of the solution of the virulence indicator molecule AFT-YJFZ01 with the series of concentrations in the kit into each hole, placing the mixture at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid after the reaction, washing an enzyme label plate, then adding AFT-YJFZ01 rabbit polyclonal antibody in the kit, adding 100-200 muL of rabbit polyclonal antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme label plate with the liquid, then adding 200 mu L of horse radish peroxidase labeled goat anti-rabbit antibody 100-, and then adding the ELISA developing solution and the stop solution in the kit in sequence, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-linked immunosorbent assay (ELISA) reader.
According to the results of the above documents, the virulence producing capability of 10 strains such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, Jcnt-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 and the like are as follows: 0 mug/L, 3.8 mug/L, 4.9 mug/L, 67.2 mug/L, 81.7 mug/L, 192.0 mug/L, 204.4 mug/L, 297.4 mug/L and 1027.5 mug/L, which shows that HBZHX-21 and HBXY-36 are non-toxigenic strains, HBHA-1-4 and GDZJ-6 are weak-toxigenic strains, HBHA-8-17 and GDZJ-108-19 are strong-toxigenic strains.
Thirdly, judging the identification result: the higher the aflatoxin toxigenic bacteria in the strain to be identified produces the virulence indicator molecule AFT-YJFZ01, the stronger the aflatoxin production capability of the strain to be identified, namely the virulence. According to the kit and the application technical scheme thereof, the results of measuring the AFT-YJFZ01 concentration of 10 strains of bacteria such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, Jcnt-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 and the like are as follows in sequence: 0ng/mL, 8ng/mL, 12ng/mL, 49ng/mL, 51ng/mL, 104ng/mL, 115ng/mL, 175ng/mL, 536ng/mL, again indicating: HBZHX-21 and HBXY-36 are non-toxigenic strains, HBHA-1-4 and GDZJ-6 are weak toxigenic strains, and HBHA-8-17 and GDZJ-108-19 are strong toxigenic strains. The determination result is consistent with the strain virulence generating sequence published in the published literature, i.e., the distribution, virulence generating capacity and infection research of aspergillus flavus in typical peanut producing areas in China, and the identification results of strains with strong virulence generating capacity and strains with weak virulence generating capacity are consistent with the literature.
Example 8 identification of whether a highly virulent strain containing aflatoxin is present in a farm field by using aflatoxin toxigenic bacteria virulence indicator AFT-YJFZ01 immune rapid detection kit
Step one, preparing a to-be-detected liquid of a to-be-identified sample: selecting 4 parts of peanut flowering phase rhizosphere soil samples such as Jilin, Liaoning, Jiangxi, Fujian and the like, and sequentially naming the samples as a soil sample-1, a soil sample-2, a soil sample-3 and a soil sample-4. Sequentially weighing farmland soil samples to be detected, uniformly crushing, transferring into the sample diluent of the kit, wherein the concentration is 0.5g/mL, and vibrating at room temperature until the sample diluent is uniform to prepare a uniform dispersion liquid of the samples to be detected. And adding 50 mu L of the uniform dispersion liquid of the sample to be detected into 30mL of the Chao's medium of the kit, placing the mixture in a shaking culture at the temperature of 28 ℃ and at 200rpm, culturing for 24h, and then sampling to form the liquid to be detected of the sample to be identified.
Step two, the determination of the sample to be identified and the solution to be determined: the liquid to be detected is added into the hole of the enzyme label plate in the kit by 100-, or adding 100-200 muL of the solution of the virulence indicator molecule AFT-YJFZ01 with the series of concentrations in the kit into each hole, placing the mixture at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid after the reaction, washing an enzyme label plate, then adding AFT-YJFZ01 rabbit polyclonal antibody in the kit, adding 100-200 muL of rabbit polyclonal antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme label plate with the liquid, then adding 200 mu L of horse radish peroxidase labeled goat anti-rabbit antibody 100-, and then adding the ELISA developing solution and the stop solution in the kit in sequence, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-linked immunosorbent assay (ELISA) reader.
Thirdly, judging the identification result: if the aflatoxin toxigenic bacteria in the sample to be identified has high concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01, the sample to be identified contains the aflatoxin-producing virulent strain. According to the kit and the application technical scheme thereof, the results of measuring the concentration of AFT-YJFZ01 in the soil sample-1, the soil sample-2, the soil sample-3 and the soil sample-4 are as follows in sequence: 0.3ng/mL, 0.2ng/mL, 16ng/mL, 19 ng/mL. The results show that the soil sample-1 and the soil sample-2 both contain AFT-YJFZ01 in an amount of less than 1.0ng per milliliter of culture solution, do not contain aflatoxin-containing strains with strong virulence, and have low pollution risk after flower production in corresponding farmlands; the amount of AFT-YJFZ01 contained in each milliliter of culture solution in the soil sample-1 and the soil sample-2 is more than 10ng/mL, the aflatoxin-containing bacterial strain has strong virulence, and the pollution risk after the production of flowers in corresponding farmlands is higher.
Example 9 identification of Strong virulence-producing Strain containing Aflatoxin in agricultural products Using Aflatoxin toxigenic bacteria virulence-producing indicator molecule AFT-YJFZ01 ImmunoRapid detection kit
Step one, preparing a to-be-detected liquid of a to-be-identified sample: selecting 4 parts of agricultural product samples such as peanut, corn, rice, wheat and the like, sequentially weighing the farmland soil sample to be detected, crushing the farmland soil sample, transferring the crushed farmland soil sample to the sample diluent of the kit, wherein the concentration of the sample diluent is 0.5g/mL, and shaking the mixture to be uniform at room temperature to prepare a uniform dispersion liquid of the sample to be detected. And adding 100 mu L of the uniform dispersion liquid of the sample to be detected into 50mL of the Chao's medium containing the kit, placing the mixture in a shaking culture at the temperature of 28 ℃ and 200rpm, culturing for 6h, and then sampling to form the liquid to be detected of the sample to be identified.
Step two, the determination of the sample to be identified and the solution to be determined: the liquid to be detected is added into the hole of the enzyme label plate in the kit by 100-, or adding 100-200 muL of the solution of the virulence indicator molecule AFT-YJFZ01 with the series of concentrations in the kit into each hole, placing the mixture at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid after the reaction, washing an enzyme label plate, then adding AFT-YJFZ01 rabbit polyclonal antibody in the kit, adding 100-200 muL of rabbit polyclonal antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme label plate with the liquid, then adding 200 mu L of horse radish peroxidase labeled goat anti-rabbit antibody 100-, and then adding the ELISA developing solution and the stop solution in the kit in sequence, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-linked immunosorbent assay (ELISA) reader.
Thirdly, judging the identification result: if the aflatoxin toxigenic bacteria in the sample to be identified has high concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01, the sample to be identified contains the aflatoxin-producing virulent strain. According to the kit and the application technical scheme thereof, the measured AFT-YJFZ01 concentration results in peanut, corn, rice and wheat samples are as follows in sequence: 12ng/mL, 13ng/mL, 0.7ng/mL, 0 ng/mL. The results show that the culture solution of peanut and corn samples contains more than 10ng of AFT-YJFZ01 in each milliliter, and the peanut and corn samples contain aflatoxin-containing strains with strong virulence, so that the pollution risk is high; the amount of AFT-YJFZ01 contained in each milliliter of culture solution in the rice sample is below 1.0, the rice sample does not contain a strong virulence-producing strain of aflatoxin, and the pollution risk is very low; the measured wheat sample contains 0 amount of AFT-YJFZ01 in each milliliter of culture solution, does not contain aflatoxin toxigenic strains, and basically has no aflatoxin pollution risk.
Example 10 identification of Strong virulence-producing Strain containing Aflatoxin in agricultural products Using Aflatoxin toxigenic bacteria virulence-producing indicator molecule AFT-YJFZ01 ImmunoRapid detection kit
Step one, preparing a to-be-detected liquid of a to-be-identified sample: selecting 3 parts of traditional Chinese medicine samples such as ginseng, angelica, medlar and the like, sequentially weighing the traditional Chinese medicine samples to be detected, crushing the traditional Chinese medicine samples, transferring the crushed traditional Chinese medicine samples to a sample diluent of the kit, wherein the concentration of the sample diluent is 0.5g/mL, and shaking the sample diluent to be uniform at room temperature to prepare a uniform dispersion liquid of the samples to be detected. And (3) adding 50 mu L of the uniform dispersion liquid of the sample to be detected into 30mL of the Chaudou culture medium containing the kit, placing the mixture in a shaking culture at the temperature of 28 ℃ and at 200rpm, culturing for 6h, and then sampling to form the liquid to be detected of the sample to be identified.
Step two, the determination of the sample to be identified and the solution to be determined: the liquid to be detected is added into the hole of the enzyme label plate in the kit by 100-, or adding 100-200 muL of the solution of the virulence indicator molecule AFT-YJFZ01 with the series of concentrations in the kit into each hole, placing the mixture at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid after the reaction, washing an enzyme label plate, then adding AFT-YJFZ01 rabbit polyclonal antibody in the kit, adding 100-200 muL of rabbit polyclonal antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme label plate with the liquid, then adding 200 mu L of horse radish peroxidase labeled goat anti-rabbit antibody 100-, and then adding the ELISA developing solution and the stop solution in the kit in sequence, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-linked immunosorbent assay (ELISA) reader.
Thirdly, judging the identification result: if the aflatoxin toxigenic bacteria in the sample to be identified has high concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01, the sample to be identified contains the aflatoxin-producing virulent strain. According to the kit and the application technical scheme thereof, the measured AFT-YJFZ01 concentration results in traditional Chinese medicine samples such as ginseng, angelica, medlar and the like are as follows in sequence: 0.3ng/mL, 0 ng/mL. The result shows that the concentration of AFT-YJFZ01 in the measured ginseng sample is below 1.0, the ginseng sample does not contain aflatoxin-producing virulent strains, and the pollution risk is very low; the measured concentration of AFT-YJFZ01 in the angelica and medlar samples is 0, no aflatoxin toxigenic strain is contained, and the aflatoxin pollution risk is basically avoided.
Example 11 identification of whether a highly virulent strain producing aflatoxin is contained in a feed by using an aflatoxin toxigenic indicator molecule AFT-YJFZ01 immune rapid detection kit
Step one, preparing a to-be-detected liquid of a to-be-identified sample: selecting 4 parts of feed samples to be detected from the market, and sequentially naming the feed samples as feed-1, feed-2, feed-3 and feed-4. Sequentially weighing farmland soil samples to be detected, crushing the farmland soil samples, transferring the crushed farmland soil samples to the sample diluent of the kit, shaking the diluted farmland soil samples at room temperature until the concentration of the diluted farmland soil samples is 0.5g/mL, and preparing the uniform dispersion of the samples to be detected. And adding 100 mu L of the uniform dispersion liquid of the sample to be detected into 200mL of the Chao's medium containing the kit, placing the mixture in a shaking culture at the temperature of 28 ℃ and 200rpm, culturing for 24h, and then sampling to form the liquid to be detected of the sample to be identified.
Step two, the determination of the sample to be identified and the solution to be determined: the liquid to be detected is added into the hole of the enzyme label plate in the kit by 100-, or adding 100-200 muL of the solution of the virulence indicator molecule AFT-YJFZ01 with the series of concentrations in the kit into each hole, placing the mixture at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid after the reaction, washing an enzyme label plate, then adding AFT-YJFZ01 rabbit polyclonal antibody in the kit, adding 100-200 muL of rabbit polyclonal antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme label plate with the liquid, then adding 200 mu L of horse radish peroxidase labeled goat anti-rabbit antibody 100-, and then adding the ELISA developing solution and the stop solution in the kit in sequence, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-linked immunosorbent assay (ELISA) reader.
Thirdly, judging the identification result: if the aflatoxin toxigenic bacteria in the sample to be identified has high concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01, the sample to be identified contains the aflatoxin-producing virulent strain. According to the above-mentioned kit and its application technical scheme, the result of determining AFT-YJFZ01 concentration in feed-1, feed-2, feed-3 and feed-4 is as follows in turn: 0.6 ng/mL, 22ng/mL, 13ng/mL, 69 ng/mL. The result shows that the feed-1 sample contains AFT-YJFZ01 in an amount of less than 1.0ng/mL in each milliliter of culture solution, does not contain aflatoxin, and has low pollution risk; taking the example that 50 mul of sample uniform dispersion liquid is added into a culture medium containing 30mL, each milliliter of culture liquid in feed-2, feed-3 and feed-4 contains more than 10ng/mL of AFT-YJFZ01, and the strain containing aflatoxin has strong virulence producing strain, and has higher pollution risk after the production of flowers in corresponding farmlands.
< 110 > institute of oil crops of Chinese academy of agricultural sciences
Rapid detection kit for aflatoxin toxigenic bacteria virulence indicator molecule immunity and application
＜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 (10)

1. An immune rapid detection kit for aflatoxin toxigenic bacteria virulence indicator molecules is characterized in that: the kit comprises an enzyme label plate, a polyclonal antibody, an aflatoxin toxigenic indicator AFT-YJFZ01, a pure aflatoxin toxigenic indicator AFT-YJFZ01 solution, a Chao's medium or other medium suitable for growth of toxigenic aspergillus flavus, a horseradish peroxidase-labeled antibody which is in binding reaction with the aflatoxin toxigenic indicator AFT-YJFZ01 polyclonal antibody, an ELISA developing solution, a stop solution and a sample diluent, wherein the bottom of the enzyme label plate is coated with a nano antibody or a monoclonal antibody of the aflatoxin toxigenic indicator AFT-YJFZ 01;
the aflatoxin toxigenic bacteria virulence indicator molecule is AFT-YJFZ01 peptide, and the amino acid sequence of the aflatoxin toxigenic bacteria virulence indicator molecule is shown in SEQ ID NO. 1.
2. The aflatoxin toxigenic bacteria virulence indicator molecule immunoassay rapid detection kit of claim 1, which is characterized in that: the ELISA plate with the hole bottom coated with the nano antibody or the monoclonal antibody of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 is prepared by the following method: dissolving the nano antibody or monoclonal antibody of AFT-YJFZ01 in an ELISA coating buffer solution to form a coating solution of 0.2-8.0 mu g/mL, adding the coating solution into an ELISA plate, standing overnight at 4 ℃ or standing for not less than 2h at 37 ℃, removing the coating solution in the ELISA plate, and washing the ELISA plate by using an ELISA conventional washing solution; then adding ELISA conventional blocking solution, standing at room temperature or 37 deg.C for at least 1h, discarding the blocking solution, and washing the ELISA plate with ELISA conventional washing solution.
3. The aflatoxin toxigenic bacteria virulence indicator molecule immunoassay rapid detection kit of claim 1, which is characterized in that: the polyclonal antibody of aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01 is rabbit-derived polyclonal antibody of aflatoxin toxigenic bacteria virulence indicator molecule AFT-YJFZ01, and the horse radish peroxidase labeled antibody is horse radish peroxidase labeled goat anti-rabbit antibody.
4. The aflatoxin toxigenic bacteria virulence indicator molecule immunoassay rapid detection kit of claim 1, which is characterized in that: the ELISA developing solution refers to ELISA conventional hydrogen peroxide and TMB developing solution;
the stop solution is ELISA conventional chromogenic stop solution: 2mol/L sulfuric acid aqueous solution.
5. The aflatoxin toxigenic bacteria virulence indicator molecule immunoassay rapid detection kit of claim 1, which is characterized in that: the sample diluent is 0.01mol/L phosphate buffer containing 0.1% sorbitol and 0.1% sugar.
6. The use method of the aflatoxin toxigenic bacteria virulence indicator molecule immune rapid detection kit of claim 1, which comprises the following steps:
culturing by using a culture medium suitable for growth of the toxigenic aspergillus flavus in a Chao's culture medium in the kit and preparing a strain to be identified or a liquid to be detected of a sample to be identified;
diluting a solution to be detected of a strain to be identified by using a sample diluent, adding the diluted solution to be detected of the strain to be identified or the solution to be detected of the sample to be identified into a hole of an ELISA plate in a kit by 100-200 mu L per hole, placing the hole at room temperature or 37 ℃ for reaction for not less than 1h, removing the liquid after the reaction, and washing the ELISA plate;
then adding AFT-YJFZ01 polyclonal antibody in the kit, adding 100-200 mu L of antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, and washing the ELISA plate;
then adding 200 mu L of horseradish peroxidase labeled antibody 100-,
and then adding the ELISA developing solution and the stop solution in the kit in sequence, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-linked immunosorbent assay (ELISA) reader.
7. The method as claimed in claim 6, wherein the aflatoxin toxigenic bacteria with a series of concentrations, which act as standard substances, are used to produce pure solutions of virulence indicator molecules AFT-YJFZ01, to replace the liquid to be tested, and the solutions are used to make a standard curve, and the concentration of AFT-YJFZ01 in the sample to be tested is calculated.
8. The method of claim 6, further comprising: and (3) evaluation of a detection result: according to the result of an enzyme-labeling instrument, obtaining the content of aflatoxin toxigenic indicator molecules AFT-YJFZ01 in the aflatoxin solution to be detected in unit volume, analyzing the aflatoxin toxigenic property of the strain to be identified, wherein the higher the concentration of aflatoxin toxigenic indicator molecules AFT-YJFZ01 in the strain to be identified is, the stronger the aflatoxin production capability of the identification strain is, namely, the toxigenic property is;
according to the result of the enzyme-labeling instrument, the content of aflatoxin toxigenic indicator AFT-YJFZ01 in unit volume of sample liquid to be identified is obtained, and whether the sample to be identified contains a strong toxigenic strain of aflatoxin or not is judged.
9. The method according to claim 6, wherein the preparation of a test solution of the strain to be identified: culturing and diluting the strain to be identified to obtain a solution to be detected of the strain to be identified; the method specifically comprises the following steps: culturing the strain to be identified in a conventional Chaudou culture medium or other culture media suitable for the growth of the strain, wherein the culture environment temperature is 15-35 ℃, and the culture time is not less than 12 hours, then fully homogenizing the mixture of the culture medium and the culture, and diluting by 1-10 times with sterile water to obtain a solution to be detected of the strain to be identified; preparing a sample solution to be identified: culturing a sample to be identified to obtain a liquid to be detected of the sample to be identified;
the method specifically comprises the following steps: weighing a sample to be identified, transferring the sample to a sample diluent, shaking the sample to be detected uniformly at room temperature to prepare a uniform dispersion liquid of the sample to be detected, adding 10-1000 mu L of the uniform dispersion liquid of the sample to a conventional Homeji culture medium containing 6-600mL or other culture medium suitable for growth of the toxigenic aspergillus flavus, placing the mixture in a shaking culture at 200 +/-50 rpm at 15-35 ℃, and sampling the mixture after culturing for 6-24h to form the liquid to be detected of the sample to be identified.
10. The method of claim 6, wherein said sample is selected from the group consisting of agricultural fields, agricultural products, herbs, and feed.
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CN113480624A (en) * 2021-06-01 2021-10-08 中国农业科学院油料作物研究所 Aspergillus flavus toxigenic bacteria reference substance containing aflatoxin early warning molecules, and preparation method and application thereof

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