CN113607959B

CN113607959B - Rapid detection kit for aflatoxin toxigenic bacteria toxigenic indicator molecule immunity and application thereof

Info

Publication number: CN113607959B
Application number: CN202110594331.2A
Authority: CN
Inventors: 张奇; 李培武; 姜俊
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2023-06-27
Anticipated expiration: 2041-05-28
Also published as: CN113607959A

Abstract

The invention relates to a quick detection kit for aflatoxin toxigenic bacteria toxigenic indicator molecule immunity. The kit comprises a nano antibody or monoclonal antibody ELISA plate with a hole bottom coated with an aflatoxin-producing virulence indicator, a polyclonal antibody of the aflatoxin-producing virulence indicator, a pure product solution of the aflatoxin-producing virulence indicator which plays a role of a standard substance, a Chlamydia medium, a horseradish peroxidase labeled antibody, ELISA chromogenic solution, stop solution and sample diluent; the aflatoxin toxigenic bacteria toxigenic indicator molecule refers to AFT-YJFZ01 peptide, and the amino acid sequence of the aflatoxin toxigenic bacteria toxigenic indicator molecule is shown as SEQ ID NO. 1. The method can be used for rapidly and quantitatively detecting the aflatoxin-producing strain virulence indicator molecule AFT-YJFZ01, can be used for identifying and comparing the aflatoxin-producing capability of the aflatoxin-producing strain of aspergillus, is easy to operate, has strong practicability and is easy to popularize and apply.

Description

Rapid detection kit for aflatoxin toxigenic bacteria toxigenic indicator molecule immunity and application thereof

Technical Field

The invention relates to a kit for rapidly detecting toxicity indicating molecule immunity of aflatoxin toxigenic bacteria and application thereof.

Background

Aflatoxin has strong toxicity and great harm, is the pollutant with the largest variety of polluted foods, generally presents a pollution aggravating trend in recent years, and seriously threatens the food safety and the health of people. Aflatoxin is the most toxic mycotoxin in nature, wherein aflatoxin B1 is a class I carcinogen identified by International cancer research organization (International Agency for Research on Cancer, IARC), and has caused excessive poisoning events of human and animal populations, and becomes one of the main causes of high incidence of liver cancer cases. Statistics of data retrieved according to the last 5 years Web of Science: the aflatoxin pollutes food and raw materials more than 110 kinds, and the high-concentration pollutants are first. However, no molecular early warning research example before the pollution of the microbial toxins such as aflatoxin exists at home and abroad, and urgent needs of early warning in advance are difficult to meet.

The existing aflatoxin early warning method is mainly established based on aflatoxin detection technology and is used for toxin pollution level evaluation or postpartum pollution degree and consumption risk evaluation, and once detection finds that pollution often occurs, urgent requirements of early warning, guidance, prevention and control in advance 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. The quick early warning system (Rapid Alert System for Food and Feed, RASFF) for European Union food and feed utilizes limit standard and detection to obtain the aflatoxin content in the food and feed, and can quickly early warn the food and feed imported into European Union in various countries. The American research institution establishes a multi-element rogestin regression analysis and overlapping Gaussian treatment early warning model based on aflatoxin detection technology and pollution monitoring data, and is mainly used for evaluating the pollution degree and consumption risk of agricultural products such as post-partum corn and the like.

The research progress in the last twenty years at home and abroad is integrated, and the lack of the current early warning molecular detection technology of aflatoxin is the root cause. Aiming at the bottleneck problem, the inventor group successfully discovers an aflatoxin toxigenic bacteria toxigenic indicator molecule and an early warning effect thereof through over ten years of attack researches, becomes the first early warning molecule, successfully invents an aflatoxin toxigenic bacteria toxigenic indicator molecule immunity rapid detection kit based on the early warning molecule, and is used for identifying the aflatoxin producing capability of a strain, namely toxigenic capability, and discovering whether a strong toxigenic aflatoxin producing strain exists in farmlands, agricultural products and feeds, thereby providing scientific basis for timely discovering the pollution risk of the aflatoxins and early prevention and early control.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a quick detection kit for aflatoxin toxigenic indicator molecule immunity and application thereof, which is used for identifying the aflatoxin producing capability of aspergillus strains and identifying whether the aflatoxin toxigenic strains with strong toxigenic capability exist in products such as farmlands, agricultural products, chinese medicinal materials, feeds and the like, and is easy to popularize and apply.

In order to solve the technical problems, the invention adopts the following technical scheme:

Providing an aflatoxin toxigenic bacteria toxigenic indicator molecule immunity rapid detection kit, comprising a nano antibody or a monoclonal antibody ELISA plate with a hole bottom coated with the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01, a polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01, a pure product solution of the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 serving as a standard substance, a Czochralski culture medium or other culture mediums suitable for growing aflatoxin-producing aflatoxin, a horseradish peroxidase labeled antibody which is combined with the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 polyclonal antibody, an ELISA color development liquid, a termination liquid and a sample diluent;

the aflatoxin toxigenic bacteria toxigenic indicator molecule refers to AFT-YJFZ01 peptide, and the amino acid sequence of the aflatoxin toxigenic bacteria toxigenic indicator molecule is shown as SEQ ID NO. 1.

According to the scheme, the ELISA plate coated with the nanometer antibody or monoclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 at the bottom of the hole is prepared by the following method: dissolving the AFT-YJFZ01 nano antibody or monoclonal antibody in ELISA coating buffer solution to form coating solution of 0.2-8.0 mug/mL, adding the coating solution into an ELISA plate (100-200 mug/hole), standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2 h, removing the coating solution in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; then adding ELISA routine blocking solution (200-300 mu L per well), standing at room temperature or 37deg.C to block at least 1 h, discarding the blocking solution, and washing the ELISA plate with ELISA routine washing solution.

The ELISA coating buffer solution refers to a conventional carbonate buffer solution, and the preparation method comprises the following steps: weighing NaHCO ₃ 1.465g、Na ₂ CO ₃ 0.795 And g, adding deionized water to fix the volume to 500 mL.

In the scheme, the polyclonal antibody of the aflatoxin-producing virulence indicator molecule AFT-YJFZ01 is an aflatoxin-producing virulence indicator molecule AFT-YJFZ01 rabbit-derived polyclonal antibody, and the horseradish peroxidase labeled antibody is a horseradish peroxidase labeled goat anti-rabbit antibody.

In the scheme, the polyclonal antibody of the aflatoxin-producing virulence indicator molecule AFT-YJFZ01 is different from the animal source of the nanometer antibody or the monoclonal antibody of the aflatoxin-producing virulence indicator molecule AFT-YJFZ01, and the obtained aflatoxin-producing virulence indicator molecule can be directly used as an antigen to prepare and obtain the nanometer antibody or the monoclonal antibody and the rabbit-source polyclonal antibody;

the nanometer antibody or monoclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 can be prepared by using AFT-YJFZ01 as an immune antigen, immunizing alpaca or Balb/c mice in a conventional manner, and then using a known conventional nanometer antibody or murine monoclonal antibody preparation technical scheme.

The rabbit-derived polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic 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 then using a known conventional rabbit polyclonal antibody preparation technical scheme.

The culture medium is a conventional culture medium, can be self-matched, and can be obtained by directly purchasing commodity.

The horseradish peroxidase labeled goat anti-rabbit antibody can be obtained by directly purchasing commercial products.

According to the scheme, the ELISA chromogenic liquid refers to conventional hydrogen peroxide and TMB chromogenic liquid for ELISA.

The stop solution refers to a conventional chromogenic stop solution for ELISA: 2mol/L sulfuric acid aqueous solution, the preparation method is as follows: adding 44 and mL concentrated sulfuric acid into 300 and mL deionized water, stirring to cool, and finally fixing the volume to 400 and mL.

The sample diluent is 0.01 mol/L phosphorus containing 0.1% sorbitol and soft sugarThe preparation method of the acid salt buffer solution comprises the following steps: sorbitol and sugar 0.5. 0.5 g each, naCL 4.0. 4.0 g, na ₂ HPO ₄ ∙12H ₂ O 1.45 g、KCL 0.1g、KH ₂ PO ₄ 0.1 And g, adding deionized water to a volume of 500 mL.

The application method of the aflatoxin toxigenic bacteria toxigenic indicator molecule immunity rapid detection kit is provided:

Culturing with a culture medium suitable for the growth of the toxic Aspergillus flavus in a reagent kit and preparing a to-be-identified strain or a to-be-identified sample to be tested liquid;

diluting the to-be-identified strain to be-identified solution with sample diluent, adding 100-200 mu L of the diluted to-be-identified strain to be-identified solution or the to-be-identified sample to an ELISA plate hole in a kit, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding the reacted solution, and washing the ELISA plate;

then adding AFT-YJFZ01 polyclonal antibody into the kit, adding 100-200 mu L of the polyclonal antibody into each hole, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding liquid, and washing the ELISA plate;

then adding 100-200 mu L of horseradish peroxidase labeled antibody into each hole of the kit, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding liquid, washing the ELISA plate,

and then sequentially adding ELISA chromogenic liquid and stop solution in the kit, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by an enzyme-labeling instrument.

According to the scheme, the method further comprises the following steps: judging the detection result: according to the result of the enzyme-labeled instrument, the content of aflatoxin toxigenic indicator molecule AFT-YJFZ01 in a unit volume of aflatoxin toxigenic bacteria in the aspergillus flavus to-be-detected liquid is obtained, and the aflatoxin toxigenic power of the strain to be identified is analyzed. If the concentration of the aflatoxin-producing virulence indicator molecule AFT-YJFZ01 in the strain to be identified is higher, the aflatoxin-producing ability, namely the virulence of the identified strain is stronger;

And according to the result of the enzyme-labeled instrument, obtaining the content of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 in the unit volume of sample liquid to be identified, and judging whether the sample to be identified contains the strong toxigenic strain of aflatoxin. If the concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 in the sample to be identified is high, the sample to be identified is proved to contain the strong toxigenic strain of aflatoxin.

According to the scheme, a series of concentration AFT-YJFZ01 pure product solution (100-200 mu L per hole) serving as a standard substance of aflatoxin toxigenic bacteria is used for replacing a liquid to be tested, and is used for manufacturing a standard curve, and the concentration of AFT-YJFZ01 in a sample to be tested is calculated.

According to the scheme, the preparation of the to-be-identified strain to be tested is as follows: culturing the strain to be identified to obtain a solution to be detected of the strain to be identified; the method comprises the following steps: culturing the strain to be identified in a conventional Chlamydia medium or other culture mediums suitable for strain growth, wherein the culture environment temperature is 15-35 ℃, the culture time is not less than 12 h, fully homogenizing a mixture of the culture medium and the culture, and diluting 1-10 times by using sterile water to obtain a solution to be detected of the strain to be identified;

Preparing the sample liquid to be identified: culturing and diluting a sample to be identified to obtain a sample to be identified to be tested;

the method comprises the following steps: weighing a sample to be identified, transferring the sample to a sample diluent, vibrating at room temperature until the sample is uniform, preparing a sample uniform dispersion liquid to be identified, adding 10-1000 mu L of the sample uniform dispersion liquid into a culture medium containing 6-600 mL conventional Chlamydomonas or other culture mediums suitable for the growth of toxic aspergillus flavus, placing the culture medium to be subjected to shake culture at 200+/-50 rpm at 15-35 ℃, and sampling after culturing 6-24 h to form the sample to be identified.

According to the scheme, the samples are farmland, agricultural products, chinese herbal medicines, feeds and other products.

The kit can be used for identifying the aflatoxin production capability of the aspergillus strain, and the higher the content of the aflatoxin production virulence indicator molecules in the strain to be identified is, the stronger the aflatoxin production capability, namely the virulence of the identified strain is; the kit can be used for identifying whether the aflatoxin toxigenic strains with strong toxigenic power exist in products such as farmlands, agricultural products, traditional Chinese medicinal materials and feeds, and if the content of the aflatoxin toxigenic bacteria toxigenic indicator molecules in a sample to be identified is high, the sample to be identified contains the aflatoxin strong toxigenic strains;

The virulence of the aspergillus flavus strain is an index for measuring the capability of the strain to produce aflatoxin, and the stronger the virulence of the strain is, the more the amount of the aflatoxin the strain can produce under the same time and culture condition is shown. The invention utilizes the toxicity indicating molecule of the aflatoxin-producing bacteria obtained by research, combines the nanometer antibody or the monoclonal antibody of the toxicity indicating molecule AFT-YJFZ01 of the aflatoxin-producing bacteria and the rabbit-source polyclonal antibody, and constructs a sandwich immunodetection method by the antibodies, and finally assembles the kit; in the application of the kit, the characteristic that the concentration is positively correlated with the bacterial strain virulence is determined by utilizing the aflatoxin virulence indicator molecules, so that the kit is used for identifying the aflatoxin producing capability of the aspergillus strain and the use of the aflatoxin producing strain for identifying whether strong virulence exists in farmlands, agricultural products and feeds, is practical and easy to popularize, provides a key grip and scientific basis for timely finding the aflatoxin pollution risk and early prevention and early control, and has important significance for promoting the high-quality development of the agricultural industry and guaranteeing the food safety.

The invention has the beneficial effects that:

1. the kit can be used for rapidly and quantitatively detecting aflatoxin-producing strain virulence indicator molecule AFT-YJFZ01, can be used for identifying and comparing aflatoxin-producing ability of aflatoxin-producing strains of aspergillus, can be used for identifying whether aflatoxin-producing strains with strong virulence exist in farmlands, agricultural products and feeds, is easy to operate, has strong practicability, is easy to popularize and apply, and has important significance for promoting high-quality development of agricultural industry and guaranteeing food safety.

Drawings

FIG. 1 is a standard curve of an AFT-YJFZ01 immune rapid detection method of an aflatoxin toxigenic bacterium toxigenic indicator molecule.

Detailed Description

Preparation of aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 capable of functioning as a Standard substance in the kit of example 1

The preparation of the culture medium was carried out as follows: 3% (w/v) sucrose, 0.3% (w/v) NaNO3,0.1% (w/v) K2HPO4,0.05% (w/v) MgSO4.7H2O, 0.05% (w/v) KCl,0.001% (w/v) FeSO4, pH6.5 were prepared to obtain a Chlamydomonas medium. Randomly selecting 10 strains of published open literature (Aspergillus flavus distribution, toxicity and infection research in China typical peanut production area), namely national institute of agriculture, namely Shuoshi institute of China, author Zhang Xing, page 33, namely published toxigenic strains HLJ-1, heNZY-2, huBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-122-2, jcnt-1, huNdx-7, HBHA-8-17 and the like, respectively inoculating the 10 strains into the Boehmeria nivea culture medium, culturing for 5 days at 28 ℃ at 200 rpm/min, fully homogenizing and crushing cells by a conventional method, and purifying to obtain the aflatoxin toxigenic strain toxicity indicator molecule AFT-YJFZ01 by using a conventional protein purification system, protein electrophoresis, immunoaffinity and other methods. Test results show that AFT-YJFZ01 can be prepared in the culture of the toxigenic strain, under the same culture conditions, the amount of AFT-YJFZ01 prepared by HBHA-8-17 is the largest, and the amount of AFT-YJFZ01 prepared by HLJ-1 is the smallest.

The immunoaffinity method is to dilute the cell disruption solution of aflatoxin-producing bacteria with a sample solution, filter the cell disruption solution with a filter paper, continuously add the cell disruption solution into the immunoaffinity column, wash the column with a conventional eluent of the immunoaffinity column when the cell disruption solution is basically drained, finally elute the cell disruption solution with a glycine buffer solution with the pH of 2.2 or a 70% methanol aqueous solution, collect the eluent, timely remove the solution by a conventional ultrafiltration centrifugation method, and dissolve the protein remained in the ultrafiltration centrifuge tube from the ultrafiltration centrifuge tube with sterile water to obtain the AFT-YJFZ01 aqueous solution of the aflatoxin-producing bacteria toxicity-producing indicator molecule.

Initial acquisition of aflatoxin-producing virulence indicator molecule AFT-YJFZ01 by using a mining method:

the method for exploring the toxicity indicator molecule AFT-YJFZ01 of the aspergillus flavus strain is as follows:

(1) Taking an aspergillus flavus strong virulence strain, and culturing to obtain a strain culture and extracellular secretion protein mixture; then breaking the cells of the strain culture to obtain an intracellular protein mixture; combining the extracellular secretion protein mixture and the intracellular protein mixture, and adding carbodiimide for coupling to obtain an aspergillus flavus antigen;

(2) Immunizing a test animal with the aspergillus flavus antigen to obtain a nano antibody library or a monoclonal antibody library;

(3) Obtaining protein combined solutions of aspergillus flavus strains with different virulence, detecting the proteins of the aspergillus flavus strains with different virulence by using the antibodies in the antibody library obtained in the step (2), and obtaining a series of detection signals;

(4) Finding out a nano antibody with a detection signal positively correlated with the aspergillus flavus strain virulence, namely an aspergillus flavus strain virulence indicator molecule antibody, and a protein corresponding to the aspergillus flavus strain virulence indicator molecule antibody, namely an discovered aspergillus flavus strain virulence indicator molecule.

In the scheme, the aspergillus flavus strong virulence strain in the step (1) is separated and identified from the natural world by a conventional method or is obtained by artificial transformation, and the virulence is identified to be not less than 10 mug/kg by a NY/T2311-2013 standard method.

And (3) the aspergillus flavus strains with different virulence in the step (3) are not less than 3 strains, and the virulence is at least 3 layers higher, middle and lower as the result of the identification by the NY/T2311-2013 standard method.

The culture medium adopted in the culture of the aspergillus flavus strong virulence strain is a Chlamydia medium or other nutrients for the normal growth of the aspergillus flavus, the culture time is not less than 12 h, and the culture environment temperature is 15-35 ℃.

The cell disruption of the strain culture is carried out by a conventional liquid nitrogen grinding method or a cell disruption instrument method.

The amount of the carbodiimide is 0.005-0.1 g per 1.0 mL of the extracellular secretion protein mixture and the intracellular protein mixture combined.

The coupling reaction is carried out at 15-37 ℃ for 2-6 h and at 4-10 ℃ for overnight.

The immunization is a conventional immunization mode, and Aspergillus flavus antigens are inoculated. The test animal refers to a white mouse or alpaca or other test animals with similar effects.

According to the scheme, the antibody preparation process refers to a conventional nanobody preparation process or a conventional hybridoma monoclonal antibody preparation process based on cell fusion.

According to the scheme, the detection of the proteins of the aspergillus flavus strains with different virulence is realized by adopting a conventional Western Blot technical process, namely, the proteins of the aspergillus flavus strains with different virulence are transferred onto a nitrocellulose membrane, and then the antibodies in the antibody library are used for detection by a direct method or an indirect method, or other technical processes with similar effects are adopted.

According to the scheme, the direct method refers to coupling the antibodies in the antibody library with a signal material by a conventional method, and then performing an immune binding reaction with the corresponding proteins transferred onto the nitrocellulose membrane.

According to the scheme, the indirect method is that the antibodies in the antibody library are subjected to immune binding reaction with the corresponding proteins transferred onto the nitrocellulose membrane, and then the second antibodies and the conjugate of the signal material are subjected to immune binding reaction with the antibodies bound onto the nitrocellulose membrane.

The signal material in the detection is horseradish peroxidase, colloidal gold, fluorescent material or other materials with similar effects. The detection signal is a chromogenic reaction signal or a spot signal or a fluorescent signal.

Example 2 preparation of nanobody of toxicity indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria

AFT-YJFZ01 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then the preparation technical scheme of known conventional nano antibodies or mouse monoclonal antibodies is utilized to develop and obtain the alpaca or Balb/c mice.

Dissolving AFT-YJFZ01 obtained by the preparation method in conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1 mg/mL, mixing and emulsifying with Freund's complete adjuvant in an equal volume, immunizing alpaca by subcutaneous or intradermal multipoint injection at back, and enhancing immunity for 1 time every 2-4 weeks, wherein Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during enhancing immunity. The immune effect is monitored by adopting a conventional ELISA flow until serum titer of alpaca is not increased any more, then the operations of venous blood collection, total RNA extraction, cDNA synthesis, VHH gene amplification, VHH gene fragment recovery, connection of the VHH gene and a double enzyme digestion pCANTAB 5E (his) carrier, electric conversion of a connection product, construction of a nanobody gene library, rescue of the nanobody gene library and the like of the alpaca are completed according to the method of a patent document CN103866401A, and finally the rescued nanobody gene library is obtained.

Fixing AFT-YJFZ01 obtained by the preparation on solid-phase carriers such as 96-well ELISA plates according to gradients of 8 mug/well, 2 mug/well, 0.5 mug/well and 0.1 mug/well, panning the saved nanobody gene library for 2-4 times according to a method of patent document CN103866401A, identifying antibodies generated by each phage clone by using AFT-YJFZ01 and indirect non-competitive ELISA, identifying phage corresponding to positive results as phage positive clones, preparing the nanobody by the positive clones through a conventional method of nanobody preparation, namely the nanobody of AFT-YJFZ01, for further application research work, and preferably characterizing the nanobody with strong specificity and high affinity through ELISA method.

EXAMPLE 3 preparation of monoclonal antibody of aflatoxin-producing virulence indicator molecule AFT-YJFZ01

The AFT-YJFZ01 obtained by the preparation method is dissolved in a conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1 mg/mL, and is mixed and emulsified with Freund's complete adjuvant in an equal volume, BALB/c mice are subjected to boost immunization 1 time every 2-4 weeks through a back subcutaneous or intradermal multipoint injection mode, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during boost immunization. And (3) monitoring the immune effect by adopting a conventional ELISA flow, after the serum titer of the BALB/c mice is no longer increased, then separating immune mouse spleen cells, fusing the spleen cells with mouse myeloma cells SP2/0, completing the selective culture operation of a semisolid culture medium on hybridoma cells by using a method of reference patent document CN103849604A, and after a needle point white spot grows on the semisolid culture medium, respectively picking the white spots into 96-hole culture plates with the built-in hybridoma conventional culture medium, thereby obtaining a monoclonal hybridoma resource library.

The monoclonal antibody obtained by the culture supernatant of the monoclonal hybridoma is obtained by the method of the reference CN103849604A, AFT-YJFZ01 obtained by the preparation is fixed on a solid-phase carrier such as a 96-well ELISA plate according to the gradient of 8 mug/well, 2 mug/well, 0.5 mug/well and 0.1 mug/well, each monoclonal antibody is identified by an indirect non-competitive ELISA program, positive clones are picked up, and the AFT-YJFZ01 monoclonal antibody is obtained and used for further application research work, and the AFT-YJFZ01 monoclonal antibody with the characteristics of strong specificity and high affinity is preferably detected.

Example 4 preparation of Rabbit-derived polyclonal antibody of aflatoxin-producing virulence indicator molecule AFT-YJFZ01

AFT-YJFZ01 is used as an immune antigen, test rabbits such as New Zealand white rabbits are immunized by a conventional mode, and a known conventional rabbit polyclonal antibody preparation technical scheme is utilized to develop the immune antigen.

The AFT-YJFZ01 prepared by the method is directly used as an antigen, the solution with the concentration not lower than 0.1mg/mL is mixed and emulsified with Freund's complete adjuvant in an equal volume, new Zealand white rabbits are subjected to subcutaneous or intradermal multipoint injection at the back, then the immunization is enhanced for 1 time every 2-4 weeks, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during the enhancement. And (3) monitoring the immune effect by adopting a conventional ELISA flow, and preparing and obtaining serum of the immune animal by a conventional method after the serum titer of the immune animal is not increased, namely the rabbit-derived polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ 01.

Example 5 establishment of an immunoassay Rapid detection method for an Aft-YJFZ01 Acidogenic indicator molecule of an Aflatoxin

Basic operation procedure of AFT-YJFZ01 immune rapid detection method, namely double-antibody sandwich indirect non-competitive ELISA method: coating a nanometer antibody of AFT-YJFZ01 in the ELISA plate, and washing the plate; adding a sealing liquid for sealing and washing the plate; adding AFT-YJFZ01 or a liquid to be tested for reaction, and washing the plate; adding AFT-YJFZ01 rabbit-source polyclonal antibody for reaction, and washing the plate; adding horseradish peroxidase labeled goat anti-rabbit antibody for reaction, and washing the plate; adding a color development liquid for reaction; and adding a stop solution, and reading and calculating a result by using an enzyme label instrument. The following work is accomplished using this basic procedure.

Determination of optimal concentration of antibody: a plurality of parallel experiments are simultaneously carried out by adopting a chessboard titration method, the nanometer antibodies with different concentrations are used for coating, in addition, the rabbit polyclonal antibodies are set to be different concentrations, and finally, the optimal working concentrations of the nanometer antibodies and the rabbit polyclonal antibodies are determined according to the result, namely, the concentrations of the two antibodies corresponding to the point with the OD450nm value of approximately 1.0 are selected under the principle of saving the dosage of the antibodies, and ELISA result researches show that although other concentrations of the nanometer antibodies and the rabbit polyclonal antibodies can be detected, the optimal coating concentration of the nanometer antibodies is 2.0 mug/mL, and the optimal concentration of the rabbit polyclonal antibodies is 3.0 mug/mL.

Determination of optimal antibody coating conditions: coating is the first step in ELISA method research, and the quality of the coating effect has a very critical effect on the ELISA result. In order to determine the influence of different coating conditions on the detection result, three different conditions of coating at 4 ℃ overnight, constant temperature coating at 37 ℃ for 2h and constant temperature coating at 37 ℃ for 1h are selected to be coated in the hole, and the detection result shows that the three coating modes can be used for coating, and the coating at 4 ℃ is the optimal coating condition.

Determination of optimal blocking agent: after the antibody is coated, in order to avoid interference of unoccupied sites of the ELISA plate hole on subsequent steps of ELISA, inert proteins, namely blocking agents, need to be used for occupying the sites, and improper blocking agents can be combined with the secondary antibody in a non-specific way, so that false positive conditions are caused. The study adopts three different blocking agents of 3% BSA/PBST, 3% skimmed milk powder/PBST and 5% skimmed milk powder/PBST for blocking, and the study results show that although the three blocking agents can achieve the purpose of blocking in different degrees, the blocking effect of 5% skimmed milk powder/PBST is optimal, and the blocking agent is the optimal blocking agent.

Determination of the closing time: the study sets up three kinds of different closure duration of constant temperature closure 1h, 2h, 3h respectively and seals, and the result of detection shows that the numerical value of positive hole OD450nm value/negative hole OD450nm value is the biggest under the setting of 37 ℃ and closure 2h, and positive sample OD450nm value is > 1, so that constant temperature closure 2h at 37 ℃ is the best closure time.

Determination of the reaction time of the rabbit polyclonal antibody: the study adopts three reaction durations of 30min, 50min and 1h of constant temperature reaction at 37 ℃ to carry out reaction, and the detection result shows that: the optimal reaction time of the rabbit polyclonal antibody is obtained by reacting for 50min at the constant temperature of 37 ℃.

ELISA standard curve of aflatoxin toxigenic indicator molecule AFT-YJFZ01 is drawn: AFT-YJFZ01 molecules are respectively diluted to 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30 and 300 ng/mL, 200 mu L of each hole is filled in the hole, and ELISA standard curves of AFT-YJFZ01 are drawn by adopting the optimal conditions, and the results are shown in figure 1. The correlation coefficient of the double-antibody sandwich ELISA method established under the optimal condition reaches 0.9980, and the detection limit of AFT-YJFZ01 molecules reaches 0.1 ng/mL, which shows that the detection method has good detection sensitivity and accuracy.

Method specificity evaluation: in order to evaluate the specificity of the immunodetection method of the aflatoxin toxigenic indicator molecule AFT-YJFZ01, several strains of fungi with certain homology with the aflatoxin are researched and selected, and cell disruption solutions of fungi cultures are detected, the results are shown in a table 1, and the method has no obvious cross reaction on proteins of fungi with homology with the aflatoxin, so that the established aflatoxin toxigenic indicator molecule AFT-YJFZ01 immunorapid detection method has good specificity.

TABLE 1 specificity determination result of aflatoxin toxigenic bacteria toxigenic indicator molecule immunity rapid detection method

Method repeatability evaluation: in order to evaluate the repeatability of the established aflatoxin toxigenic indicator molecule AFT-YJFZ01 immune rapid detection method, randomly taking the non-virulent strains of 4 aflatoxins, namely positive 1, positive 2, positive 3, positive 4 and 1 aflatoxins, namely negative 5, and analyzing the data variation in and among the plates of the measurement result. The results of the above study are shown in tables 2 and 3, and the calculated intra-plate variation coefficient is 0.5% -3.5%, and the calculated inter-plate variation coefficient is 0.9% -5.7%, which are below 7%, so that the method has good repeatability.

TABLE 2 in-board reproducibility assay results of virulence indicator AFT-YJFZ01 immunorapid assay

TABLE 3 results of determination of the inter-plate reproducibility of AFT-YJFZ01 immune rapid detection method for virulence indicator molecules

Evaluation of accuracy of the method: in order to evaluate the detection accuracy of the method and also to examine the practicability of the method, peanut and corn samples are selected as examples for research and evaluation, aflatoxin-producing strain virulence indicator molecule AFT-YJFZ01 is added into the corn and peanut samples, and the more the recovery rate of the detection result is close to 100%, the more accurate and practical the method is described. The research results are shown in Table 4, and the results show that the established aflatoxin toxigenic bacteria toxigenic indicator molecule immunity rapid detection method is used for detecting the toxigenic indicator molecule AFT-YJFZ01 in peanuts and corns, the recovery rate of the method reaches 82.5% -109.5%, and the method is high in accuracy, can be applied to actual sample detection, and has good practicability.

TABLE 4 addition recovery test results of the fast detection method of the virulence indicator molecules of aflatoxin toxigenic bacteria

Example 6 composition and shelf-life of AFT-YJFZ01 immune Rapid detection kit

The rapid immune detection kit for the aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01 mainly comprises an ELISA plate coated with a nano antibody or a monoclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01 at the bottom of a hole, a rabbit-derived polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01, a pure product solution of the aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01 serving as a standard substance, a Czochralski culture medium, a horseradish peroxidase marked goat anti-rabbit antibody, ELISA chromogenic liquid, a stop solution and a sample diluent.

The preparation method of the ELISA plate coated with the nanometer antibody or monoclonal antibody of aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01 at the bottom of the hole comprises the following steps: dissolving the nanometer antibody or the monoclonal antibody of the AFT-YJFZ01 in an ELISA coating buffer carbonate solution to form 2 mug/mL coating liquid, adding 100-200 mug/hole into a 96-hole ELISA plate, standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2 h, removing the coating liquid in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; and adding 200-300 mu L of ELISA conventional blocking solution into each hole, standing at room temperature or 37 ℃ to block at least 1 h, discarding the blocking solution, and washing the ELISA plate with ELISA conventional washing solution to obtain the ELISA plate with the hole bottom coated with the aflatoxin-producing virulence indicator molecule AFT-YJFZ 01.

The nanobody or monoclonal antibody of aflatoxin-producing virulence indicator molecule AFT-YJFZ01 as described above can be prepared by the manner of example 2 and example 3.

The rabbit-derived polyclonal antibody of the aflatoxin-producing virulence indicator molecule AFT-YJFZ01 can be prepared by the method of the example 4.

The ELISA chromogenic liquid refers to conventional hydrogen peroxide and TMB chromogenic liquid for ELISA.

The stop solution is a conventional chromogenic stop solution 2mol/L sulfuric acid aqueous solution for ELISA, and the preparation method comprises the following steps: adding 44 and mL concentrated sulfuric acid into 300 and mL deionized water, stirring to cool, and finally fixing the volume to 400 and mL.

The sample diluent is 0.01 mol/L phosphate buffer solution containing 0.1% sorbitol and sugar, and the preparation method comprises the following steps: sorbitol and sugar 0.5. 0.5 g each, naCL 4.0. 4.0 g, na ₂ HPO ₄ ∙12H ₂ O 1.45 g、KCL 0.1g、KH ₂ PO ₄ 0.1 And g, adding deionized water to a volume of 500 mL.

The shelf life research result of the kit shows that the kit can be stored for at least 6 months at 4 ℃.

Example 7 aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 Immunity rapid detection kit for identifying and comparing bacterial strain toxigenic power of aflatoxin

Firstly, preparing a to-be-identified strain to be tested liquid: according to the toxicity of the strain, 10 strains of the published toxicity-producing strains HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, heNZY-2, huBha-24, JSON-1, huNdx-7, GDZJ-108-19, HBHA-8-17 and the like of the published literature of the research on Aspergillus flavus distribution, toxicity and infection in a China typical peanut production area, namely, the national institute of agriculture, namely, the national institute of Chinese agricultural science, are selected, the author Zhang Xing, page 33, the published toxicity-producing strains HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, heNZY-2, huBha-24, JSNT-1, huNdx-7, GDZJ-108-19, HBHA-8-17 and the like are cultured in a Chlrabi culture medium in the kit, the culture medium and the culture medium are fully homogenized after the culture time is not less than 12 h, and the sample diluent of the kit is diluted for 1-10 times, so as to obtain the solution to be tested.

Second, the measurement of the strain to be identified is carried out: adding 100-200 mu L of the solution to be tested into the wells of the enzyme-labeled plate of the kit, or adding 100-200 mu L of the solution of the virulence indicator molecule AFT-YJFZ01 of the invention with serial concentration into the kit, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding the reacted liquid, washing the enzyme-labeled plate, adding the AFT-YJFZ01 rabbit-derived polyclonal antibody into the kit, adding 100-200 mu L of the solution into each well, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding the liquid, washing the enzyme-labeled plate, adding 100-200 mu L of the horseradish peroxidase-labeled goat anti-rabbit antibody into the kit into each well, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding the liquid, washing the enzyme-labeled plate, then sequentially adding ELISA color-developing solution and stop solution into the kit, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be tested through an enzyme-labeled instrument.

According to the results of the above documents, the virulence 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, etc., are as follows: 0. mu g/L, 0 mu g/L, 3.8 mu g/L, 4.9 mu g/L, 67.2 mu g/L, 81.7 mu g/L, 192.0 mu g/L, 204.4 mu g/L, 297.4 mu g/L and 1027.5 mu g/L, which indicate that HBZHX-21 and HBXY-36 are non-virulent strains, HBHA-1-4 and GDZJ-6 are weak virulent strains, and HBHA-8-17 and GDZJ-108-19 are strong virulent strains.

Thirdly, judging the identification result: the higher the content of the aflatoxin-producing virulence indicator molecule AFT-YJFZ01 in the strain to be identified, the stronger the aflatoxin-producing ability, i.e. the virulence of the identified strain. According to the above kit and the application technical scheme, AFT-YJFZ01 concentration results 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 sequentially determined: 0 ng/mL, 0 ng/mL, 8 ng/mL, 12 ng/mL, 49 ng/mL, 51 ng/mL, 104 ng/mL, 115 ng/mL, 175 ng/mL, 536 ng/mL, the results also indicate that: HBZHX-21 and HBXY-36 are non-virulent strains, HBHA-1-4 and GDZJ-6 are weak virulent strains, HBHA-8-17 and GDZJ-108-19 are strong virulent strains. The determination result is consistent with the sequence of the strain virulence intensity published in the publication of the distribution, virulence and infection research of the Aspergillus flavus in the China typical peanut production area, and the identification result of the strain with strong virulence and the strain with weak virulence is consistent with the publication, so that the kit and the application technical scheme thereof provided by the invention can be used for identifying the strain virulence of the aflatoxin, and the method is simple, easy to operate and strong in practicability.

Example 8 identification of highly virulent Strain containing Aflatoxin in agricultural fields Using A fast detection kit for AFT-YJFZ01, an indicator molecule for virulence of Aflatoxin-producing bacteria

Firstly, preparing a sample to be identified and a liquid to be tested: the peanut flowering period rhizosphere soil samples such as Jilin, liaoning, jiangxi, fujian and the like are selected for 4 parts, and are named as soil sample-1, soil sample-2, soil sample-3 and soil sample-4 in sequence. Sequentially weighing farmland soil samples to be measured, uniformly crushing, transferring the farmland soil samples to be measured into sample diluent of the kit, and vibrating the farmland soil samples to be measured at room temperature until the concentration is 0.5g/mL, so as to prepare uniform dispersion liquid of the samples to be measured. And 50 mu L of the uniform dispersion liquid of the sample to be detected is taken, added into a Chlamydia medium of the kit of 30 mL, placed at 28 ℃ for shake culture at 200 rpm, and sampled after 24 h culture, so as to form the sample to be detected of the sample to be identified.

Step two, the determination of the sample to be identified is carried out: adding 100-200 mu L of the solution to be tested into an enzyme-labeled plate hole of the kit, or adding 100-200 mu L of the solution of the virulence indicator molecule AFT-YJFZ01 of the invention with a series concentration into the kit, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding the reacted liquid, washing the enzyme-labeled plate, adding the AFT-YJFZ01 rabbit-derived polyclonal antibody into the kit, adding 100-200 mu L of the solution into each hole, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding the liquid, washing the enzyme-labeled plate, adding 100-200 mu L of the horseradish peroxidase labeled goat anti-rabbit antibody into each hole of the kit, standing at room temperature or 37 ℃ for reaction of not less than 1 h, washing the enzyme-labeled plate, then adding ELISA color development liquid and stop liquid into the kit, and finally reading and calculating the concentration of AFT-YJFZ01 in the sample to be tested by an enzyme-labeled instrument.

Thirdly, judging the identification result: if the concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 in the sample to be identified is high, the sample to be identified is proved to contain the strong toxigenic strain of aflatoxin. According to the kit and the application technical scheme thereof, the concentration results of AFT-YJFZ01 in the soil sample-1, the soil sample-2, the soil sample-3 and the soil sample-4 are sequentially: 0.3 ng/mL, 0.2 ng/mL, 16 ng/mL, 19 ng/mL. The results show that the amount of AFT-YJFZ01 contained in each milliliter of culture solution in the soil sample-1 and the soil sample-2 is below 1.0 and ng, and the strong virulence strain without aflatoxin is low in risk of post-partum pollution of peanuts in corresponding farmland; the amount of AFT-YJFZ01 contained in each milliliter of culture solution in each soil sample-1 and each soil sample-2 is more than 10 ng/mL, and the strong virulence strain containing aflatoxin has higher risk of postpartum peanut pollution in corresponding farmlands.

Example 9 identification of highly virulent Strain containing Aflatoxin in agricultural products Using A Rapid test kit for AFT-YJFZ01, an indicator of virulence of Aflatoxin producing bacteria

Firstly, preparing a sample to be identified and a liquid to be tested: and selecting 4 agricultural product samples such as peanut, corn, rice and wheat, sequentially weighing the samples to be detected, crushing the samples, transferring the crushed samples to a sample diluent of the kit, and vibrating the samples at room temperature until the concentration is 0.5g/mL, so as to prepare a uniform dispersion of the samples to be detected. And taking 100 mu L of the uniform dispersion liquid of the sample to be detected, adding the uniform dispersion liquid into a Nahnia culture medium containing 50mL of the kit, placing the culture medium at 28 ℃ for shake culture at 200 rpm, and sampling after culturing 6 h to form the sample to be detected of the sample to be identified.

Thirdly, judging the identification result: if the concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 in the sample to be identified is high, the sample to be identified is proved to contain the strong toxigenic strain of aflatoxin. According to the kit and the application technical scheme thereof, the concentration results of AFT-YJFZ01 in the peanut, corn, rice and wheat samples are sequentially: 12 ng/mL, 13 ng/mL, 0.7 ng/mL, 0 ng/mL. The result shows that the amount of AFT-YJFZ01 contained in each milliliter of culture solution in the peanut and corn samples is more than 10 ng, and the peanut and corn samples contain strong virulence strains of aflatoxin, so that the pollution risk is high; the rice sample contains AFT-YJFZ01 in an amount of below 1.0 in each milliliter of culture solution, does not contain strong virulent strain of aflatoxin, and has low pollution risk; the measured wheat sample contains AFT-YJFZ01 in an amount of 0 per milliliter of culture solution, does not contain aflatoxin toxigenic strains, and basically has no risk of aflatoxin pollution.

Example 10 identification of highly virulent Strain containing Aflatoxin in agricultural products Using A Rapid test kit for AFT-YJFZ01, an indicator of virulence of Aflatoxin producing bacteria

Firstly, preparing a sample to be identified and a liquid to be tested: selecting 3 parts of Chinese medicinal material samples such as ginseng, angelica, medlar and the like, sequentially weighing the Chinese medicinal material samples to be detected, crushing, transferring to a sample diluent of the kit, and vibrating at room temperature until the concentration is 0.5g/mL, thereby preparing a uniform dispersion of the samples to be detected. And 50 mu L of the uniform dispersion liquid of the sample to be detected is taken, added into a Chlamydia medium containing 30mL of the kit, placed at 28 ℃ for shake culture at 200 rpm, and sampled after 6 h culture, so as to form the sample to be detected of the sample to be identified.

Thirdly, judging the identification result: if the concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 in the sample to be identified is high, the sample to be identified is proved to contain the strong toxigenic strain of aflatoxin. According to the above kit and the application technical scheme thereof, the determined AFT-YJFZ01 concentration results in the Chinese medicinal material samples of ginseng, angelica, medlar and the like are as follows in sequence: 0.3 ng/mL, 0 ng/mL, 0 ng/mL. The result shows that the concentration of AFT-YJFZ01 in the measured ginseng sample is below 1.0, the strong virulent strain containing no aflatoxin is not contained, and the pollution risk is low; the concentration of AFT-YJFZ01 in the determined Chinese angelica and medlar samples is 0, and the aflatoxin-producing strain is not contained, so that the risk of aflatoxin pollution is basically avoided.

Example 11 identification of highly virulent Strain containing Aflatoxin in feed Using AFT-YJFZ01 immune Rapid test kit

Firstly, preparing a sample to be identified and a liquid to be tested: 4 parts of feed samples to be tested are selected from the market, and are named feed-1, feed-2, feed-3 and feed-4 in sequence. And sequentially weighing samples to be detected, crushing, transferring the crushed samples to sample diluent of the kit, and vibrating the crushed samples at room temperature until the concentration is 0.5g/mL, thereby preparing uniform dispersion liquid of the samples to be detected. And taking 100 mu L of the uniform dispersion liquid of the sample to be detected, adding the uniform dispersion liquid into a Czochralski culture medium containing 200 mL of the kit, placing the culture medium at 28 ℃ for shake culture at 200 rpm, and sampling after culturing for 24 h to form the sample to be detected of the sample to be identified.

Thirdly, judging the identification result: if the concentration of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 in the sample to be identified is high, the sample to be identified is proved to contain the strong toxigenic strain of aflatoxin. According to the kit and the application technical scheme thereof, the concentration results of AFT-YJFZ01 in the feed-1, the feed-2, the feed-3 and the feed-4 are as follows in sequence: 0.6 ng/mL, 22 ng/mL, 13 ng/mL, 69 ng/mL. The result shows that the amount of AFT-YJFZ01 contained in each milliliter of culture solution in a feed-1 sample is below 1.0 ng/mL, and the feed-1 sample does not contain strong virulence strain of aflatoxin and has low pollution risk; taking 50 mu L of sample uniform dispersion liquid as an example, adding the sample uniform dispersion liquid into a 30 mL-containing culture medium, wherein the amount of AFT-YJFZ01 contained in each milliliter of culture liquid in feed-2, feed-3 and feed-4 is above 10 ng/mL, and the sample uniform dispersion liquid contains strong virulence strains of aflatoxin.

< 110 > institute of oil crop and oil crop of national academy of agricultural sciences

Kit for rapidly detecting toxicity indicating molecule immunity of aflatoxin-producing bacteria less than 120 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

1. The aflatoxin toxigenic bacteria toxigenic indicator molecule immunity rapid detection kit is characterized in that: the ELISA kit comprises an ELISA plate coated with a nano antibody or a monoclonal antibody of an aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01 at the bottom of a hole, a polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01, a pure product solution of the aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01 serving as a standard substance, a Chlamydia medium or other medium suitable for growing of the aflatoxin-producing aspergillus flavus, a horseradish peroxidase labeled antibody which is combined with the aflatoxin toxigenic bacteria toxigenic indicator AFT-YJFZ01 polyclonal antibody, ELISA color development liquid, a termination liquid and a sample diluent;

2. The rapid detection kit for aflatoxin-producing virulence indicator molecule immunity according to claim 1, wherein the kit comprises: the ELISA plate coated with the nanometer antibody or monoclonal antibody of aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 at the bottom of the hole is prepared by the following method: dissolving the AFT-YJFZ01 nano antibody or monoclonal antibody in ELISA coating buffer solution to form 0.2-8.0 mug/mL coating solution, adding the coating solution into an ELISA plate, standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2 h, removing the coating solution in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; then adding ELISA routine blocking solution, standing at room temperature or 37 ℃ to block at least 1 h, discarding the blocking solution, and washing the ELISA routine washing solution.

3. The rapid detection kit for aflatoxin-producing virulence indicator molecule immunity according to claim 1, wherein the kit comprises: the polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 is an aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ01 rabbit-derived polyclonal antibody, and the horseradish peroxidase labeled antibody is a horseradish peroxidase labeled goat anti-rabbit antibody.

4. The rapid detection kit for aflatoxin-producing virulence indicator molecule immunity according to claim 1, wherein the kit comprises: the ELISA chromogenic liquid refers to conventional hydrogen peroxide and TMB chromogenic liquid for ELISA;

the stop solution refers to a conventional chromogenic stop solution for ELISA: 2mol/L sulfuric acid aqueous solution.

5. The rapid detection kit for aflatoxin-producing virulence indicator molecule immunity according to claim 1, wherein the kit comprises: the sample diluent is 0.01 mol/L phosphate buffer solution containing 0.1% sorbitol and 0.1% sugar.

6. The method for using the aflatoxin toxigenic bacteria toxigenic indicator molecule immunity rapid detection kit of claim 1, which comprises the following steps:

Diluting the strain to be identified or the sample to be identified with sample diluent, adding 100-200 mu L of the diluted strain to be identified or the sample to be identified into an ELISA plate hole in a kit, standing at room temperature or 37 ℃ for reaction of not less than 1 h, discarding the reacted liquid, and washing the ELISA plate;

7. The method according to claim 6, wherein the concentration of AFT-YJFZ01 in the sample to be measured is calculated by replacing the sample solution with a pure AFT-YJFZ01 solution of a series of concentrations of aflatoxin-producing bacteria which act as a standard substance.

8. The method as recited in claim 6, further comprising: judging the detection result: according to the result of an enzyme-labeled instrument, the content of aflatoxin toxigenic indicator molecules AFT-YJFZ01 in a unit volume of aflatoxin toxigenic bacteria to be detected in the aspergillus flavus liquid is obtained, the aflatoxin toxigenic power of the strain to be identified is analyzed, and the higher the concentration of the aflatoxin toxigenic indicator molecules AFT-YJFZ01 in the strain to be identified is, the stronger the aflatoxin producing capacity, namely the virulence of the strain to be identified is;

And according to the result of the enzyme-labeled instrument, obtaining the content of the aflatoxin toxigenic indicator molecule AFT-YJFZ01 in the unit volume of sample liquid to be identified, and judging whether the sample to be identified contains the strong toxigenic strain of aflatoxin.

9. The method according to claim 6, wherein the preparation of the test solution of the strain to be identified is: culturing and diluting the strain to be identified to obtain a solution to be detected of the strain to be identified; the method comprises the following steps: culturing the strain to be identified in a conventional Chlamydia medium or other culture mediums suitable for strain growth, wherein the culture environment temperature is 15-35 ℃, the culture time is not less than 12 h, fully homogenizing a mixture of the culture medium and the culture, and diluting 1-10 times by using sterile water to obtain a solution to be detected of the strain to be identified;

preparing sample liquid to be identified: culturing a sample to be identified to obtain a sample to be identified to be tested;

10. The method of claim 6, wherein the sample is a farm field, agricultural product, chinese herbal medicine, or feed.