CN114097459B - Method for preventing and controlling aflatoxin and increasing number of active root nodules of nitrogen fixation enzyme and application - Google Patents

Abstract

The invention relates to a method for preventing and controlling aspergillus flavus and toxins thereof and increasing the number of root nodules of nitrogen fixation enzyme activity of leguminous roots. Comprising the following steps: obtaining bacillus amyloliquefaciens, brevibaciens laterosporus, bacillus mucilaginosus and escherichia coli with the effects of preventing and controlling aspergillus flavus and toxins thereof; 2 or 3 of bacillus amyloliquefaciens, bacillus laterosporus and bacillus mucilaginosus are mixed with escherichia coli, and then the mixture is applied to the field of leguminous crops, so that the number of root nodules with nitrogen fixation enzyme activity of the roots of the leguminous crops is increased. The method is applied to increasing the number of root nodules with the activity of the azotase of the root of leguminous crops such as peanuts and the like, simultaneously reducing the levels of aspergillus flavus and aflatoxin, and has simple use, remarkable yield and ecological benefits and easy popularization and application.

Description

Method for preventing and controlling aflatoxin and increasing number of active root nodules of nitrogen fixation enzyme and application

Technical Field

The invention relates to a method for preventing and controlling aflatoxin and increasing the number of root nitrogen fixation enzyme active nodules of leguminous crops and application thereof.

Background

The aflatoxin has strong toxicity and great harm, is the pollutant with the largest variety of polluted foods, is one of the most serious aflatoxin pollution countries in the world in China, 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, wherein agricultural products such as peanuts and the like are most easily polluted. Therefore, the development of aflatoxin prevention and control technology has important significance for promoting green production.

The existing aflatoxin prevention and control bacteria screening method is summarized, and the prevention and control bacteria with good screening effect are evaluated by detecting the content of the processed aflatoxin and comparing the content difference of the aflatoxins. However, two important questions remain unsolved about the biocontrol bacteria screened by such conventional methods: (1) does biocontrol bacteria have an effect of inhibiting the growth of Aspergillus flavus? (2) Is the biocontrol bacterium able to block toxin synthesis from the source of aflatoxin biosynthesis? To solve the two important questions, there is still a need to further determine the bacteriostatic effect of the biocontrol bacteria on the growth of aflatoxin-producing bacteria by means of a bacteriostatic test on the one hand, and to determine the biosynthesis pathway of aflatoxins, such as a series of precursor substances, on the other hand.

How can one-step efficient screening of aflatoxin-control bacteria be achieved?

In order to solve the bottleneck problem, the inventor groups successfully find an aflatoxin toxigenic bacteria toxigenic indicator molecule through years of attack research, wherein the indicator molecule content is positively related to the aflatoxin toxigenic power of the strain for different toxigenic strains cultured under the same toxigenic condition. Further studies have also found that for the same strain of aflatoxin-producing bacteria, the indicator molecule content is positively correlated with the production of the bacteria over a certain incubation time. Intensive studies have found that the source of the aflatoxin synthesis pathway must be inhibited as long as the production of the indicator molecule is inhibited. On the basis, a method for efficiently screening aflatoxin prevention and control bacteria is successfully established, and is applied to efficiently screening out effective prevention and control bacteria capable of inhibiting aflatoxin synthesis from the source and inhibiting aflatoxin toxigenic bacteria growth, and a core material is provided for efficient green prevention and control of aflatoxin.

When leguminous crops such as peanuts and the like grow in the field, roots can be infected by rhizobium to form rhizobium, if the rhizobium has the activity of nitrogen fixation enzyme, free nitrogen can be fixed, nitrogen-containing compounds can be synthesized, and the leguminous crops can be utilized, so that high yield is obtained, and the leguminous crop cultivation method has very important practical significance for fertilizer application reduction, carbon peak reduction and carbon neutralization. If the root nodule count is relatively dense, it is known as super nodulation. Super-nodulation is still very rare at present. Therefore, how are legumes such as peanuts to produce a large number of nodules having a nitrogen-fixing enzyme activity? This has been a hotspot problem for worldwide research.

In actual production, crops such as peanuts and the like generate few root nodules due to long-term and large-scale use of chemical fertilizers in farmlands, and usually only few root nodules are visible in main roots. In order to increase the number of crop nodules, much research effort has focused on rhizobia, developing good rhizobia, but few are currently available for use in production and play a significant role.

Aiming at a series of problems, the inventor groups have conducted intensive researches and established a peanut aflatoxin prevention and control strain library, namely peanut-containing endophytes and ectobacteria, by independently establishing a high-efficiency peanut aflatoxin prevention and control method, and on the basis, the method for preventing and controlling aflatoxins and toxins thereof and increasing the number of root nodules of leguminous crops, namely root nodules of leguminous crops, has been successfully invented through the combined approach researches of the endophytes and the ectobacteria, and has important significance in increasing the number of root nodules of leguminous crops, such as leguminous crops, and the like, promoting yield increase and guaranteeing safety, and providing a key method support for reducing fertilizer and pesticide application, peak carbon and neutralization in farmlands.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides the method for preventing and controlling the aflatoxin and increasing the number of the root nodules of the root nitrogenase activity of the leguminous crops and the application thereof, which is applied to increasing the number of the root nodules of the leguminous crops such as peanuts and the like, and simultaneously reduces the level of the aflatoxin and the aflatoxin, has simple use and obvious ecological benefit, and is easy to popularize and apply.

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

a method for preventing and controlling aflatoxin and increasing the number of root nodules of nitrogen fixation enzyme of root of leguminous plants comprises the following steps: obtaining bacillus amyloliquefaciens with aspergillus flavus and toxin prevention and control effectsBacillusamyloliquefaciens) Brevibacillus laterosporus @Brevibacilluslaterosporu) Bacillus mucilaginosus @BacillusmucilaginosusKrass ilnikov) And Ledebark enterobacterEnterobacterludwigii) The method comprises the steps of carrying out a first treatment on the surface of the Mixing 2 or 3 of Bacillus amyloliquefaciens, brevibaciens and Bacillus mucilaginosus with Enterobacter ludwigii, applying to field of leguminous crops, increasing root nodule quantity of leguminous crops with nitrogen fixation enzyme activity, and preventing and controlling Aspergillus flavus and Aspergillus flavusIts toxin.

According to the scheme, the method comprises the following steps: obtaining bacillus amyloliquefaciens, brevibaciens laterosporus, bacillus mucilaginosus and escherichia coli with the effects of preventing and controlling aspergillus flavus and toxins thereof; batch culture of 4 microbial strains: the method comprises the steps of (1) degrading bacillus amyloliquefaciens, (2) side spore bacillus brevis, (3) bacillus mucilaginosus and (4) ludwigia bowel, mixing any 2 or 3 of microbial strains (1), (2) and (3) with the microbial strain (4), and applying the mixture to the field of leguminous crops, so that the aspergillus flavus and toxin level thereof are effectively prevented and controlled, and the number of nitrogen-fixing enzyme active nodules of peanut roots is increased. The method not only effectively reduces the prevention and control of the aflatoxin level of the peanut, but also obviously increases the number of active root nodules of the nitrogen fixation enzyme of the peanut root and promotes the yield increase.

According to the scheme, the acquisition method of the bacillus amyloliquefaciens, the bacillus laterosporus, the bacillus mucilaginosus and the escherichia coli ludwigii with the aspergillus flavus and toxin prevention and control effects comprises the following steps of: and detecting the aflatoxin content and/or the aflatoxin content of the strain to be screened after treatment, and comparing the difference of the aflatoxin content and/or the aflatoxin content of the treated strain and the untreated strain to obtain the control bacteria with the control effect on the aflatoxin and the toxins thereof. Preferably, a strain is selected which has an aflatoxin inhibition of more than 90% and an inhibition of more than 80% against the toxigenic bacteria.

According to the scheme, the acquisition method of the bacillus amyloliquefaciens, the bacillus laterosporus, the bacillus mucilaginosus and the escherichia coli ludwigii with the aspergillus flavus and toxin prevention and control effects comprises the following steps of: the aflatoxin prevention and control bacteria with the aflatoxin prevention and control effects are obtained by a one-step high-efficiency screening method of the aflatoxin prevention and control bacteria:

the method for efficiently screening aflatoxin prevention and control bacteria in one step comprises the following steps: determining the AFT-YJFZ01 content of a candidate aflatoxin biocontrol bacterium test treatment group and a control group, calculating the inhibition rate of biocontrol bacterium to the indicator molecule AFT-YJFZ01, and efficiently screening aflatoxin control bacterium according to the result of the inhibition rate of the indicator molecule AFT-YJFZ01, wherein the amino acid sequence of the indicator molecule AFT-YJFZ01 is shown as SEQ ID NO. 1; the higher the inhibition rate is, the better the effect of inhibiting the synthesis of aflatoxin source and the growth of toxigenic bacteria is, so that the aflatoxin prevention and control bacteria with better effect are screened out: (1) Bacillus amyloliquefaciens, (2) Brevibacillus laterosporus, (3) Bacillus mucilaginosus, and (4) Enterobacter ludwigii.

According to the scheme, the aflatoxin prevention and control bacteria are efficiently screened in one step, and preferably, the strain with the inhibition rate of the indicator molecule AFT-YJFZ01 being more than or equal to 90% is selected.

According to the scheme, the aflatoxin biocontrol bacteria test treatment group and the control group can be realized by the following steps: mixing peanut kernel powder, aspergillus flavus toxigenic fungus spores and a series of candidate prevention and control bacteria, and culturing in a culture medium for a certain time to obtain a test treatment group, wherein the culture medium is used for replacing the candidate prevention and control bacteria to culture for a certain time to obtain a control group.

According to the scheme, the peanut kernel powder can be, but is not limited to, a powdery product prepared by shelling varieties of Zhonghua No. 6, luhua No. 8 and the like and grinding.

According to the above scheme, the Aspergillus flavus spore can be, but is not limited to, 5×10 ⁵ And the cfu/mL of aspergillus flavus toxigenic spore liquid. Aspergillus flavus toxigenic bacteria refer to Aspergillus flavus toxigenic bacteria capable of producing aflatoxin, such as Aspergillus flavus standard toxigenic strain 3.4408, etc., but are not limited thereto.

According to the scheme, the culture medium can be a liquid state sand culture medium or other culture media with similar effects.

According to the above scheme, the mixture is cultured in the culture medium for a certain period of time, which can be, but is not limited to, 24 h and above.

The quantitative detection of the concentration of the aflatoxin indicator molecules can be realized by adopting the amino acid sequence of the indicator molecules AFT-YJFZ01 or partial sequences thereof, preparing antibodies corresponding to the proteins through a conventional antibody preparation flow, realizing the quantitative detection of the indicator molecule peptides, and also realizing the quantitative detection of the indicator molecule peptides in one-to-one correspondence through other detection technical means, thereby realizing the detection, wherein the partial sequences refer to a part of the whole sequences having one correspondence with the aflatoxin indicator molecule proteins.

Specifically, the content of the indicator molecule AFT-YJFZ01 can be realized by the following steps:

a, after the treatment group and the control group are cultured for a certain time, the culture is fully homogenized, a solution to be detected is prepared, the solution is added into an enzyme-labeled plate hole of which the bottom is coated with a nanometer antibody or a monoclonal antibody of an indicator molecule AFT-YJFZ01, the reaction is carried out, and the plate is washed;

b, adding an indicator molecule AFT-YJFZ01 polyclonal antibody for reaction, and washing the plate;

c, adding a horseradish peroxidase labeled antibody which is subjected to a binding reaction with the indicator molecule AFT-YJFZ01 polyclonal antibody, reacting, and washing the plate;

d, adding a color development liquid for reaction; adding a stop solution, and reading and calculating the content of AFT-YJFZ01 in the solution to be measured by an enzyme-labeled instrument;

And replacing the solution to be tested with a series of concentration indicator molecule AFT-YJFZ01 pure product solution serving as a standard substance for making a standard curve, and calculating the content of the indicator molecule AFT-YJFZ01 according to the reading of an enzyme-labeled instrument.

The inhibition rate of the biocontrol bacteria to AFT-YJFZ01 can be calculated by the following method: inhibition ratio = (control AFT-YJFZ01 content-treatment AFT-YJFZ01 content)/control AFT-YJFZ01 content x 100%.

According to the scheme, the polyclonal antibody of AFT-YJFZ01 is different from the nanometer antibody or monoclonal antibody of AFT-YJFZ01 in animal source, and the indicator molecule AFT-YJFZ01 can be directly used as an antigen to prepare and obtain the nanometer antibody or monoclonal antibody and rabbit polyclonal antibody, specifically:

the nanometer antibody or monoclonal antibody of the indicator molecule AFT-YJFZ01 can be obtained by the following method: AFT-YJFZ01 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then a known conventional nano antibody or murine monoclonal antibody preparation technical scheme is utilized to develop and obtain the alpaca or Balb/c mice;

the polyclonal antibody of the indicator molecule AFT-YJFZ01 can be obtained by the following method: the AFT-YJFZ01 is used as an immune antigen, a conventional mode is adopted to immunize test rabbits such as New Zealand white rabbits, and a known conventional polyclonal antibody preparation technical scheme is utilized to develop and obtain a rabbit-derived polyclonal antibody of aflatoxin indicator molecule AFT-YJFZ 01;

The horseradish peroxidase-labeled antibody which is subjected to a binding reaction with the polyclonal antibody of the indicator molecule AFT-YJFZ01 is a horseradish peroxidase-labeled goat anti-rabbit antibody, and can be directly purchased.

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

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.

According to the scheme, the batch culture can be a conventional method for fermenting bacillus or a conventional bacterial culture medium amplifying culture method.

According to the scheme, the 4 microorganism strains can be obtained by separating from peanut pods or rhizosphere soil by a conventional method, and can also be obtained by separating from roots or rhizosphere soil of other crops such as soybeans and the like.

According to the scheme, the mixing proportion refers to the proportion of any strain after mixing is more than or equal to 1%.

According to the scheme, the mixed microorganism strain can be applied to the field along with the crop base fertilizer, or can be applied to the field in the growing period of crops, and the application amount is more than or equal to 1000 hundred million viable bacteria per mu.

According to the scheme, the leguminous crops comprise peanut, soybean, alfalfa, astragalus sinicus and the like.

The invention also provides application of the method in increasing the number of the root nodules with the activity of the azotase of the leguminous crops and simultaneously reducing the aflatoxin and the toxin-producing bacteria level of the leguminous crops.

Further, there is provided the use of the above method for inducing the super nodulation phenomenon in leguminous plants and simultaneously reducing the aflatoxin and the level of toxigenic bacteria thereof in leguminous crops.

The invention has the beneficial effects that:

1. the method can be used for reducing aflatoxin and the level of toxin-producing bacteria of leguminous crops such as peanuts, and the like, 2, can be used for increasing the number of nitrogen-fixing enzyme active root nodules of the roots of leguminous crops such as peanuts, soybeans, alfalfa, astragalus sinicus and the like, and inducing the occurrence of super nodulation phenomenon of the roots, 2, the method is easy to operate, high in practicability and easy to popularize and apply, 3, is favorable for promoting the yield increase of crops, has economic benefits, 4, is favorable for improving the soil environment of farmlands, has ecological benefits, 5, is favorable for reducing the application of farmland fertilizers and pesticides, and has important significance for peak carbon and carbon neutralization.

Drawings

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

FIG. 2 shows the root nodule condition of a peanut treated by the method of the invention, and the upper graph shows untreated peanut root nodule condition; the following figure shows the supernodulation phenomenon after treatment by the method of the present invention.

FIG. 3 is a graph of peanut crop growth after treatment using the method of the present invention. The left panel shows peanut plants not treated with the method of the invention (control zone); the right panel shows peanut plants (demonstration area) treated by the method of the present invention.

Fig. 4 is a plot of peanut crop growth in a control zone.

FIG. 5 is a plot of peanut crop growth in an exemplary region.

Detailed Description

Initial acquisition of the indicator molecule AFT-YJFZ01 was developed by:

(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.

The indicator molecule AFT-YJFZ01 antibody can be prepared by using all peptide fragments or part of peptide fragments of the indicator molecule AFT-YJFZ01 after the total sequence of the indicator molecule AFT-YJFZ01 of aflatoxin is known.

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, published toxigenic fungus strains HLJ-1, heNZY-2, huBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-108-19, jcnt-1, huNdx-7, HBHA-8-17 and the like, respectively inoculating the 10 strains into the 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 by using a conventional protein purification system, protein electrophoresis, immunoaffinity and other methods to obtain the AFT-YJFZ01. Test results show that AFT-YJFZ01 can be prepared in the strain culture of the toxigenic fungi, under the same culture conditions, the amount of AFT-YJFZ01 prepared by HBHA-8-17 is the largest, and the amount of AFT-YJFZ01 prepared by HLJ-1 is the smallest.

The immunoaffinity method is to prepare an immunoaffinity column by using a nanometer antibody or a monoclonal antibody of an indicator molecule AFT-YJFZ01 through a conventional method, and then to enrich and purify from aflatoxin toxigenic fungus cell disruption liquid by using the immunoaffinity method, and dissolve with deionized water to obtain the product. Specifically, the aflatoxin toxigenic fungi cell disruption solution can be diluted by using a sample solution, filtered by using filter paper, continuously added into the immunoaffinity column, and after the immunoaffinity column is basically completely drained, the immunoaffinity column is washed by using a conventional eluent, and finally the column is eluted by using a glycine buffer solution with the pH of 2.2 or a 70% methanol aqueous solution, the eluent is collected and then the solution is timely removed by a conventional ultrafiltration centrifugation method, and then the protein remained in the ultrafiltration centrifuge tube is dissolved by using sterile water from the ultrafiltration centrifuge tube, so that the indicator molecule AFT-YJFZ01 aqueous solution can be obtained.

Example 1 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 by a method of a patent document CN103866401A, identifying antibodies generated by each phage clone by using AFT-YJFZ01 and indirect non-competitive ELISA, identifying phage corresponding to positive results as phage positive clones, preparing the nanobody by the positive clones through a conventional method of nanobody preparation, namely the nanobody of AFT-YJFZ01, for further application research work, and preferably characterizing the nanobody with strong specificity and high affinity by ELISA method.

EXAMPLE 2 preparation of monoclonal antibody of aflatoxin-producing virulence indicator molecule AFT-YJFZ01

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

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 according to a method of 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 conventional culture medium of the built-in hybridoma, thereby obtaining the monoclonal hybridoma resource library.

The monoclonal antibody obtained by the culture supernatant of the monoclonal hybridoma described above was obtained by the method described in patent document CN103849604a, the obtained AFT-YJFZ01 was immobilized on a solid support such as a 96-well ELISA plate at a gradient of 8 μg/well, 2 μg/well, 0.5 μg/well, 0.1 μg/well, and each monoclonal antibody was identified by an indirect non-competitive ELISA procedure, and positive clones were picked up to obtain an AFT-YJFZ01 monoclonal antibody, which was used for further application studies, preferably to detect an AFT-YJFZ01 monoclonal antibody having a strong specificity and a high affinity.

Example 3 preparation of Rabbit-derived polyclonal antibody of toxicity indicator molecule AFT-YJFZ01 of Aflatoxin toxigenic bacteria

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 specific rabbit-derived polyclonal antibody of the aflatoxin toxigenic bacteria toxigenic indicator molecule AFT-YJFZ 01.

Example 4 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 specific nano antibody of AFT-YJFZ01 in the ELISA plate, and washing the plate; adding a sealing liquid for sealing and washing the plate; adding AFT-YJFZ01 or a liquid to be tested for reaction, and washing the plate; adding AFT-YJFZ01 rabbit-source polyclonal antibody for reaction, and washing the plate; adding horseradish peroxidase labeled goat anti-rabbit antibody for reaction, and washing the plate; adding a color development liquid for reaction; adding stop solution, enzyme-labeled instrument toxin and calculating the result. The following work is accomplished using this basic procedure.

Determination of optimal concentration of antibody: a plurality of parallel experiments are simultaneously carried out by adopting a chessboard titration method, the specific nano-antibody coatings with different concentrations are adopted, in addition, the rabbit polyclonal antibody is set to be different concentrations, and finally, the optimal working concentrations of the nano-antibody and the rabbit polyclonal antibody are determined according to the result, namely, the concentrations of the two antibodies corresponding to the point with the OD450nm value of approximately 1.0 are selected under the principle of saving the antibody consumption, and ELISA result researches show that although other concentrations of the nano-antibody and the rabbit polyclonal antibody can be detected, the optimal coating concentration of the nano-antibody is 2.0 mug/mL, and the optimal concentration of the rabbit polyclonal antibody 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 all three coating modes can be studied to realize coating, but 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-plate reproducibility assay results of the indicator AFT-YJFZ01 immunorapid assay method

Table 3 shows the results of the repeat measurements between the plates of the AFT-YJFZ01 immunorapid assay

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 5 construction of peanut endophytic and exophyte bacterial Strain library

Representative peanut pod and rhizosphere soil samples are collected from 15 nationwide peanut main production provinces such as jaw, robust, and Yu, bacterial strains are separated from peanut pods or rhizosphere soil or bacterial strains are separated from roots or rhizosphere soil of other crops such as soybeans and the like through a conventional bacterial separation method, and a series of bacterial strains such as bacillus amyloliquefaciens, brevibacterium laterosporus, bacillus mucilaginosus, escherichia coli and the like, including peanut endophytic strains and exogenous strains, are identified through a conventional 16s rDNA method, so that peanut endophytic and exogenous bacterial strain libraries are obtained.

Example 6 screening of Aflatoxin control bacteria

1) Preparation of bacterial strains to be screened: randomly selecting bacterial strains to be screened from the strain library, wherein the inhibition results represented by 20 bacterial strains to be screened are shown in Table 5, respectively activating the bacterial strains in LB plates, culturing 24 h in a 37 ℃ incubator, picking single colonies of the activated strains to be screened by a picking needle, transferring the single colonies into a triangular flask filled with 15 mL of LB liquid medium, and culturing 12 h at 28 ℃ in 200 r min < -1 > in an oscillating manner. Transferring 1% of the culture solution into a triangular flask filled with 15 mL of LB liquid culture medium, and shake culturing at 28deg.C and r.min-1 for 12 h to obtain fermentation broth of strain to be screened with final concentration of 1×10 ⁷ CFU/mL。

2) Preparation of aflatoxin toxigenic bacteria: taking Aspergillus flavus distribution, toxicity and infection research of China typical peanut production area, national academy of agricultural sciences, author Zhang Xing, page 33, published strain HuNdx-7 of aflatoxin production as an example, selecting a small amount of bacterial liquid of HuNdx-7 strain, inoculating on DG18 solid culture medium for activation, culturing in inverted condition at constant temperature of 28 ℃ until the DG18 culture medium is full of yellow-green spores, washing spores with 0.1% Tween water, collecting to obtain spore suspension, counting under a high-power microscope, calculating spore concentration in the spore suspension, transferring the spore suspension into 200ml of Sa liquid culture medium until the final spore concentration is 5×10 ⁵ cfu/ml。

3) Prevention and control test: the fermentation liquid of the strain to be screened is respectively cultivated together with the spore suspension of the aflatoxin toxigenic strain in a Saccharum sinensis Roxb liquid culture medium containing Zhonghua No. 6 peanut powder at the temperature of 28 ℃ for 1 day at 200 rpm, 3 repetitions are arranged for each treatment, and the Saccharum sinensis Roxb culture medium is used for replacing the strain to be screened to be cultivated for a certain time to obtain a control group.

4) Determining the content of an indicator molecule AFT-YJFZ01 and selecting prevention and control bacteria: the method established in example 4 is adopted to measure the content of the indicator molecule AFT-YJFZ01 in the test treatment group and the control group, and the inhibition rate of the strain to be screened on the AFT-YJFZ01 is calculated by the following method:

AFT-YJFZ01 inhibition ratio= (AFT-YJFZ 01 content of control group-AFT-YJFZ 01 content of treatment group)/AFT-YJFZ 01 content of control group x 100%

The research results are shown in Table 5, and the results show that the significant difference exists in AFT-YJFZ01 inhibition rate between different strains, and the higher the AFT-YJFZ01 inhibition rate is, the better the effect of simultaneously inhibiting aflatoxin source synthesis and the growth of toxigenic bacteria is, so that strains such as aflatoxin prevention and control bacillus amyloliquefaciens (strain 01), brevibaciens laterosporus, enterobacter lubilis, flavobacterium breve and the like with good effects are deduced and screened out, and the method can be used for further preparation development and development.

TABLE 5 inhibitory Effect of 20 strains to be screened on AFT-YJFZ01

5) Verification confirmation: in order to further verify the reliability of the screening result of the one-step method, a conventional multi-step method, namely a bacteriostasis test and a toxicity inhibition test, is adopted.

Co-culturing the fermentation broth of the final strain to be screened obtained in the step 1) and the final spore suspension of the step 2) in a Saccharum liquid culture medium at 28 ℃ for 1 day at 200 rpm, and setting 3 repetitions for each treatment, wherein the Saccharum liquid culture medium is used for replacing the strain to be screened to be cultured for a certain time to obtain a control group. The method comprises the steps of determining the aflatoxin levels of a treatment group and a control group by adopting a high performance liquid chromatography-mass spectrometry method in determination of aflatoxins B and G in national food safety standard food of GB 5009.22-2016, and calculating the inhibition rate of strains to be screened on aflatoxins by the following modes:

Aflatoxin inhibition = (control group aflatoxin content-treatment group aflatoxin content)/control group aflatoxin content x 100%

The results of the aflatoxin inhibition calculation are shown in Table 6.

The mycelium is cleaned and dried, the mycelium dry weight of a treatment group and a control group is measured, and the inhibition rate of strains to be screened on the toxigenic bacteria is calculated by the following method:

inhibition ratio of toxigenic bacteria = (dry weight of toxigenic bacteria of control group-dry weight of toxigenic bacteria of treatment group)/dry weight of toxigenic bacteria of control group x 100%

The effect of inhibiting the growth of the toxigenic bacteria was calculated, and the results are shown in Table 6.

TABLE 6 inhibition of aflatoxin and toxigenic bacteria growth by the 20 strains to be screened

According to the research results, the effect of the strains such as bacillus amyloliquefaciens (strain 01), bacillus laterosporus, escherichia coli, flavobacterium breve and the like on inhibiting the growth of aflatoxin and toxigenic bacteria is obviously better than that of other strains, so that the result of the screening technical scheme is confirmed to be correct and reliable.

Example 7 establishment of a method for controlling Aspergillus flavus and toxins thereof and increasing the number of root nodules of peanut root Azotobacter activity

(1) The novel idea of combining peanut endophytic control strains and ectobiocontrol strains is adopted, strains with control effects on the indicator molecule AFT-YJFZ01 in the embodiment 6 are selected, and every 2, every 3 and every 4 strains are mixed to form a series of control bacteria combinations, wherein the proportion of the viable count of any strain in each control bacteria combination is greater than or equal to 1%.

(3) The fungus prevention and control combination is respectively applied to the field along with the peanut sowing base fertilizer or applied to the field in the peanut growing period, and the application amount is more than or equal to 1000 hundred million viable bacteria per mu.

(4) The results of investigation tests on days 3-45 before peanut harvest show that the combination 01 (or 03) -08 (or 09) -15 (or 16) -18 (or 19) -bacillus amyloliquefaciens, brevibacillus laterosporus, enterobacter ludwigii and bacillus mucilaginosus, the combination 08 (or 09) -15 (or 16) -18 (or 19) -Brevibacillus laterosporus, enterobacter ludwigii and bacillus mucilaginosus, the combination 01 (or 03) -15 (or 16) -18 (or 19) -bacillus amyloliquefaciens, enterobacter ludwigii and bacillus mucilaginosus, the combination 01 (or 03) -08 (or 09) -15 (or 16) -bacillus amyloliquefaciens, brevibaciens laterosporus and Enterobacter ludwigii, and the 4-microorganism-species combined treated field peanuts have the excellent characteristics of preventing and controlling the peanut aflatoxin and increasing the root nitrogen-fixing enzyme root nodule quantity of the peanut and promote the peanut growth, and realize the remarkable yield increase compared with the control field peanuts which are not treated by preventing and controlling bacteria.

Example 8 application in peanut production

The above fungus control combinations 01 (or 03) -08 (or 09) -15 (or 16) -18 (or 19) -bacillus amyloliquefaciens, brevibaciens laterosporus, enterobacter ludwigii and Bacillus mucilaginosus of the embodiment 7 are applied to 15 main peanut producing areas nationally, so that good application effects are obtained: firstly, compared with a control area, the peanut rhizosphere soil aflatoxin toxin-producing bacteria in the demonstration area are reduced by 67% on average, and aflatoxin is reduced by 63% on average; the number of root nodules of peanuts in the second and demonstration areas is increased by more than 30 times, even the phenomenon of super nodulation of large-area peanuts occurs, and the root nodules have the activity of the azotase as measured by a conventional azotase activity measuring method, as shown in figure 2. Third, see fig. 3, 4 and 5, the demonstrated zone peanut growth vigor is significantly better than the control zone, and the demonstrated zone leaf spot is also significantly less than the control zone; fourth, the peanut fruits in demonstration areas are plump, the effective full fruit number is obviously increased, the yield of the peanut is increased, and the yield increase rate per mu in most demonstration areas exceeds 10%.

Example 9 application in the production of other leguminous crops

The combination of the prevention and control bacteria of the embodiment 7 of 01 (or 03) -08 (or 09) -15 (or 16) -18 (or 19) -bacillus amyloliquefaciens, bacillus laterosporus, escherichia coli ludwigii and bacillus mucilaginosus is used on crops such as soybeans, alfalfa, milk vetch and the like, has excellent characteristics of preventing and controlling aspergillus flavus and toxins thereof and increasing the number of root nodules of the nitrogen fixation enzyme of peanut roots, promotes the growth of the peanuts, and realizes extremely obvious yield increase.

In conclusion, the research results show that the technical scheme of the invention can be used for simultaneously reducing the aflatoxin and the toxin-producing bacteria level of leguminous crops such as peanuts and the like and increasing the number of nitrogen-fixing enzyme active root nodules of leguminous crops such as peanuts, soybeans, alfalfa, astragalus sinicus and the like, is easy to operate, has strong practicability, is easy to popularize and apply, is favorable for promoting the yield increase of crops, has economic benefits, is favorable for improving the farmland soil environment, has ecological benefits, is favorable for reducing the application of farmland fertilizers and pesticides, and has important significance for carbon peak and carbon neutralization. The obtaining areas of the bacillus amyloliquefaciens, the bacillus laterosporus, the bacillus mucilaginosus and the escherichia coli of the ludwigi are not limited, and the obtaining mode is screening or commercial purchase.

The invention is based on (1) the aflatoxin-producing virulence indicator molecule AFT-YJFZ01 peptide, once inhibited, the source of the aflatoxin biosynthesis pathway is also inhibited; (2) For the same toxigenic strain, the toxigenic strain toxigenic indicator molecule AFT-YJFZ01 peptide of the aflatoxin has a positive correlation with the growth of the strain, so that the one-step efficient screening can be successfully invented, the effective prevention and control bacteria capable of inhibiting the synthesis of the aflatoxin from sources and the growth of the aflatoxin toxigenic strain can be inhibited, and the method can be applied to screening out the aflatoxin prevention and control bacteria such as bacillus amyloliquefaciens, brevibaciens, bacillus mucilaginosus and escherichia coli; (3) The method and the application thereof which are used for preventing and controlling peanut aspergillus flavus and toxins thereof and increasing the nitrogen fixation enzyme active root nodule quantity of peanut root are successfully invented, and the method and the application thereof are applied to leguminous crops such as peanuts, soybeans, alfalfa, astragalus sinicus and the like to reduce the aflatoxin and the toxin-producing bacteria level thereof, increase the root nodule quantity, promote the yield increase and provide key technical support for farmland weight loss and medicine reduction, carbon peak and carbon neutralization.

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

Method for preventing and controlling aspergillus flavus and toxins thereof and increasing root nitrogen fixation enzyme activity root nodule number of leguminous crops and application thereof

＜160＞ 1

＜210＞ 1

＜211＞ 33172

＜212＞ PRT

< 213 > Aspergillus flavus

＜400＞ 1

AIGVEEPEAD PTYYHNAIGV EEPEADPTYY HNNKAIGVEE PEADPTYYHN NKTTASMVWE 60

EAQQVSGKAS VETPASIEAA SELSKAVSPS FEDVWSQPRD GAGQMFIPLN PNAYSPNTLN 120

KDGAGQMFIP LNPNAYSPNT LNKGSPKDGV YVDKSVTSGF VDGIKDGLRD VGGPIEDQNS 180

LQVGDRDVHG FATRFEQLPI NQPRFGKPVG AVGSAAFGKP VGAVGSAATA LKGDNEIPQA 240

ATAHDSAWDF FSQQPSGDNE IPQAATAHDS AWDFFSQQPS SLHGPNFEQL PINQPRGPTL 300

LEDFIFRGVD FTEDPLLQGR GVGAHGVFTS YHGGPNFEQL PINQPRHVDG FGIHTFRLAS 360

VETPASIEAA SELSKLFSYL DTQLNRLFYN SLTPAEQQFV VDAIRNAGIQ TSRNAGIQTS 420

RDGVYVDKNS LTPAEQQFVV DAIRNYFAET EQVMFQPGHN YFAETEQVMF QPGHIVRPEE 480

YVPITKPLQI VIDGFRPNAY SPNTLNKPVG AVGSAATALK QDLFEAIEAG RQLSEDGVDV 540

VVVAERSEDG VDVVVVAERS LTPAEQQFVV DAIRSMVWEE AQQVSGKSPS FEDVWSQPRS 600

VETPASIEAA SELSKSVTSG FVDGIKDGLR TTDVGTFGQK VDFTEDPLLQ GRVETPASIE 660

AASELSKVGF LASVETPASI EAASELSKYP EWELGVQIMD EEDQLKFDHE RVPERFGFDL 720

FDPTKFDLFD PTKFGKPVGA VGSAATNPDF MRQDLFEAIE AGRFVTDNGD SKLVKFVTDN 780

GDSKSVTSGF VDGIKGFDLF DPTKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKIVPEEY 840

VPITKDGVYV DKSVTSGFVD GIKFENSNVK SSVVRVGGPI EDQNSLQVGD RGDNEIPQAA 900

TAHDSVTSGF VDGIKDTTDV GTFGQKLKGV GAHGVFTGDN EIPQAATAHF VVDAIRTLLE 960

DFIFRFTEDP LLQGRPDLIH AVKPRVDGFG IHTFRGVGAH GVFYLDTQLN RNVIIQLNRK 1020

PVGAVGSAAT ALKDGVYVDK NNVIIQLNRI VPEEYVPITK LGKTPAEQQF VVDAIRFENS 1080

NVKEQVMFQP GHIVRGVDFT EDPLLQGRAI GVEEPEADPT YYHNAIGVEE PEADPTYYHN 1140

ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKASMVWEEA QQVSGKASMV 1200

WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKASMVWEEA 1260

QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS 1320

GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS 1380

MVWEEAQQVS GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE 1440

EAQQVSGKAS MVWEEAQQVS GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ 1500

VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKAVSPSFED VWSQPRAVSP SFEDVWSQPR 1560

AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED VWSQPRAVSP 1620

SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED 1680

VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ 1740

PRAVSPSFED VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV 1800

SPSFEDVWSQ PRAVSPSFED VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF 1860

EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED VWSQPRDGAG QMFIPLNPNA YSPNTLNKDG 1920

AGQMFIPLNP NAYSPNTLNK DGAGQMFIPL NPNAYSPNTL NKDGAGQMFI PLNPNAYSPN 1980

TLNKDGAGQM FIPLNPNAYS PNTLNKDGAG QMFIPLNPNA YSPNTLNKDG AGQMFIPLNP 2040

NAYSPNTLNK GSPKDGAGQM FIPLNPNAYS PNTLNKGSPK DGVYVDKDGV YVDKDGVYVD 2100

KSVTSGFVDG IKDGVYVDKS VTSGFVDGIK FENSNVKSSV VRFGFDLFDP TKFGFDLFDP 2160

TKFGKPVGAV GSAATALKFG KPVGAVGSAA TALKFGKPVG AVGSAATALK FGKPVGAVGS 2220

AATALKFGKP VGAVGSAATA LKFGKPVGAV GSAATALKFG KPVGAVGSAA TALKFGKPVG 2280

AVGSAATALK FGKPVGAVGS AATALKFGKP VGAVGSAATA LKFGKPVGAV GSAATALKFG 2340

KPVGAVGSAA TALKFGKPVG AVGSAATALK FVTDNGDSKF VTDNGDSKLV KFVTDNGDSK 2400

LVKFVTDNGD SKLVKGPTLL EDFIFRGVDF TEDPLLQGRG VDFTEDPLLQ GRGVDFTEDP 2460

LLQGRGVDFT EDPLLQGRGV DFTEDPLLQG RGVDFTEDPL LQGRHGGPNF EQLPINQPRH 2520

GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2580

GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2640

GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2700

GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2760

GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2820

GGPNFEQLPI NQPRHGGPNF EQLPINQPRH VDGFGIHTFR HVDGFGIHTF RHVDGFGIHT 2880

FRHVDGFGIH TFRHVDGFGI HTFRHVDGFG IHTFRHVDGF GIHTFRHVDG FGIHTFRHVD 2940

GFGIHTFRHV DGFGIHTFRH VDGFGIHTFR HVDGFGIHTF RHVDGFGIHT FRHVDGFGIH 3000

TFRHVDGFGI HTFRHVDGFG IHTFRHVDGF GIHTFRHVDG FGIHTFRHVD GFGIHTFRHV 3060

DGFGIHTFRI VPEEYVPITK IVPEEYVPIT KIVPEEYVPI TKLFSYLDTQ LNRLFSYLDT 3120

QLNRLFSYLD TQLNRLFSYL DTQLNRLFYN SLTPAEQQFV VDAIRLFYNS LTPAEQQFVV 3180

DAIRNAGIQT SRNAGIQTSR DGVYVDKNNV IIQLNRNYFA ETEQVMFQPG HNYFAETEQV 3240

MFQPGHIVRN YFAETEQVMF QPGHIVRNYF AETEQVMFQP GHIVRNYFAE TEQVMFQPGH 3300

IVRNYFAETE QVMFQPGHIV RNYFAETEQV MFQPGHIVRN YFAETEQVMF QPGHIVRNYF 3360

AETEQVMFQP GHIVRNYFAE TEQVMFQPGH IVRNYFAETE QVMFQPGHIV RNYFAETEQV 3420

MFQPGHIVRN YFAETEQVMF QPGHIVRNYF AETEQVMFQP GHIVRNYFAE TEQVMFQPGH 3480

IVRNYFAETE QVMFQPGHIV RQDLFEAIEA GRQDLFEAIE AGRQLSEDGV DVVVVAERQL 3540

SEDGVDVVVV AERQLSEDGV DVVVVAERQL SEDGVDVVVV AERSLQGKAS MVWEEAQQVS 3600

GKSLQGKASM VWEEAQQVSG KSPSFEDVWS QPRSVTSGFV DGIKSVTSGF VDGIKDGLRS 3660

VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS 3720

VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS 3780

VTSGFVDGIK DGLRTTDVGT FGQKTTDVGT FGQKTTDVGT FGQKTTDVGT FGQKTTDVGT 3840

FGQKLKTTDV GTFGQKLKVG FLASVETPAS IEAASELSKV GFLASVETPA SIEAASELSK 3900

VGFLASVETP ASIEAASELS KVGFLASVET PASIEAASEL SKVGFLASVE TPASIEAASE 3960

LSKVGFLASV ETPASIEAAS ELSKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKVPVHNN 4020

NRDGAGQMFI PLNPNAYSPN TLNKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKYPEWEL 4080

GVQIMDEEDQ LKYPEWELGV QIMDEEDQLK ALFNRDIATG KANNYCSNQV EGPYSLYSGR 4140

DIATGKVSIA KDYACPWNGG EEVSLKDYAC PWNGGEEVSL KVEYSDAAKE GDPEMYGNNE 4200

TVNKVCAKAN NYCSNQVEGP YSLYSGREPG ICETTPGVKE QTASVVNGTA VIKGFSATGD 4260

YPRGGPGSSS MIGLMQENGP CRGYLEDIAY VLDSGIKGYY DISHFDPDIG LMQENGPCRL 4320

AAEGDPEMYG NNETVNKNAP LSIWMNGGPG SSSMIGLMQE NGPCRNQVEG PYSLYSGRNY 4380

CSNQVEGPYS LYSGRPGGCK DQIIECRQVE GPYSLYSGRQ YGNFSFTRSN QVEGPYSLYS 4440

GRTNASYVGG LVRTVYDMAM EAWSKTVYDM AMEAWSKPGG TVYDMAMEAW SKPGGCKVAL 4500

VYGDRDYACP WNGGEEVSLK VFEAGHEVPA YQPETAYEIF VFEAGHEVPA YQPETAYEIF 4560

HRVSIWTESY GGRYCSNQVE GPYSLYSGRY GPSFTAFFQE QNEKVALVYG DRVEYSDAAK 4620

FRISYKEPGI CETTPGVKFT AFFQEQNEKS IWTESYGGRN YIVVDADSSF WFFESRISYK 4680

EPGIHDDRVS IWTESYGGRY GPSFTAFFQE QNEKVALVYG DRDYAANNYC SNQVEGPYSL 4740

YSGRANNYCS NQVEGPYSLY SGRANNYCSN QVEGPYSLYS GRANNYCSNQ VEGPYSLYSG 4800

RANNYCSNQV EGPYSLYSGR ANNYCSNQVE GPYSLYSGRA NNYCSNQVEG PYSLYSGRAN 4860

NYCSNQVEGP YSLYSGRANN YCSNQVEGPY SLYSGRANNY CSNQVEGPYS LYSGRANNYC 4920

SNQVEGPYSL YSGRANNYCS NQVEGPYSLY SGRANNYCSN QVEGPYSLYS GRANNYCSNQ 4980

VEGPYSLYSG RANNYCSNQV EGPYSLYSGR ANNYCSNQVE GPYSLYSGRA NNYCSNQVEG 5040

PYSLYSGRAN NYCSNQVEGP YSLYSGRANN YCSNQVEGPY SLYSGRDIAT GKVSIAKDYA 5100

CPWNGGEEVS LKDYACPWNG GEEVSLKGDP EMYGNNETVN KVCAKANNYC SNQVEGPYSL 5160

YSGRGGPGSS SMIGLMQENG PCRIGLMQEN GPCRIGLMQE NGPCRISYKE PGICETTPGV 5220

KISYKEPGIC ETTPGVKISY KEPGICETTP GVKISYKEPG ICETTPGVKI SYKEPGICET 5280

TPGVKISYKE PGICETTPGV KISYKEPGIC ETTPGVKNAP LSIWMNGGPG SSSMIGLMQE 5340

NGPCRNAPLS IWMNGGPGSS SMIGLMQENG PCRNAPLSIW MNGGPGSSSM IGLMQENGPC 5400

RNAPLSIWMN GGPGSSSMIG LMQENGPCRN APLSIWMNGG PGSSSMIGLM QENGPCRNAP 5460

LSIWMNGGPG SSSMIGLMQE NGPCRNAPLS IWMNGGPGSS SMIGLMQENG PCRNAPLSIW 5520

MNGGPGSSSM IGLMQENGPC RNAPLSIWMN GGPGSSSMIG LMQENGPCRN APLSIWMNGG 5580

PGSSSMIGLM QENGPCRNAP LSIWMNGGPG SSSMIGLMQE NGPCRPGGCK DQIIECRTVY 5640

DMAMEAWSKT VYDMAMEAWS KTVYDMAMEA WSKTVYDMAM EAWSKTVYDM AMEAWSKTVY 5700

DMAMEAWSKT VYDMAMEAWS KTVYDMAMEA WSKPGGTVYD MAMEAWSKPG GTVYDMAMEA 5760

WSKPGGCKTV YDMAMEAWSK PGGCKTVYDM AMEAWSKPGG CKTVYDMAME AWSKPGGCKT 5820

VYDMAMEAWS KPGGCKTVYD MAMEAWSKPG GCKTVYDMAM EAWSKPGGCK TVYDMAMEAW 5880

SKPGGCKVAL VYGDRDYACP WNGGEEVSLK VALVYGDRDY ACPWNGGEEV SLKVALVYGD 5940

RDYACPWNGG EEVSLKVALV YGDRDYACPW NGGEEVSLKV ALVYGDRDYA CPWNGGEEVS 6000

LKVALVYGDR DYACPWNGGE EVSLKVALVY GDRDYACPWN GGEEVSLKVA LVYGDRDYAC 6060

PWNGGEEVSL KVALVYGDRD YACPWNGGEE VSLKVALVYG DRDYACPWNG GEEVSLKVFE 6120

AGHEVPAYQP ETAYEIFHRV FEAGHEVPAY QPETAYEIFH RVFEAGHEVP AYQPETAYEI 6180

FHRVFEAGHE VPAYQPETAY EIFHRVFEAG HEVPAYQPET AYEIFHRVFE AGHEVPAYQP 6240

ETAYEIFHRV FEAGHEVPAY QPETAYEIFH RVFEAGHEVP AYQPETAYEI FHRVFEAGHE 6300

VPAYQPETAY EIFHRVSIWT ESYGGRVSIW TESYGGRVSI WTESYGGRVS IWTESYGGRV 6360

SIWTESYGGR VSIWTESYGG RVSIWTESYG GRVSIWTESY GGRVSIWTES YGGRVSIWTE 6420

SYGGRVSIWT ESYGGRVSIW TESYGGRYGP SFTAFFQEQN EKYGPSFTAF FQEQNEKYGP 6480

SFTAFFQEQN EKAAWLFEDS QAKADEINQI FDAISYMKAD VPSGSTNITH GRAFPCFDEP 6540

ALKAGMIADA GALASSGYQS TSGLLSLLKA VEQSLDAIRD GHILQQFKFA AGETSAIHPN 6600

IRGFDNEAEF IVWNEIVARI VDVLLDEKIV DVLLDEKNSG ASRLNADHSA IYRLTFTGIL 6660

NDNMAGFYRN GGEKEYNVVY DRNQDIYMPL GGLRNVGFPV VTVAEDAASS SIKSSHPIEV 6720

PVKSSHPIEV PVKRTGDVRP EEDTTLYPVM LGLRTHEIGW EFSEKTKQGL DENTMLTERT 6780

LGLALSDEVK VYATPDQDIE HGRYLASTQM EPTDARYLGE DVFIQGVRQG LLTVEDRGSV 6840

FSIVLKAAQE MFQRQGLDEN TMLTERTDVE SWLKAGMIAD AGALASSGYQ STSGLLSLLK 6900

FAAGETSAIH PNIRLTFTGI LNDNMAGFYR NGGEKEYNVV YDRNGGEKEY NVVYDRNGGE 6960

KEYNVVYDRN GGEKEYNVVY DRNQDIYMPL GGLRVYATPD QDIEHGRYLA STQMEPTDAR 7020

AWYENGITNC VGDNTRCCDS GVEQLVSFSD VSDFKDADAC NGGGIEYDSP ADTPLEFKDN 7080

TCNAPIPVSF PVAPTDTKDP YMFHQANLRD QCNYSLQYTI GNKFAANGNY GSETTAAVIN 7140

NFNGRFTTSA SDGFDGMQVN PRGNGVIEAA AGKGNVAGNI LVIAKITTAD MDGISSWLPT 7200

INGKIYYNCD TPACTVQEWI DTSAGSGDFS NLLATEKKPL GTGTDLWPKL DDLFVWWTTP 7260

ANRLVDWPIV TITHQEMSAN MNAGSSYFVE VGHNMNAGSS YFVEVGHNGN GVIEAAAGKQ 7320

AWVETMVQEF VRSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 7380

VNPRVIANGN VAGNILVIAK YFTSNGIIPP AITGLHNGDA LRQITGVTLS AKSASDGFDG 7440

MQVNPRDPYM FHQANSDVSD FKEAGLKGII PPAITGLHNG DALRYFTSNG IIPPAITGLH 7500

NEISFNQAWL RLVDWPIVTI THQEMSANFL DRKPLGTGTD LANGNVAGNI LVIAKHQEMS 7560

ANFLDRYNRD QCNYSLQYTI GNKAWYENGI TNCVGDNTRA WYENGITNCV GDNTRAWYEN 7620

GITNCVGDNT RAWYENGITN CVGDNTRAWY ENGITNCVGD NTRAWYENGI TNCVGDNTRA 7680

WYENGITNCV GDNTRAWYEN GITNCVGDNT RDADACNGGG IEYDSPADTP LEFKDADACN 7740

GGGIEYDSPA DTPLEFKDAD ACNGGGIEYD SPADTPLEFK DNTCNAPIPV SFPVAPTDTK 7800

DNTCNAPIPV SFPVAPTDTK DPYMFHQAND PYMFHQANLR DPYMFHQANL RDPYMFHQAN 7860

LRDPYMFHQA NLRDQCNYSL QYTIGNKEIS FNQAWLREIS FNQAWLREIS FNQAWLREIS 7920

FNQAWLREIS FNQAWLREIS FNQAWLREIS FNQAWLRFAA NGNYGSETTA AVINNFNGRF 7980

AANGNYGSET TAAVINNFNG RFAANGNYGS ETTAAVINNF NGRFAANGNY GSETTAAVIN 8040

NFNGRFAANG NYGSETTAAV INNFNGRFAA NGNYGSETTA AVINNFNGRF AANGNYGSET 8100

TAAVINNFNG RFAANGNYGS ETTAAVINNF NGRFAANGNY GSETTAAVIN NFNGRITTAD 8160

MDGISSWLPT INGKITTADM DGISSWLPTI NGKITTADMD GISSWLPTIN GKITTADMDG 8220

ISSWLPTING KITTADMDGI SSWLPTINGK KPLGTGTDLW PKKPLGTGTD LWPKKPLGTG 8280

TDLWPKKPLG TGTDLWPKKP LGTGTDLWPK KPLGTGTDLW PKKPLGTGTD LWPKKPLGTG 8340

TDLWPKKPLG TGTDLWPKKP LGTGTDLWPK KPLGTGTDLW PKKPLGTGTD LWPKLDDLFV 8400

WWTTPANRLD DLFVWWTTPA NRLDDLFVWW TTPANRLDDL FVWWTTPANR LVDWPIVTIT 8460

HQEMSANLVD WPIVTITHQE MSANFLDRLV DWPIVTITHQ EMSANFLDRM NAGSSYFVEV 8520

GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA 8580

AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS 8640

YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN 8700

GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKTPLLNQ QNSMWPYFTT SASDGFDGMQ 8760

VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8820

VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8880

VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8940

VNPRVIANGN VAGNILVIAK VIANGNVAGN ILVIAKYFTS NGIIPPAITG LHNGDALRAS 9000

HTVDKNGIWS SEVKATDNYI ANAAAAVAKD LLQDIVTWDD KEAGIYLIAR GPLNEGGLYA 9060

ERGTNYVALS LWALESDGAK HTDYSSQEST SYKLGSFELS YTTPVLTGYG NVESPEQPKL 9120

SGQDASAITW KLTGNLGGED YQDKLTGNLG GEDYQDKVRN SAYNYWVPEL PTEGTSPGFS 9180

TSKPGIGFYT AQFDLDLPKQ GFHQPQPPSE SWESGSPLEG LSKSPGSFFV VRSSYDDSAW 9240

VSADLPKTSY DYGSPITETR YPDADYMQYV MDQARNGIWS SEVKVLVLYG GPKASPSYLT 9300

ATPRYLDTLP EIKTLHLEQS PSTPYAQLYV NGYQYGKKAD IVVPFPWGGP GFEKAQLYVN 9360

GYQYGKAQLY VNGYQYGKAT DNYIANAAAA VAKLSGQDAS AITWKLTGNL GGEDYQDKVR 9420

NGIWSSEVKQ GFHQPQPPSE SWESGSPLEG LSKSSYDDSA WVSADLPKYP DADYMQYVMD 9480

QARYPDADYM QYVMDQARAL TNGAGAIKEA IADVLEHLGE NDEDIAVYAP NPFYKGFAPL 9540

EYLGSNFENG ELPKGFDNAG FVMGTSSSLF NQFILRGKMP MPILVADGRH FQLINTAAYW 9600

KIPNVAIAVS GGGYRLNGTD IPNFLKLNLS SFDASGYIDR LPDICNTCFK MPMPILVADG 9660

RNSILEGPDV KSAAALSTSE KDWLQVRSSS LFNQFILRTF LNLGLNKTNT KLPDICNTCF 9720

KTSLTDYWGR SSFDASGYID RSTSEKDWLQ VRGTDIPNFL KLNLGLNKGF DNAGFVMGTS 9780

SSLFNQFEYL GSNFENGELP KMPILVADGR DLYDAVKINT AAYWKSIALG DDFKKALTNG 9840

AGAIKALTNG AGAIKALTNG AGAIKALTNG AGAIKGKMPM PILVADGRGK MPMPILVADG 9900

RGKMPMPILV ADGRGKMPMP ILVADGRGKM PMPILVADGR GKMPMPILVA DGRHFQLINT 9960

AAYWKHFQLI NTAAYWKLPD ICNTCFKLPD ICNTCFKMPI LVADGRMPMP ILVADGRMPM 10020

PILVADGRMP MPILVADGRM PMPILVADGR MPMPILVADG RMPMPILVAD GRMPMPILVA 10080

DGRMPMPILV ADGRMPMPIL VADGRNSILE GPDVKNSILE GPDVKNSILE GPDVKSAAAL 10140

STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS 10200

AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL 10260

QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE 10320

KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL 10380

STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS 10440

AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL 10500

QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL STSEKDWLQV RTNTKLPDIC 10560

NTCFKTNTKL PDICNTCFKT NTKLPDICNT CFKAASLPAS FSGFKALVSH DGTFVADAKE 10620

NSLVWHQQVL GWLNKFNAVF SGTLKGDAGS PVFSPDSKGD AGSPVFSPDS KKIAYWQMAD 10680

ESYEADHRIQ AFVIYPENFD KLFSIPADAG DDYKPKLNPE GLISAPRLPV SEGLSLFNIL 10740

QERMINWIQG SDLGRNAESP YPPFGGASDY DLSPDGKRSE AIPNPSGDVA VFSQSQYSFK 10800

SEAIPNPSGD VAVFSQSQYS FKTAVPINGP DSPGTPEGVK TAVPINGPDS PGTPEGVKGD 10860

AGSPVFSPDS KTLATANKID PELKTLYVYT VGSEETIPSL AADWDRTTSQ WNVLDLKVVT 10920

TDSGDVRWIQ GSDLGRALVS HDGTFVADAK ALVSHDGTFV ADAKGDAGSP VFSPDSKKIA 10980

YWQMADESYE ADHRIAYWQM ADESYEADHR IAYWQMADES YEADHRLFSI PADAGDDYKP 11040

KLFSIPADAG DDYKPKLNPE GLISAPRRSE AIPNPSGDVA VFSQSQYSFK RSEAIPNPSG 11100

DVAVFSQSQY SFKTAVPING PDSPGTPEGV KTAVPINGPD SPGTPEGVKT AVPINGPDSP 11160

GTPEGVKGDA GSPVFSPDSK TTSQWNVLDL KTTSQWNVLD LKAASNFDGD TLVLGYDSGN 11220

GNPETSFMTL MRAGSTIAVT DVQITGGAVG IKASGTCSGP IQSAPTSYWL ADQDHSGDAR 11280

DAGSPKPVVQ IGHEGDVGVA EIQNMRFSVA EILPGAKGDG STDDSASLNA ILANNAANCK 11340

GTCSGPIQSA PTSYWLADQD HSGDARIVGE AWAVITGAGD AFKNSQILIQ NLSHDNSNAI 11400

AVDSKNSQIL IQNLSHDNSN AIAVDSKDNI KNVVLDTTAL SANTKSAPTS YWLADQDHSG 11460

DARVGTIITG DPLDPPVLKV TNSPSNLVWY SISTRYPAEV FLPGGTYQLG KSLGSLVLLD 11520

SSSINSGPVV RDTLVIPPGS RAQPTYAEYS NDQIVNVKSG TCSGPIQSAP TSYWLADQDH 11580

SGDARAGSTI AVTDVQITGG AVGIKAQPTY AEYSNDQIVN VKAQPTYAEY SNDQIVNVKD 11640

AGSPKPVVQI GHEGDVGVAE IQNMRFSVAE ILPGAKNSQI LIQNLSHDNS NAIAVDSKNS 11700

QILIQNLSHD NSNAIAVDSK NSQILIQNLS HDNSNAIAVD SKNSQILIQN LSHDNSNAIA 11760

VDSKDNIKNS QILIQNLSHD NSNAIAVDSK DNIKNSQILI QNLSHDNSNA IAVDSKDNIK 11820

NVVLDTTALS ANTKSAPTSY WLADQDHSGD ARVTNSPSNL VWYSISTRVT NSPSNLVWYS 11880

ISTRAGALLL GKAGVIPESL HQDTVGTFGK DRLETTAGSW ALLGSVVPRG IMDETYYQAL 11940

EFCQRGKTPE GGYAQFLTNK GPLHGIPFIV KIHQTQPYLN AILQVNPDAF KLETTAGSWA 12000

LLGSVVPRNS VVGIKPTVGL TSRTIVSPDG FNWDYGSTRT PEGGYAQFLT NKTTREEGID 12060

AALKVDFYNN LKDYLSEVEN TKAALSEWAD MRALGTETDG SVINPAQREE GIDAALKDAV 12120

YALDAIYGID ARAALSEWAD MRAALSEWAD MRAGVIPESL HQDTVGTFGK AGVIPESLHQ 12180

DTVGTFGKAG VIPESLHQDT VGTFGKAGVI PESLHQDTVG TFGKGIMDET YYQALEFCQR 12240

IHQTQPYLNA ILQVNPDAFK NSVVGIKPTV GLTSRNSVVG IKPTVGLTSR NSVVGIKPTV 12300

GLTSRTIVSP DGFNWDYGST RTTREEGIDA ALKAVWPGDM GVAVPAAFVS TGDLESVKAY 12360

QGYFHSNDDL LNRDGSASYV MPDKDRAVWP GDMGVAVPAA FVSTGDLESV KDSAFPGLWE 12420

ENIYAPSSRG GSGALGLAFS EAKGVYYVDD TATITVSGGG SHRIWYSGAY TLQTNAVPVN 12480

TGRNALQTMY DTQDKNALQT MYDTQDKTTG AFDESGPPLS QKSPDGYTLQ FSVPPGTKSS 12540

EKPSITIDGN NINKTTGAFD ESGPPLSQKI APQFGDLKAL ELIRRFQASW DKSPDGYTLQ 12600

FSVPPGTKIS SFEIQGHFKA VWPGDMGVAV PAAFVSTGDL ESVKAYQGYF HSNDDLLNRA 12660

YQGYFHSNDD LLNRAYQGYF HSNDDLLNRA YQGYFHSNDD LLNRDGSASY VMPDKDGSAS 12720

YVMPDKDRAV WPGDMGVAVP AAFVSTGDLE SVKGVYYVDD TATITVSGGG SHRGVYYVDD 12780

TATITVSGGG SHRISSFEIQ GHFKNALQTM YDTQDKNALQ TMYDTQDKNA LQTMYDTQDK 12840

NALQTMYDTQ DKTTGAFDES GPPLSQKNAL QTMYDTQDKT TGAFDESGPP LSQKNALQTM 12900

YDTQDKTTGA FDESGPPLSQ KNALQTMYDT QDKTTGAFDE SGPPLSQKSP DGYTLQFSVP 12960

PGTKSPDGYT LQFSVPPGTK SSEKPSITID GNNINKSSEK PSITIDGNNI NKSSEKPSIT 13020

IDGNNINKTT GAFDESGPPL SQKDNILPEN LDDGLPSQFV YEKFGPHEYN GDQYTSIIRL 13080

VIDLSGNGGG YILQGYDTFR NVGLVSVSLD GKPSSDPMQG IGGIKQLFPS IVQDGYTRSD 13140

KYAGEYEFQA DLFKSFEPST PAEFQAVLEK YAGEYEFQAD LFKPFAASTP GFDGYFSGSA 13200

RAASTPGFDG YFSGSARYDL NLENKAFNLA HDGHFRHFTS LEEKFFPDLL TKSIAIGGRP 13260

SSDPMQGIGG IKFGPHEYNG DQYTSIIRFG PHEYNGDQYT SIIRQLFPSI VQDGYTRQLF 13320

PSIVQDGYTR QLFPSIVQDG YTRSDKYAGE YEFQADLFKE PGAEGVCETT PGVKFANQMP 13380

NGCQDLISTC KGCQDLISTC KQLPKNPTGV KSAGYTPLKV NGVEYGETRS YSGYVDTSPE 13440

SHTFTALADY ALCAEATNMC RTIFGWDIAE GQKTIFGWDI AEGQKKVYEA GHEVPYYQPI 13500

ASLYKEPGAE GVCETTPGVK LSGLPSLDSR SYSGYVDTSP ESHTFFHNPE TAPITLWLNK 13560

IWPSYKVYEA GHEVPYYQPI ASVNGVEYGE TRTALADYAL CAEATNMCAE GVCETTPGVK 13620

FANQMPNGCQ DLISTCKFAN QMPNGCQDLI STCKFANQMP NGCQDLISTC KFANQMPNGC 13680

QDLISTCKFA NQMPNGCQDL ISTCKFANQM PNGCQDLIST CKKIWPSYKK IWPSYKSAGY 13740

TPLKVNGVEY GETRSAGYTP LKVNGVEYGE TRSAGYTPLK VNGVEYGETR SAGYTPLKVN 13800

GVEYGETRSA GYTPLKVNGV EYGETRSAGY TPLKVNGVEY GETRSAGYTP LKVNGVEYGE 13860

TRSAGYTPLK VNGVEYGETR SAGYTPLKVN GVEYGETRSA GYTPLKVNGV EYGETRSAGY 13920

TPLKVNGVEY GETRTALADY ALCAEATNMC RTALADYALC AEATNMCRTA LADYALCAEA 13980

TNMCRTALAD YALCAEATNM CRTALADYAL CAEATNMCRT ALADYALCAE ATNMCRTALA 14040

DYALCAEATN MCRTALADYA LCAEATNMCR TALADYALCA EATNMCRTIF GWDIAEGQKT 14100

IFGWDIAEGQ KTIFGWDIAE GQKTIFGWDI AEGQKTIFGW DIAEGQKTIF GWDIAEGQKT 14160

IFGWDIAEGQ KTIFGWDIAE GQKTIFGWDI AEGQKTIFGW DIAEGQKTIF GWDIAEGQKT 14220

IFGWDIAEGQ KTIFGWDIAE GQKKVNGVEY GETRVNGVEY GETRVNGVEY GETRYKEPGA 14280

EGVCETTPGV KYKEPGAEGV CETTPGVKYK EPGAEGVCET TPGVKYKEPG AEGVCETTPG 14340

VKYKEPGAEG VCETTPGVKA SDFWANELVT WWNKEAEPSQ EYVSYSHGVF LRFSYEEGEK 14400

FLNKGAKDDV FIKGGSILPM QEVALTTRKA TGDVLFNTKN AHGQEILLRN HNVLSAIPQE 14460

PYRQYQLSTV GLPAMQQYNT LGFHQCRSSE AEPSQEYVSY SHGVFLRTLG GSVDLTFYSG 14520

PTQAEVTKWA SVIDATKSEA EPSQEYVSYS HGVFLRAEPS QEYVSYSHGV FLRAEPSQEY 14580

VSYSHGVFLR AEPSQEYVSY SHGVFLRAEP SQEYVSYSHG VFLRASDFWA NELVTWWNKE 14640

AEPSQEYVSY SHGVFLREAE PSQEYVSYSH GVFLREAEPS QEYVSYSHGV FLRFSYEEGE 14700

KFLNKGGSIL PMQEVALTTR GGSILPMQEV ALTTRNAHGQ EILLRNAHGQ EILLRNHNVL 14760

SAIPQEPYRN HNVLSAIPQE PYRNHNVLSA IPQEPYRNHN VLSAIPQEPY RNHNVLSAIP 14820

QEPYRSEAEP SQEYVSYSHG VFLRWASVID ATKWASVIDA TKDPSINDDS VMIYAPAVRF 14880

LDEALTYPPP KGIQINDPSI NDDSVMIYAP AVRIASAMLD EEDEKYFNVK NGDQSPPSAL 14940

GPLPSVIERP SINDDSVMIY APAVRTDYSV CGETTIFKVY LTGESYAGQY IPYIASAMLD 15000

EEDEKVYLTG ESYAGQYIPY IASAMLDEED EKYFNVKIAS AMLDEEDEKT DYSVCGETTI 15060

FKNGDQSPPS ALGPLPSVIE RVYLTGESYA GQYIPYTLIA GAGLLGTAHT ERETTIFKNG 15120

DQSPPSALGP LPSVIERTDV QKALHVPRDD SVMIYAPAVR FLDEALTYPP PKGIQINDPS 15180

INDDSVMIYA PAVRGIQIND PSINDDSVMI YAPAVRGIQI NDPSINDDSV MIYAPAVRGI 15240

QINDPSINDD SVMIYAPAVR IASAMLDEED EKIASAMLDE EDEKYFNVKI ASAMLDEEDE 15300

KYFNVKIASA MLDEEDEKYF NVKIASAMLD EEDEKYFNVK IASAMLDEED EKYFNVKIAS 15360

AMLDEEDEKY FNVKIASAML DEEDEKYFNV KNGDQSPPSA LGPLPSVIER TDYSVCGETT 15420

IFKNGDQSPP SALGPLPSVI ERTDYSVCGE TTIFKNGDQS PPSALGPLPS VIERVYLTGE 15480

SYAGQYIPYI ASAMLDEEDE KYFNVKAHIL PPNGRDLNPN GSQFITPGGK DLNPNGSQFI 15540

TPGGKNDPVA VFDGSVIPKE AGLVPFQVSP TTKFHVLTAQ LSFPRFRDLN PNGSQFITPG 15600

GKLDRPPVIP LPPSDSDVTA FRNDPVAVFD GSVIPKPVAV FDGSVIPKTI SNVVDNELAR 15660

TTNGIVSTNE SGRDPVAVFD GSVIPKFALS TWARILPATS QVSTKAHILP PNGRAHILPP 15720

NGRAHILPPN GRDLNPNGSQ FITPGGKFAL STWARFALST WARFHVLTAQ LSFPRFHVLT 15780

AQLSFPRFRD LNPNGSQFIT PGGKFRDLNP NGSQFITPGG KFRDLNPNGS QFITPGGKND 15840

PVAVFDGSVI PKTISNVVDN ELARTISNVV DNELARTISN VVDNELARTT NGIVSTNESG 15900

RDWSDSYYQG PAFKFGLGAD NTLAFEVVTA DGQLVTASRG VGSDAWTVSE SGRITNEYVP 15960

QLEAVTPGSG CYQNEGNFRN VLENNPTGMA SVLRSKWDPN NFFYVLKTTA LTDLGIAYKV 16020

SAGVMGYQIL NAAHAKVSYT EYDSYYDHYN KYMGPLPYGN LAVATYQYGG RWDPNNFFYV 16080

LKDWSDSYYQ GPAFKGVGSD AWTVSESGRG VGSDAWTVSE SGRGVGSDAW TVSESGRGVG 16140

SDAWTVSESG RITNEYVPQL EAVTPGSGCY QNEGNFRNVL ENNPTGMASV LRNVLENNPT 16200

GMASVLRVSA GVMGYQILNA AHAKVSAGVM GYQILNAAHA KVSAGVMGYQ ILNAAHAKVS 16260

AGVMGYQILN AAHAKVSAGV MGYQILNAAH AKVSAGVMGY QILNAAHAKV SAGVMGYQIL 16320

NAAHAKVSYT EYDSYYDHYN KYMGPLPYGN LAVATYQYGG RALMNGAGAI KDAGYETSIT 16380

DYWGRDFFNH VTIKDGNWTT CVGCAILSRG FVPLEYVGSK HVYDAVQDKP TVYGFVPLEY 16440

VGSKTNTQVP DACTQCFQKQ ADMPMPLLVA DGRTAFSDIL AKSIALTDTF KILDSATYYK 16500

ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK 16560

ALMNGAGAIK ALMNGAGAIK ALMNGAGAIK DAGYETSITD YWGRDAGYET SITDYWGRDA 16620

GYETSITDYW GRDAGYETSI TDYWGRDAGY ETSITDYWGR DFFNHVTIKD FFNHVTIKDF 16680

FNHVTIKDFF NHVTIKGFDN AGFVMGTSSS LFNQFMPLLV ADGRMPLLVA DGRMPMPLLV 16740

ADGRMPMPLL VADGRMPMPL LVADGRMPMP LLVADGRMPM PLLVADGRMP MPLLVADGRM 16800

PMPLLVADGR QADMPMPLLV ADGRQADMPM PLLVADGRQA DMPMPLLVAD GRQADMPMPL 16860

LVADGRTSIT DYWGRAHDDT VNYLYEELKK ATAFAVATYA NDLSSIPKGG DPNNVVALGG 16920

HTDSVEAGPG INDDGSGIIS NLVIAKGPYS AIVGISLEDG QKNLGCSEAD YPSDVEGKQP 16980

QVHLWSNADQ TLKTMTYSPS VEVTADVAVV KTTYNVVAQT KVGDEEIEAK AHDDTVNYLY 17040

EELKKAHDDT VNYLYEELKK AHDDTVNYLY EELKKQPQVH LWSNADQTLK TMTYSPSVEV 17100

TADVAVVKAG QFPISANDGA TSTKAGVLSW SYTWSPADKE AASAALAAGY KFDGVTWDEE 17160

NWLLKFDPSA AIYPWTSGRF VGGASTDAFA DPKLLPEEGI YITPNLPPQI PYVKPSAAIY 17220

PWTSGRVVLT LTGIEPSTIY TAEEENQVRA YVASDSELEY VTWTVDNRGD WEVTSILSID 17280

QERTLIPADK IPTGKTLSTN EEGYETSAVR VSQTNPTVTL SLLNIASKYP ILFTPYGGPG 17340

AQEVTKDGTD GWLDNLLSMK TLSTNEEGYE TSAVRKFYDS MYTERTLIPA DKIPTGKATS 17400

GGTSAAAPVF AGLVGMLNDA RDFTDITAGS SIGCDGVNPQ TGKGFPDVAA HSLTPRPDVA 17460

AHSLTPRPNS ALPQVLSNSY GDEEQTVPEY YAKSALPQVL SNSYGDEEQT VPEYYAKSYG 17520

DEEQTVPEYY AKVCNLIGLM GLRLKDLVLS LAWYQESAVS KATSGGTSAA APVFAGLVGM 17580

LNDARATSGG TSAAAPVFAG LVGMLNDARA WYQESAVSKA WYQESAVSKD FTDITAGSSI 17640

GCDGVNPQTG KGFPDVAAHS LTPRGFPDVA AHSLTPRGFP DVAAHSLTPR SALPQVLSNS 17700

YGDEEQTVPE YYAKVCNLIG LMGLRDAEEE PYDWSNEGRG ISDGIDWQAG YSAVQKMDDA 17760

EQYEATSRNI YIQSATLDGK PYSKSLNYIP VEDFDYKTLE YSYDDFTIAQ MARTMINPQD 17820

YTGENPLWKD NGIFVNSRSI NGYPLPGGAF VRMDDAEQYE ATSRTLEYSY DDFTIAQMAR 17880

DIDFGENGDG IKDYVPNTQI PVTVAANTFP GGQEGFIDFV KFQESPEGAD FWGARTKVTI 17940

SPELSIRVFQ TAFGPAGTML TYEPIVRADA IVAAIRLAED HINWVEIRVT ISPELSIRHE 18000

LGVDEIWRDI DFGENGDGIK DIDFGENGDG IKFQESPEGA DFWGARLAED HINWVEIRAG 18060

VKPSNYVGDI FGTLGGTPDF GPGRCDVATT DVYYSGKSKY TAEGYEAATK TASNFDQPHS 18120

DESALQHLRY CASAQEDNAT LQALLRYTAE GYEAATKYVD AGGFEPSIKL QALLRSKYTA 18180

EGYEAATKTA SNFDQPHSDE SALQHLRGHL TAMTGDGVND APSLKTAALV QGASDSGHFK 18240

TGTLTANQLS IRAYGIVVAT AKTGDGVNDA PSLKMLTGDA LAIAKLAIEH EVDAHGKIEN 18300

MLSHLSKDKS ETETETEIEI SNKIKEIQEA GDVRLSDELE DDNAPIGFET TKSVEIAQLH 18360

SEVEALVEKY TVTAGELTSD FKEVEVEKEW TKTETKTEIE IERIKEIQEA GDVRASSDDS 18420

NYGWEDSKGI QDAGVIATAK IGAQSTVLLK NFGEIGDASE YVYPEGLERY TPPNFSSWTR 18480

VNEFVDVQRA VDIVSQMTLT EKDSPNWDVD SDALPAIPEG AKEIPVGYSA ADIDTNRGVD 18540

YQPGGSSNLA DPIADAEGCK RNTLAFFSGN EVINDGPSSK YGLVEIDDGK VKTLADFDAL 18600

KYGLVEIDDG KVKDLMQAMA DFGPKFPGGN NLEGDTLDGR TGEVYASAVI VSKYPSNLDA 18660

WIPVDGSALS LKYVEVGNED NLNDGLDSYK FQAFYDAIKE QTYTGSFYVK ASLGHPEPWT 18720

VKDLMQAMAD FGPKELPSGP YFVSLYTGEV FKGISPEAHQ SLTTFTRLYP DDNLAFIQAG 18780

ISDEKQLLLA GGGWDGKSLF VSVYSVGTTD YRLYYTPTAE KPLAGLRGIS PEAHQSLTTF 18840

TRQLLLAGGG WDGKDSLSEA IAYAKGGGGG TFGVVMESTH RIANECQNQE LFWALRSQGG 18900

TAVIEEFPSW YEFYQKYVVP NAVTVGNAHF AATRSSGQGT LSLWTRAVTV GNAHFAATRG 18960

GGGGTFGVVM ESTHRGGGGG TFGVVMESTH RGGGGGTFGV VMESTHRIAN ECQNQELFWA 19020

LRSSGQGTLS LWTRYVVPNA VTVGNAHFAA TRYVVPNAVT VGNAHFAATR YVVPNAVTVG 19080

NAHFAATRAS SVSGVITLSD GRTTADSDGN FSFENVRVQD ETWELSDGSY ITKYDWSDFI 19140

NSAKYEEFEV PAGTLVKIWQ IGTLDRAYTQ YTETSVYGML KDGDLVTQQN ELQGKHEGWI 19200

DEAAVQEAKL HYGAYSIKSN LFNSYSENQV LLPASVYGSY KETYGSAAGW DKLADALAAS 19260

SLPEAWWGEN YEPLLNIKGG GGGTFGVVIE STHRIANECQ NQDLFWALRV EPQLSFVAAV 19320

IKYVTSNAVS VGVTHFAGSR AVFETAEGRG GGGGTFGVVI ESTHRYVTSN AVSVGVTHFA 19380

GSRDAEVAPP NDPVDPMAPD SSTKFEGYLP DARGSTGNVL VDVSHVLPSF RVGISWLSTE 19440

KLSITATGGD GNGDSQIYVQ KGAVNWEDGY RDAEVAPPND PVDPMAPDSS TKDSVKEDDY 19500

EDLFNYICAK KFTDTPVLYG PKMLDDAGIY LITDLSSPSE SINRSDGQCS DLLKQQLSFV 19560

MNQWYEKYGA YSVCSPKTLP AIESKKFTDT PVLYGPKMLD DAGIYLITDL SSPSESINRS 19620

DGQCSDLLKA DAAGSHGEAL NEVQAKAKAD AAGSHGEALN EVQAKLEAAE QALSEARVGA 19680

LESQLSTEQD AIKEAAESAG TTHSQQLQEL RDALEAAEAA AKIQEQLKGQ TPLPILVADG 19740

RNNILEGPDV KSHLSVVDGG EDGQNIPLHP LIQPERTIDY WTELVDTVKT STTLPEVCSK 19800

AMLNGAGALK AMLNGAGALK AMLNGAGALK AMLNGAGALK AMLNGAGALK GQTPLPILVA 19860

DGRINLGLNK NNILEGPDVK SHLSVVDGGE DGQNIPLHPL IQPERTFINL GLNKTSLTDY 19920

WGRFNVDETA FTGAWGRIGS LAITDVSLPF FKIQGISNPS GALSSGGLGE PKVQNGAVTW 19980

ESDPNRATTV YGESIKSIYA INSGRELDTQ HIHPPDSYFV SPLTRGFTEI DELWNGVTAE 20040

TNAAQDLRQA QVAHDFWQKF YHQVVELNRK DAELTDAGVK LNTGAVIPVL VRELDTQHIH 20100

PPDSYFVSPL TRLNTGAVIP VLVRLNTGAV IPVLVRQAQV AHDFWQKQAQ VAHDFWQKAI 20160

NDYIDSQLDK KGVQISTNIP KSSPWIMLGG SYPGMRYQSL EYQQSLCYRL FSLALKISIP 20220

IDHEDPSMGT YQNRAINDYI DSQLDKKCSS HDDCSDELAC TDGVCACTAD SAVTCSWEGH 20280

CAGAKLNLQY QASGDAKKSL VDFSAARGDN PSILGLRSAV TCSWEGHCAG AKIGTTIDDI 20340

KCTADSAVTC SWEGHCAGAK CTADSAVTCS WEGHCAGAKC TADSAVTCSW EGHCAGAKCT 20400

ADSAVTCSWE GHCAGAKAFP DVAAQGMNFA VYDKELYNIG DYQADANSGS KIAFASYLEE 20460

YARQGLQDIT LGASIGCTGR YADLENFENY LAPWAKAFPD VAAQGMNFAV YDKAFPDVAA 20520

QGMNFAVYDK ELYNIGDYQA DANSGSKYAD LENFENYLAP WAKYADLENF ENYLAPWAKA 20580

GSSPTDIISG ISDKTDALDS AIKKVEQAID DIIAKSAADG LASAITSKSG DDISTTDALA 20640

LPEPVQALTK SPTDIISGIS DKTDALDSAI KAQNDPNAFG VVAARLGACP PGKETALLGD 20700

KPNAFGVVAA RQNDPNAFGV VAARAATYCP ENIEKIENQS DADGYSSCST LKLTGLTTLT 20760

TLSFAALTKS DKLNVIDFPK VGSIEFTALP QLQSLDFTKL NVIDFPKTVN GGFQIARGVA 20820

AWLFERLSFG SIDLENANIN RSLSFIPGVL YDGSPIGKQE STFAAVERSC FEIGKFVDPL 20880

IGSNNGGNVF AGASLPYGMA KGWTQGGSNA DVVLTDAYVK VGISYISTDR AGNWQNLYKA 20940

QHPFLTIVDP EAQSRSVFSQ NESVAAGLKT TAVLFDEGKE ICLAALGRAL VIVSDSIRTD 21000

TIHGVGQNSF YKASHPIEVP VKASHPIEVP VKIMSILLGG AIPDDLKPLV LFDSVTKTAT 21060

ESEPSLSDIE KTGYVNYNVD TTNLRKHNPL VLFDSVTKTS PFPYDSKKHN PLVLFDSVTK 21120

KHNPLVLFDS VTKKHNPLVL FDSVTKTGYV NYNVDTTNLR APTPPDFSLG YIQSKIEQDG 21180

SESLLTNEYA PLKGYAFIWN MPAQGRTSGW GGNPGGYRYQ LASYLRDYLD EYLVFPPAGV 21240

QPQKIYVTGE SYAGRVDHLP DVPFDVGEMY SGLVPIDKDD KNFQELFGIK KTPLDDFRAD 21300

DVLEVNPLAD PEVVSYFRLD ADAITAQYFG NDAPWYRSDY ASFLYGPYRL VFAPQEEKAT 21360

WDGVDADKIR FQYPGDLFDQ GTTIRFTGDA ATVNSIAARP DSDEIYFGGQ FEKNLEVLSL 21420

TKHVAAYSFG SKAWTALGGG VNGPVHKNLA LLDGKDLTDY LMKSYELPDG QVITIGNERV 21480

APEEHPVLLT EAPINPKQEY DESGPSIVHR QEYDESGPSI VHRVAPEEHP VLLTEAPINP 21540

KVAPEEHPVL LTEAPINPKV APEEHPVLLT EAPINPKAEL PEGYPESSAN PAFRDIQYLE 21600

NYQGQGYSGP AVKFVTVTEE TDPDLFWALR LSLGDSGACK DAEVEPANWG VEGRLGGAQL 21660

FTSRTVSLVE DGSNTPATLS AGTFARSLVD IYRTVSLVED GSNTPATLSA GTFARAYVEM 21720

MQCTDEKEPL VRLEKDLPGT TLSSKYYGYG GGNPLGPAQG IGFANELIAR LEKDLPGTTL 21780

SSKLSEAGHS VLLIEKTPIE SDATSYLNDR GGPMGTYLVS ASERVILSAG TFGTPKAVIT 21840

DIVNQQRLIN QVELSEDKTI ARGGSNNFGI VTRLLLSDAM WYTRERPSAQ LLSGKFDTLG 21900

TSGPTAKLAN AYTWEGGRAV ADRIPLAIHD EVSPVGDTDA LLERAPVVQY ALNRDGYMGY 21960

HGVPQIPIYA YKAIHDEVSP VGDTDALLER AIHDEVSPVG DTDALLERAI HDEVSPVGDT 22020

DALLERDGYM GYHGVPQIPI YAYKLAEESA ALGVKVNPII LTGDMWRVSD NAGLGDWVPN 22080

PDRFPDGLTP LVEDVTKALG GTSTINGMAY TRSQLSDYAA ATVKAEDVQI DVWQKALGGT 22140

STINGMAYTR EIGFTVQEDN TDGKIDFGSA PNIVNAIAED RNSPVWPDGI QQTYEYPNRA 22200

STNMITSNSG AGLVPQDENR IGGSAGTELQ SDKITGEVWP VLMAYGQKSA ISQYGDSFAK 22260

SQSDFESEFS TAKVNAGIGI GPDDLVSFIK GNAMDHFSSI MERYGPTYAA YFLDQNEKAI 22320

TETQYEEAKD LTLDLPDIGL QYAGTVKTVA ALINWRNIWD GTTAQNVKKE DLETSSYSFS 22380

GDGKVDFASL QSAATQSTTY SNAPAVKTCS LVFLFPKKED LETSSYSFSG DGKFDGILGL 22440

GFDTISVNKY GSGSLSGFVS QDTLKWYSVY DLGNGAVGLA KDAYSPHEIY SRIFEQLEGM 22500

SLSKTYEVVG NVYKDQVLKD VALIKAVPGT AQQAAAIKDA GEFDVERGTV VTNDQCGGAS 22560

SVRLHLVTAE EADIKIIYDM PDGSSCKGID VAKPTGRVNG VWTVTHSPFE GLSALKSAMT 22620

LPRVNGVWTV THSPFEGLSA LKVPTVLMSP WVGKSIDQFF NDAKNVAPDD PDHSITGGNQ 22680

QVYSTYHPNA KESTLHLVLR IQDKEGIPPD QQRIQDKEGI PPDQQRIQDK EGIPPDQQRD 22740

VSLVANHIDT VGKTAATVNT WTGGWSDSKA IGTYQNSGLS QYTVRYSEGV HFPARTVANW 22800

LVREVAGDVD NAVNPAWRLG AGVQGFEAYE AANAQGLRLD GGVIEDFAQK NPDLSSTSDT 22860

TDVIRGPDEP YSGQYDEERQ FISVTNPTGA EPVPKFLSEL LEDEYFTKFV NIWLENTDYE 22920

SAANDPHLSK LLEYDIASGT PVYRNNNQGL EALTISPDGK VYAISDQDDT GPWIRDAYQN 22980

DFAARVDPST AVDYNHYSDA ADRSGDVQTL QFAWALQHLS ERTGNQTGQL GTYFNKNINM 23040

LLYGTDDCSG KGMVFSIDAQ GEKNINMLLY GTDDCSGKNP NPDQAFLQVR FWVATGDSSK 23100

AALDALQQSI YLQPKFSDGN GLFYQYERVA VAGYDDTTGG VGPLLAQKAI AIALQSSHRQ 23160

TGSVDGYAYT DANKNDLITY LKSVVENNND GLTAAYRVAP NSGAYLNEAD FRSLPLIVGN 23220

SDQEGKANEQ PTWVYRSDYQ ECADAPGQKF GATGDEYREP SNDPNPPETY SKVALLFSER 23280

LADGSIPTRS ISSDEDSAET EQSDSSDPKV AIIDGLADPW RTMGVGYATN DDSTIRDVPI 23340

MQELNTNTIR DVDPIVIKVE TGVIKPGMVV TFAPANVTTE VKIVVIGHVD SGKDDADLQE 23400

LGAKSSAFAF RKDPSDAIPS IPSIPISYKE AIPFLKEAAE IDSHWERDDY MPFIEVPRDD 23460

YMPFIEVPRA LQGVDELVEK DALHPQFDNF YQEQPKASYG AGVTIQDRDL SVFFTRINDL 23520

LPVYVELLQK AALTNMVNAA KGEITPEQYE KIINEPTAAA IAYGLDKQLT SEEIWQAEEK 23580

LDKPAGDYTI RDYSNNWESG ALKLAPNQMT GSLDATYLKL APNQMTGSLD ATYLKCASDG 23640

SAETCSWEGH CKCASDGSAE TCSWEGHCKC ASDGSAETCS WEGHCKCASD GSAETCSWEG 23700

HCKCASDGSA ETCSWEGHCK LAAISVEPGK ASELGCSSGD LDCLCKVGEC AQMCISNMNA 23760

KGTLLSSNQG SQAADVKNDD DFLNYGISSK APSVITYDEA TKYEAAGITV HKDDYLPFID 23820

MPSEVTQIDA KIPMALDDWP TLSNMIFSGK DNFDTSSVTH RGYQINFLSN SAKDNIQGIT 23880

KPAIRDNIQG ITKPAIGPLG LSPKLQLWDT AGQERAEDYL LNPSPKNFGI GQDIQPKIDA 23940

TTNPGMRQLG LAMLGNKLVI DGLKEVTEIP ATADASRVNV DYTEVPRLAV NMVPFPRYCD 24000

LILGEWRDQI KDVLRIDYIG GGDLFRGSSP NVLYKSANWT PPEGIVRAAS TGSMAEQYTK 24060

AATGTYASST TVYKAENQAV AVGRALVEGS TFAKALYSSA ATGTYASSTT VYKATGTYAS 24120

STTVYKATTV YGESIKAYAD GYVQIVQTYA ASTGSMAEQY TKDLTWSYAA LLTANNRFNV 24180

DETAFTGAWG RGQSAQGASP GVVIASPSKI GADGQSAQGA SPGVVIASPS KIGSLAITDV 24240

SLPFFKIQGI SNPSGALSSG GLGEPKKYTV PSTCGVKPSG ALSSGGLGEP KQAILNNIGA 24300

DGQSAQGASP GVVIASPSKS DPDYFYTWTR SIYAINSGRT GTYASSTTVY KTVGSSCPYC 24360

DSQAPQVRVQ NGAVTWESDP NRVQNGAVTW ESDPNRKPDY FYTWTRYTVP STCGVKSSAA 24420

TGTYASSTTV YKGAVTWESD PNRIVGSISQ LGSWNPSSAT ALSAVQSDVW RDINTVLGSI 24480

HTFDPQNIGA DGQSAQGASP GVVIASPSKA ASTGSMAEQY TKAENQAVAV GRAENQAVAV 24540

GRALVEGSTF AKALVEGSTF AKALYSSAAT GTYASSTTVY KAYADGYVQI VQTYAASTGS 24600

MAEQYTKDLT WSYAALLTAN NRDLTWSYAA LLTANNRIQG ISNPSGALSS GGLGEPKIQG 24660

ISNPSGALSS GGLGEPKNIG ADGQSAQGAS PGVVIASPSK PDYFYTWTRQ AILNNIGADG 24720

QSAQGASPGV VIASPSKQAI LNNIGADGQS AQGASPGVVI ASPSKQAILN NIGADGQSAQ 24780

GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS KQAILNNIGA DGQSAQGASP 24840

GVVIASPSKQ AILNNIGADG QSAQGASPGV VIASPSKQAI LNNIGADGQS AQGASPGVVI 24900

ASPSKQAILN NIGADGQSAQ GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS 24960

KQAILNNIGA DGQSAQGASP GVVIASPSKQ AILNNIGADG QSAQGASPGV VIASPSKQAI 25020

LNNIGADGQS AQGASPGVVI ASPSKQAILN NIGADGQSAQ GASPGVVIAS PSKSAVQSDV 25080

WRSAVQSDVW RSAVQSDVWR SAVQSDVWRS AVQSDVWRSD PDYFYTWTRS DPDYFYTWTR 25140

SDPDYFYTWT RSDPDYFYTW TRSDPDYFYT WTRSDPDYFY TWTRSIYAIN SGRSIYAINS 25200

GRSIYAINSG RTVGSSCPYC DSQAPQVRTV GSSCPYCDSQ APQVRVQNGA VTWESDPNRV 25260

QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN RVQNGAVTWE SDPNRVQNGA 25320

VTWESDPNRV QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN RVQNGAVTWE 25380

SDPNRVQNGA VTWESDPNRV QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN 25440

RKVQNGAVTW ESDPNRKVQN GAVTWESDPN RKVQNGAVTW ESDPNRKASM VWEEAQQVSG 25500

KAVSPSFEDV WSQPRDGAGQ MFIPLNPNAY SPNTLNKDVG GPIEDQNSLQ VGDRFGFDLF 25560

DPTKGPTLLE DFIFRHGGPN FEQLPINQPR HVDGFGIHLF SYLDTQLNRL FYNSLTPAEQ 25620

QFVVDAIRNN VIIQLNRQDL FEAIEAGRSL TPAEQQFVVD AIRSVTSGFV DGIKVGFLAS 25680

VETPASIEAA SELSKTTDVG TFGQKDVHGF ATRIVPEEYV PITKFVTDNG DSKASMVWEE 25740

AQQVSGKASM VWEEAQQVSG KASMVWEEAQ QVSGKASMVW EEAQQVSGKA SMVWEEAQQV 25800

SGKASMVWEE AQQVSGKAVS PSFEDVWSQP RAVSPSFEDV WSQPRAVSPS FEDVWSQPRA 25860

VSPSFEDVWS QPRAVSPSFE DVWSQPRDGA GQMFIPLNPN AYSPNTLNKD GAGQMFIPLN 25920

PNAYSPNTLN KHGGPNFEQL PINQPRHGGP NFEQLPINQP RHGGPNFEQL PINQPRHGGP 25980

NFEQLPINQP RLFSYLDTQL NRNNVIIQLN RNNVIIQLNR NNVIIQLNRS VTSGFVDGIK 26040

SVTSGFVDGI KAAALAELVW SGNRAELVWS GNRASNSLQY VNVQVKDAYS PHEIYSREYL 26100

VANGVQAQAL VPKGIMLDTG RGVQAQALVP KHIVGATAPL WGEQVDDINV SHIVGATAPL 26160

WGEQVDDINV SSMIFEQLEG MSLSKVIPEI DMPSHSSSGW KYNVMANPDA NTPNFNYGGN 26220

GGSWCAPYKT YEVVGNVYKD IEADLQHAET VWGALHAFLV MWEDIALSAD NAHDVPKAAA 26280

LAELVWSGNR AAALAELVWS GNRAAALAEL VWSGNRAAAL AELVWSGNRA SNSLQYVNVQ 26340

VKASNSLQYV NVQVKASNSL QYVNVQVKAS NSLQYVNVQV KDAYSPHEIY SRDAYSPHEI 26400

YSRDAYSPHE IYSRDAYSPH EIYSRDAYSP HEIYSRDAYS PHEIYSRDAY SPHEIYSREY 26460

LVANGVQAQA LVPKEYLVAN GVQAQALVPK EYLVANGVQA QALVPKGIML DTGRIFEQLE 26520

GMSLSKIFEQ LEGMSLSKIF EQLEGMSLSK IFEQLEGMSL SKTYEVVGNV YKYNVMANPD 26580

ANTPNFNYGG NGGSWCAPYK YNVMANPDAN TPNFNYGGNG GSWCAPYKYN VMANPDANTP 26640

NFNYGGNGGS WCAPYKYNVM ANPDANTPNF NYGGNGGSWC APYKATSGGT SAAAPVFAGL 26700

VGMLNDARAW YQESAVSKDF TDITAGSSIG CDGVNPQTGK GFPDVAAHSL TPRPDVAAHS 26760

LTPRPNSALP QVLSNSYGDE EQTVPEYYAK SALPQVLSNS YGDEEQTVPE YYAKSNSYGD 26820

EEQTVPEYYA KSYGDEEQTV PEYYAKYLDQ QITAETKAWY QESAVSKAWY QESAVSKAWY 26880

QESAVSKAWY QESAVSKGFP DVAAHSLTPR GFPDVAAHSL TPRGFPDVAA HSLTPRGFPD 26940

VAAHSLTPRG FPDVAAHSLT PRPDVAAHSL TPRSALPQVL SNSYGDEEQT VPEYYAKYLD 27000

QQITAETKYL DQQITAETKA NEQPTWVYRF FCPTDYLIDV RGTPSVLTEQ GLVKLGVPGN 27060

ELAIEIGSTG DYNARQGTPS VLTEQGLVKS LPLIVGNSDQ EGKTFQGTPS VLTEQGLVKY 27120

PVVQNPVTLA ESSCQSVFNP NIPKNPSAGP GWDQAKPTNG PLAKFQGTPS VLTEQGLVKA 27180

NEQPTWVYRA NEQPTWVYRG TPSVLTEQGL VKNPSAGPGW DQAKPTNGPL AKSLPLIVGN 27240

SDQEGKSLPL IVGNSDQEGK SLPLIVGNSD QEGKARNHGT STVAPQVQAS VYRASVDISN 27300

VDTYSSTEVA NDDSFQQVGK ATFLVWDQQR KASVDISNVD TYSSTEVAND DSFQQVGKNH 27360

GTSTVAPQVQ ASVYRSSTEV ANDDSFQQVG KTLYLTDTDA GVPMIDPRTV YAFDVSEDGS 27420

YLKVASNGYV ITGAGKARNH GTSTVAPQVQ ASVYRARNHG TSTVAPQVQA SVYRATFLVW 27480

DQQRATFLVW DQQRATFLVW DQQRKASVDI SNVDTYSSTE VANDDSFQQV GKTVYAFDVS 27540

EDGSYLKVAS NGYVITGAGK VASNGYVITG AGKIENQSDA DGYSSCSTLK LTGLTTLTTL 27600

SFAALTKSAS SLNSIGDTFK SDKLNVIDFP KSLNSIGDTF KVGSIEFTAL PQLQSLDFTK 27660

TVNGGFQIAR LNVIDFPKGQ LGFWGNKGTY SGDLQLNGVK LNVIDFPKSD KLNVIDFPKS 27720

DKLNVIDFPK TVNGGFQIAR TVNGGFQIAR AVGSDEWTVR LGITYTTYSK MTNDYISALT 27780

KSEMLAEQDK MTNDYISALT KSTITTPWKS VVENNNDGLT AAYRVAPNSG AYLNEADFRY 27840

FYGDNYATLR SGAYLNEADF RTAVGSDEWT VRLGITYTTY SKMTNDYISA LTKMTNDYIS 27900

ALTKMTNDYI SALTKSEMLA EQDKMTNDYI SALTKSEMLA EQDKMTNDYI SALTKSEMLA 27960

EQDKMTNDYI SALTKSVVEN NNDGLTAAYR SVVENNNDGL TAAYRSVVEN NNDGLTAAYR 28020

SVVENNNDGL TAAYRETTMF VLQGFGASMA RLNALQGGRL VQNDFNTLLR QDILGGMVDS 28080

YTDPKTGETT QIHARYGLEA AVPLMYESTG LGLGDMTKVS VADVKIDYIG GGDLFRLVQN 28140

DFNTLLRQDI LGGMVDSYTD PKYGLEAAVP LMYESTGLGL GDMTKALEAY KVNGKAVDFS 28200

GHDEFQGKEP SNDPNPPETY SKFGATGDEY RSDYQECADA PGQKYIARPD IMKSTLPDLS 28260

EVIKVNGKEV GQFKSVDNFH LLTVYAVDFS GHDEFQGKFG ATGDEYRYIA RPDIMKFLDE 28320

ALTYPPPKGI QINDPSINDD SVMIYAPAVR IASAMLDEED EKYFNVKNGD QSPPSALGPL 28380

PSVIERPSIN DDSVMIYAPA VRTDYSVCGE TTIFKTVDDE EGVAAQFKIA SAMLDEEDEK 28440

FLDEALTYPP PKFLDEALTY PPPKGIQIND PSINDDSVMI YAPAVRGIQI NDPSINDDSV 28500

MIYAPAVRIA SAMLDEEDEK IASAMLDEED EKYFNVKIAS AMLDEEDEKY FNVKNGDQSP 28560

PSALGPLPSV IERNGDQSPP SALGPLPSVI ERAGAVAAVV YNNEKHGIPG GGIATGAEGI 28620

KLVLGDAVPE SAAPMGLTPP TKSDKELVSS SAFQSHVKSF EGFPKRLVAH SVATYARIAD 28680

LGKEEYNHPT RAGAVAAVVY NNEKHGIPGG GIATGAEGIK HGIPGGGIAT GAEGIKLVLG 28740

DAVPESAAPM GLTPPTKLVL GDAVPESAAP MGLTPPTKLV LGDAVPESAA PMGLTPPTKS 28800

DKELVSSSAF QSHVKFTDTP VLYGPKKFTD TPVLYGPKML DDAGIYLITD LSSPSESINR 28860

QQLSFVMNQW YEKYGAYSVC SPKSDGQCSD LLKWDVDLYS RLITDLSSPS ESINRDLSSP 28920

SESINRKFTD TPVLYGPKKF TDTPVLYGPK QQLSFVMNQW YEKQQLSFVM NQWYEKCDVA 28980

TTDVYYSGKG GTPDFGPGRS KYTAEGYEAA TKVATIGSAT FARYCASAQE DNATLQALLR 29040

YTAEGYEAAT KYVDAGGFEP SIKSKYTAEG YEAATKVATI GSATFARVAT IGSATFARYV 29100

DAGGFEPSIK DADACNGGGI EYDSPADTPL EFKDNTCNAP IPVSFPVAPT DTKEISFNQA 29160

WLRFAANGNY GSETTAAVIN NFNGRITTAD MDGISSWLPT INGKVIANGN VAGNILVIAK 29220

SDVSDFKEAG LKDADACNGG GIEYDSPADT PLEFKDADAC NGGGIEYDSP ADTPLEFKDA 29280

DACNGGGIEY DSPADTPLEF KFAANGNYGS ETTAAVINNF NGRANNYCSN QVEGPYSLYS 29340

GRVSIWTESY GGRYGPSFTA FFQEQNEKTV YDMAMEAWSK ISYKEPGICE TTPGVKSAGY 29400

APLKPGGCKD QIIECRANNY CSNQVEGPYS LYSGRANNYC SNQVEGPYSL YSGRANNYCS 29460

NQVEGPYSLY SGRISYKEPG ICETTPGVKV SIWTESYGGR AIMGAEEAAK TGGAMWPYRT 29520

LIPDVVGIFA GTPKFVTNMQ AALLKALSEM ILQSEKDYYA SMLQQPKANF EVETPRGITG 29580

TSIARANFEV ETPRDYYASM LQQPKFVTNM QAALLKFVTN MQAALLKTGG AMWPYRTGGA 29640

MWPYRAEDYL LNPSPKLSDL TGDTEYAQLS QKTDDQVSLF ETTIRTDSGF AGLTNVNAAN 29700

GGGRYDNQES FLFAEVLKIT GQEIYRAEDY LLNPSPKTDD QVSLFETTIR AGFAGDDAPR 29760

QEYDESGPSI VHRSYELPDG QVITIGNERV APEEHPVLLT EAPINPKDLT DYLMKIIAPP 29820

ERSYELPDGQ VITIGNERVA PEEHPVLLTE APINPKALLF GAAGSAEDPV VVKNVGFPVV 29880

TVAEDAASSS IKVYATPDQD IEHGRAVEQS LDAIRQGLLT VEDRGPLNEG GLYAERLSGQ 29940

DASAITWKLT GNLGGEDYQD KASPSYLTAT PRALMNGAGA IKDAGYETSI TDYWGRPTVY 30000

GFVPLEYVGS KTAFSDILAK TNTQVPDACT QCFQKMPMPL LVADGRTAFS DILAKAFPDV 30060

AAQGMNFAVY DKELYNIGDY QADANSGSKI AFASYLEEYA RLETIGDTFK QGLQDITLGA 30120

SIGCTGRAFP DVAAQGMNFA VYDKELYNIG DYQADANSGS KQGLQDITLG ASIGCTGRLV 30180

EGAAAGIVVA SPSKQGVLNN IGADGKSSSA YESLTSAVKA SALIAYGNSL ISSDKSVYGI 30240

NNGRPDYFYT WTRPDYFYTW TRSNPDYFYT WTRSNPDYFY TWTRSNPDYF YTWTRSNPDY 30300

FYTWTRSNPD YFYTWTRSNP DYFYTWTRFS VAEILPGAKN VVLDTTALSA NTKVGTIITG 30360

DPLDPPVLKY PAEVFLPGGT YQLGKADKET DIGSAIEKAS AIQLDGIIYR QLSGHVGPLT 30420

SSSSKETDIG SAIEKADKET DIGSAIEKQL SGHVGPLTSS SSKQLSGHVG PLTSSSSKIY 30480

SFFVGGAVPE NLRQTSSEQN PSLEEIQAAQ ATVLPHSPVS NVKSAGEYNT FSPEWPVPLT 30540

KTFDENDTYE IGNKSIDQFF NDAKVPTVLM SPWVGKDGQE ATFHFDRVDW SPSFRAAEVI 30600

NYYTPDHVPV FNAMSIDQFF NDAKNAFITN YPSEQRYDTA TFIDKRIDAT TNPGMRQLGL 30660

AMLGNKNMHD VIGNDGTVPS EFRIDATTNP GMRIDATTNP GMRNAFITNY PSEQRNAFIT 30720

NYPSEQRQLG LAMLGNKAQI TAVNLEARGD GGGGPTFEHL EKSMDNDNTS LLVFGKMGES 30780

VDDFFARAQI TAVNLEARMG ESVDDFFARG IVSGEGEESS DPVKVDNVVA SFKYMQQLLD 30840

QTKVVWQDSV RAQNDPNAFG VVAARDPNAF GVVAARNDPN AFGVVAARAA TYCPENIEKA 30900

QNDPNAFGVV AARAQNDPNA FGVVAARTIF GWDIAEGQKS AGYTPLKVNG VEYGETRSAG 30960

YTPLKVNGVE YGETRSAGYT PLKVNGVEYG ETRGGSILPM QEVALTTRWA SVIDATKKAT 31020

GDVLFNTKHT ADGAWAKGGS ILPMQEVALT TRGGSILPMQ EVALTTRWAS VIDATKDGLE 31080

GSFKWDNLDS AALNTKVEDG NLILTMPKVE DGNLILTMPK VEDGNLILTM PKWDNLDSAA 31140

LNTKWDNLDS AALNTKSAIS QYGDSFAKSQ SDFESEFSTA KVNAGIGIGP DDLVSFIKSA 31200

ISQYGDSFAK SQSDFESEFS TAKGFNIVVA PGLDGRHVDV PLTGEDEITI LAIHDEVSPV 31260

GDTDALLERA PVVQYALNRY LVDQLNPEGK AIHDEVSPVG DTDALLERAI HDEVSPVGDT 31320

DALLERAPVV QYALNRAPVV QYALNRDAEL TDAGVKQAQV AHDFWQKLNT GAVIPVLVRL 31380

GDLSANPIER VLPQVIEATN RIYVTGQSYA GRLGDLSANP IERVLPQVIE ATNRVLPQVI 31440

EATNRNDPVA VFDGSVIPKE AGLVPFQVSP TTKTLGIDIA RGQTPLPILV ADGRTSTTLP 31500

EVCSKNNILE GPDVKGQTPL PILVADGRIN TAAYWKEAIA DVLEHLGEND EDIAVYAPNP 31560

FYKNSILEGP DVKMPMPILV ADGRMPMPIL VADGRNDDDF LNYGISSKSP VTSEYTSVRS 31620

IFEAANEKAI NDYIDSQLDK YLTNSQALAD LPYFAEKGVQ ISTNIPKGVQ ISTNIPKEII 31680

STYSIDGLRS VYQTMTDRYN TDAELYKGGS ELGFRSAADG LASAITSKSG DDISTTDALA 31740

LPEPVQALTK AGSSPTDIIS GISDKTDALD SAIKYDYENV DSDGANKYNL SNGAPAPETV 31800

TNKSMPTSGA VDLVAKCSSH DDCSDELACT DGVCACTADS AVTCSWEGHC AGAKGDNPSI 31860

LGLRCTADSA VTCSWEGHCA GAKDSPNWDV DSDALPAIPE GAKTLADFDA LKTLADFDAL 31920

KINPGPLARL YPDDNLAFIQ AGISDEKLYT GEVFKTDEGK GQEPPAAIVE VQKVAIIDGL 31980

ADPWRSVNIV NYTPSDSYTY SDNSGSWQSV KVTTGGQGAE FTLAKDQTTW SVDGNVVRVT 32040

TGGQGAEFTL AKVTTGGQGA EFTLAKAGQF PISANDGATS TKEAASAALA AGYKQTYTSC 32100

NPLKKTLNYA DALDGENYPQ TPSRVAVAGY DDTTGGVGPL LAQKINPSSG LLEPQTPLAV 32160

SPGSGPRVAV AGYDDTTGGV GPLLAQKTTG AFDESGPPLS QKNALQTMYD TQDKAALDAL 32220

QQSIYLQPKF SDGNGLFYQY ERLGAEVVTA GRIYAVADTQ ERLINQVELS EDKAVITDIV 32280

NQQRAVITDI VNQQRGENIL SAPLITYAPA GPELDEKELG FTAVGGEGKD IQYLENYQGQ 32340

GYSGPAVKIL QYAQGRDYSN NWESGALKDD YMPFIEVPRE AAEIDSHWER TIPIDNDVDY 32400

VVTGYRVYWV DSGPRTGDGV NDAPSLKDGQ EQEILARADA IVAAIRSALI AFEKNINMLL 32460

YGTDDCSGKG MVFSIDAQGE KTGTDQASVG YYKDAVYALD AIYGIDARDN ILPENLDDGL 32520

PSQFVYEKIL VENLQDQTAK ILVENLQDQT AKAEPYVTGS SAASGSNFVA DFAEAGTDGK 32580

QISYWAFTTP AVKFEPPAVY NDELKDAEEE PYDWSNEGRL SDELEDDNAP IGFETTKDQE 32640

MAVAAFRTSD DFASQMDGRC MGCDSTSIDV SRCMGCDSTS IDVSRDASGG DQITEWQDIY 32700

LPPITKGIQD AGVIATAKGV GSDAWTVSES GRGIDGDKGL VVKDLYGNIV MSGGSTLYPG 32760

IADRAGFAGD DAPRQEYDES GPSIVHRSYE LPDGQVITIG NERSYELPDG QVITIGNERL 32820

SGGVAVIKTT AVLFDEGKIG GSAGTELQSD KDLALVDPGL ELSYNTKLVG GSDFGEDEAK 32880

TLSTNEEGYE TSAVRASYGA GVTIQDRSAY VVYDLSNNEI SLANTKVPYL IGANTDEGTS 32940

FAIRLPVEAF QALASSTSET KDAGNAATND PLFPFSRAAL PGTEVLFADS VAKVTSAQYY 33000

VNPKIQAFVI YPENFDKVGA GVNVGELYAF ADKALTQYSV KTTYNVVAQT KTYANLPQAL 33060

VNSGAIKVIP LQGCDADEYG RDNIQGITKP AIRLANAYTW EGGRYQGASQ CPFRTMGVGY 33120

ATNDDSTIRC ASDGSAETCS WEGHCKCASD GSAETCSWEG HCKLILPGEL AK 33172

Claims (10)

1. The method for preventing and controlling aflatoxin and increasing the number of root nodules of the root nitrogen fixation enzyme of the leguminous plant is characterized by comprising the following steps: comprising the following steps: obtaining bacillus amyloliquefaciens, brevibaciens laterosporus, bacillus mucilaginosus and escherichia coli with the effects of preventing and controlling aspergillus flavus and toxins thereof; 2 or 3 of bacillus amyloliquefaciens, bacillus laterosporus and bacillus mucilaginosus are mixed with escherichia coli, and then the mixture is applied to the field of leguminous crops, so that the number of root nitrogen fixation enzyme active root nodules of the leguminous crops is increased.

2. The method according to claim 1, characterized in that: the method comprises the following specific steps: obtaining bacillus amyloliquefaciens, brevibaciens laterosporus, bacillus mucilaginosus and escherichia coli with the effects of preventing and controlling aspergillus flavus and toxins thereof; 4 microbial strains of bacillus amyloliquefaciens, bacillus laterosporus, bacillus mucilaginosus and escherichia coli are cultivated in batches, and any 2 or 3 of bacillus amyloliquefaciens, bacillus laterosporus and bacillus mucilaginosus are mixed with the escherichia coli and applied to the field of leguminous crops, so that the aspergillus flavus and toxin level thereof are effectively prevented and controlled, and the number of nitrogen fixing enzyme active root nodules of roots of the leguminous crops is increased.

3. The method according to claim 1, characterized in that: the method comprises the following specific steps: the method for obtaining the bacillus amyloliquefaciens, the Brevibaciens laterosporus, the bacillus mucilaginosus and the escherichia coli of the ludwigia has the effects of preventing and controlling the aflatoxin and the toxin of the aspergillus flavus comprises the following steps: detecting the content of aflatoxin and/or the content of aflatoxin after the treatment of the strain to be screened, and comparing the difference of the content of the aflatoxin and/or the content of the aflatoxin after the treatment with the non-treatment to obtain the prevention and control bacteria with the prevention and control effects of the aflatoxin and the toxins thereof;

or is: through a method of efficiently screening aflatoxin prevention and control bacteria in one step, the prevention and control bacteria with the aflatoxin prevention and control effect are obtained,

the method for efficiently screening aflatoxin prevention and control bacteria in one step comprises the following steps: determining the AFT-YJFZ01 content of a candidate aflatoxin biocontrol bacterium test treatment group and a control group, calculating the inhibition rate of biocontrol bacterium to the indicator molecule AFT-YJFZ01, and efficiently screening aflatoxin control bacterium according to the result of the inhibition rate of the indicator molecule AFT-YJFZ01, wherein the amino acid sequence of the indicator molecule AFT-YJFZ01 is shown as SEQ ID NO. 1; the higher the inhibition rate is, the better the effect of inhibiting the synthesis of aflatoxin source and the growth of toxigenic bacteria is, so that the aflatoxin prevention and control bacteria with better effect are screened out: bacillus amyloliquefaciens, brevibaciens laterosporus, bacillus mucilaginosus and Enterobacter ludwigii.

4. A method according to claim 3, characterized in that: in the method for efficiently screening the aflatoxin prevention and control bacteria in one step, an aflatoxin biocontrol bacteria test treatment group and a control group are realized by the following steps: mixing peanut kernel powder, aspergillus flavus toxigenic fungus spores and a series of candidate prevention and control bacteria in a culture medium, and culturing for a certain time to obtain a test treatment group, wherein the culture medium is used for replacing the candidate prevention and control bacteria to culture for a certain time to obtain a control group;

the peanut kernel powder is a powdery product prepared by shelling and grinding Chinese flowering No. 6 and Luhua No. 8 varieties;

the Aspergillus flavus spore is 5×10 ⁵ cfu/mL and above Aspergillus flavus toxigenic spore liquid; the culture medium is a liquid state sand culture medium;

the content of the indicator molecule AFT-YJFZ01 is realized by the following steps:

a, after the treatment group and the control group are cultured for a certain time, fully homogenizing the culture to prepare a solution to be detected, adding the solution to be detected into a hole of an ELISA plate coated with a nanometer antibody or a monoclonal antibody of an indicator molecule AFT-YJFZ01 at the bottom of the hole, reacting, and washing the plate;

b, adding an indicator molecule AFT-YJFZ01 polyclonal antibody for reaction, and washing the plate;

c, adding a horseradish peroxidase labeled antibody which is subjected to a binding reaction with the indicator molecule AFT-YJFZ01 polyclonal antibody, reacting, and washing the plate;

d, adding a color development liquid for reaction; adding a stop solution, and reading and calculating the content of AFT-YJFZ01 in the solution to be measured by an enzyme-labeled instrument;

and replacing the solution to be tested with a series of concentration indicator molecule AFT-YJFZ01 pure product solution serving as a standard substance for making a standard curve, and calculating the content of the indicator molecule AFT-YJFZ01 according to the reading of an enzyme-labeled instrument.

5. The method according to claim 2, characterized in that: the batch culture is a conventional bacterial culture medium amplifying culture method; the 4 microorganism strains are obtained by separating peanut pods or rhizosphere soil thereof or soybean roots or rhizosphere soil thereof by a conventional method.

6. The method according to claim 1, characterized in that: the mixing ratio refers to the ratio of the number of viable bacteria of any strain after mixing to be more than or equal to 1%.

7. The method according to claim 1, characterized in that: the microbial strain is applied to the field of leguminous crops, and the mixed microbial strain is applied to the field along with the crop base fertilizer or applied to the field in the growing period of the crops, wherein the application amount is more than or equal to 1000 hundred million viable bacteria per mu.

8. The method according to claim 1, characterized in that: the leguminous crops comprise peanut, soybean, alfalfa and astragalus sinicus.

9. Use of the method of claim 1 for increasing the number of active nodules of a leguminous crop nitrogen-fixing enzyme while simultaneously reducing the levels of aflatoxins and its toxigenic bacteria in the leguminous crop.

10. Use of the method of claim 1 for inducing super nodulation in leguminous plants while simultaneously reducing aflatoxin and its toxigenic levels in leguminous crops.

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