An aflatoxin-producing fungus toxigenicity-indicating molecule and its application

Abstract

The present invention relates to an aflatoxin-producing fungus toxigenicity-indicating molecule and its application. The amino acid sequence of the aflatoxin-producing fungus toxigenicity-indicating molecule AFT-YJFZ01 is shown in SEQ ID NO.1. It is used to identify the ability of Aspergillus aflatoxin-producing strains to produce aflatoxin and to identify whether there are highly toxigenic Aspergillus aflatoxin-producing strains in farmland, agricultural products and feeds, and is easy to popularize and apply.

Description

Technical Field

[0001] The present invention relates to a toxigenicity indicator molecule of aflatoxin-producing fungi and its application. Background Art

[0002] Aflatoxin is highly toxic and harmful, and it is the pollutant that contaminates the most types of food. In recent years, the overall pollution trend has been increasing, seriously threatening food safety and people's health. Aflatoxin is one of the most toxic mycotoxins in nature. Among them, aflatoxin B1 is a class I carcinogen identified by the International Agency for Research on Cancer (IARC), and it has caused many poisoning incidents in human and livestock groups, becoming one of the main reasons for the high incidence of liver cancer cases. According to the statistics of Web of Science retrieval data in the past 5 years: the types of foods and raw materials contaminated by aflatoxin exceed 110, ranking first among pollutants. However, so far, there is still no example of molecular early warning research before the contamination of microbial toxins such as aflatoxin at home and abroad, which is difficult to meet the urgent need for early warning.

[0003] The existing aflatoxin early warning methods are mainly established based on aflatoxin detection technology, and are used for the evaluation of toxin pollution levels or the assessment of postnatal pollution degree and consumption risk. Once detected, the pollution has often occurred, which is difficult to meet the urgent requirements of early warning and guiding prevention and control. The Rapid Alert System for Food and Feed (RASFF) in the EU uses limit standards and detections to obtain the aflatoxin content in foods and feeds, and quickly warns the foods and feeds imported by various countries into the EU. Research institutions in the United States have established early warning models such as multiple logistic regression analysis and superimposed Gaussian processing based on aflatoxin detection technology and pollution monitoring data, mainly used for evaluating the pollution degree and consumption risk of mycotoxins in agricultural products such as corn after production.

[0004] Based on the research progress at home and abroad in the past two decades, the unclear early warning molecule of aflatoxin is the fundamental reason. To address this bottleneck problem, the inventor's team has conducted research for more than a decade, constructed a library of aflatoxin-producing strains in China, a database of strain toxigenicity, a protein antibody library of highly toxigenic strains, established an antibody library method for exploring aflatoxin-producing strain toxigenicity indicator molecules, and successfully invented a toxigenicity indicator molecule of aflatoxin-producing fungi, which is used to identify the ability of strains to produce aflatoxin, and to discover whether there are highly toxigenic aflatoxin-producing strains in farmland, agricultural products and feeds, providing a scientific basis for timely discovering aflatoxin pollution risks and early prevention and control. Summary of the Invention

[0005] The present invention aims at the deficiencies existing in the prior art, and provides a mycotoxin-producing ability-indicating molecule AFT-YJFZ01 for aflatoxin-producing fungi, as well as a method for identifying the mycotoxin-producing ability of aflatoxin-producing fungi in a discrimination system. It is used to identify the ability of Aspergillus strains to produce aflatoxins and to identify whether there are aflatoxin-producing strains with strong mycotoxin-producing ability in farmland, agricultural products and feeds, and is easy to promote and apply.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] Provide a mycotoxin-producing ability-indicating molecule AFT-YJFZ01 for aflatoxin-producing fungi, characterized in that the amino acid sequence of the mycotoxin-producing ability-indicating molecule for aflatoxin-producing fungi is as shown in SEQ ID NO.1.

[0008] Provide a method for identifying the mycotoxin-producing ability of aflatoxin-producing fungi in a discrimination system. Based on the detection of the content of the mycotoxin-producing ability-indicating molecule AFT-YJFZ01 for aflatoxin-producing fungi, the mycotoxin-producing ability of aflatoxin-producing fungi in the system is identified. The amino acid sequence of the mycotoxin-producing ability-indicating molecule AFT-YJFZ01 for aflatoxin-producing fungi is as shown in SEQ ID NO.1.

[0009] Specifically, by using the amino acid sequence of the mycotoxin-producing ability-indicating molecule AFT-YJFZ01 for aflatoxin-producing fungi or a partial sequence thereof, through the conventional antibody preparation process, an antibody corresponding to this protein can be prepared, and quantitative detection of this indicator molecule protein can be achieved. Alternatively, quantitative detection of these substances that have a one-to-one correspondence with this protein can also be achieved through other detection technical means, so as to achieve the above uses. The partial sequence refers to a part of the full sequence that has a one-to-one correspondence with this indicator molecule protein.

[0010] Application for identifying the ability of Aspergillus aflatoxin-producing strains to produce aflatoxins. The higher the content of the mycotoxin-producing ability-indicating molecule for aflatoxin-producing fungi in the strain to be identified, the stronger the ability of the identified strain to produce aflatoxins, that is, the stronger the mycotoxin-producing ability;

[0011] The specific application method includes:

[0012] (1) Provide a nanobody or monoclonal antibody of the mycotoxin-producing ability-indicating molecule AFT-YJFZ01 for aflatoxin-producing fungi:

[0013] (2) Provide a polyclonal antibody of the mycotoxin-producing ability-indicating molecule AFT-YJFZ01 for aflatoxin-producing fungi;

[0014] (3) Preparation of the test solution of the strain to be identified: Cultivate and dilute the strain to be identified to obtain the test solution of the strain to be identified;

[0015] Specifically, the strain to be identified can be cultured in a conventional Czapek medium or other medium suitable for the growth of the strain at an ambient temperature of 15 to 35°C for at least 12 hours, and then the mixture of the medium and the culture is fully homogenized and diluted 1 to 10 times with sterile water to obtain a test solution of the strain to be identified;

[0016] (4) Determination of the aflatoxin-producing ability of the strain to be identified:

[0017] An indirect non-competitive double-antibody sandwich method is used to identify the aflatoxin-producing ability of aflatoxin-producing strains of the genus Aspergillus. The steps include: coating an AFT-YJFZ01 nanoantibody or monoclonal antibody in an ELISA plate, washing the plate; adding a blocking solution for blocking, washing the plate; adding a test solution for reaction, washing the plate; adding an AFT-YJFZ01 polyclonal antibody for reaction, washing the plate; adding a horseradish peroxidase-labeled antibody that reacts with the polyclonal antibody for the toxicity indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria, reacting, washing the plate; adding a color developing solution for reaction; adding a stop solution, and reading and calculating the results with an ELISA reader.

[0018] The specific steps are:

[0019] The nanobody or monoclonal antibody of AFT-YJFZ01 is prepared into a coating solution of 0.2-8.0 μg / mL using ELISA coating buffer, and then added to the ELISA plate (200 μL / well). After being placed at 4°C overnight or at 37°C for not less than 2 hours, the coating solution in the ELISA plate is removed, and the ELISA plate is washed with a conventional ELISA washing solution. Then, skim milk powder with a concentration of not less than 1% is used as a blocking solution (300 μL is added to each well), and the plate is blocked at room temperature or at 37°C for not less than 1 hour. The blocking solution is then discarded, and the ELISA plate is washed with a conventional ELISA washing solution.

[0020] Then, dilute the test solution appropriately with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells (add 200 μL to each well), place it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with conventional ELISA washing solution;

[0021] Then, dilute the AFT-YJFZ01 polyclonal antibody appropriately with a conventional phosphate buffer with a pH close to neutral, add 200 μL to the ELISA plate wells, place at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with conventional ELISA washing solution;

[0022] Then, dilute the commercial horseradish peroxidase-labeled antibody as needed with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells (200 μL per well), place it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with a conventional ELISA wash solution;

[0023] Then, add the chromogenic solution and stop solution routinely used in ELISA successively, and finally read and calculate the content result of AFT-YJFZ01 by an enzyme-labeled instrument.

[0024] According to the above scheme, a standard curve is made with solutions of the toxin-producing ability indicator molecule AFT-YJFZ01 with a series of concentration gradients.

[0025] Application for identifying whether there are Aspergillus flavus toxin-producing strains with strong toxin-producing ability in a sample. The specific application method and steps are as follows:

[0026] (1) Provide a nanobody or monoclonal antibody of the toxin-producing ability indicator molecule AFT-YJFZ01 of Aspergillus flavus toxin-producing bacteria:

[0027] (2) Provide a polyclonal antibody of the toxin-producing ability indicator molecule AFT-YJFZ01 of Aspergillus flavus toxin-producing bacteria;

[0028] (3) Preparation of the sample to be identified: Culture and dilute the sample to be identified to obtain the test solution of the sample to be identified;

[0029] Specifically, it can be: Weigh the sample to be identified, transfer it to an appropriate amount of sterile water, shake it at room temperature until it is uniform to make a uniformly dispersed solution of the test sample. Take 10 - 1000 μL of the uniformly dispersed solution of the sample and add it to a conventional Czapek medium containing 6 - 600 mL or other suitable medium for the growth of Aspergillus flavus producing toxins. Incubate it at 15 - 35 °C with shaking at 200 ± 50 rpm. After incubating for 6 - 24 h, take a sample to form the test solution of the sample to be identified.

[0030] (4) Used for identifying whether there are Aspergillus flavus toxin-producing strains with strong toxin-producing ability in a sample: The steps include:

[0031] Coat the nanobody or monoclonal antibody of AFT-YJFZ01 on an enzyme-labeled plate, wash the plate; add a blocking solution to block, wash the plate; add the test solution of the sample to be identified to react, wash the plate; add the polyclonal antibody of AFT-YJFZ01 to react, wash the plate; add a horseradish peroxidase-labeled antibody that binds to the polyclonal antibody of the toxin-producing ability indicator molecule AFT-YJFZ01 of Aspergillus flavus toxin-producing bacteria to react, wash the plate; add a chromogenic solution to react; add a stop solution, and read and calculate the result with an enzyme-labeled instrument;

[0032] The specific steps are as follows:

[0033] Prepare a coating solution of 0.2 - 8.0 μg / mL of the above-mentioned nanobody or monoclonal antibody of AFT-YJFZ01 with ELISA coating buffer, and then add it to the ELISA plate (200 μL / well). After placing it at 4°C overnight or at 37°C for at least 2 hours, remove the coating solution in the ELISA plate, and then wash the ELISA plate with ELISA conventional washing solution; then, use skim milk powder with a concentration of not less than 1% as the blocking solution (add 300 μL per well), place it at room temperature or 37°C for at least 1 hour, then discard the blocking solution, and then wash the ELISA plate with ELISA conventional washing solution;

[0034] Then, appropriately dilute the sample solution to be identified with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells (add 200 μL per well), place it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with ELISA conventional washing solution;

[0035] Then, appropriately dilute the polyclonal antibody of the above-mentioned AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells (add 200 μL per well), place it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with ELISA conventional washing solution;

[0036] Then, dilute the commercially available horseradish peroxidase-labeled antibody as needed with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells (add 200 μL per well), place it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with ELISA conventional washing solution;

[0037] Then, add the ELISA conventional chromogenic solution and stop solution successively, and finally read and calculate the content result of AFT-YJFZ01 by an ELISA reader.

[0038] (5) Judgment of identification results:

[0039] The higher the content of the aflatoxin-producing fungus toxin-producing ability indicator molecule AFT-YJFZ01 in the sample to be identified, the stronger the aflatoxin-producing ability, that is, the toxin-producing ability, of the sample to be identified. According to the calculation result of the above ELISA reader, obtain the content of the aflatoxin-producing fungus toxin-producing ability indicator molecule AFT-YJFZ01 in the sample solution to be detected per unit volume, and determine whether there are strong toxin-producing strains of aflatoxin in the sample to be identified.

[0040] According to the above scheme, the sample to be identified is soil, agricultural products, Chinese medicinal materials or feed, etc.

[0041] According to the above solution, the polyclonal antibody of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi has a different animal origin from the nanobody or monoclonal antibody of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi. Specifically:

[0042] The nanobody or monoclonal antibody of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi can be obtained by the following method: using AFT-YJFZ01 as an immunizing antigen, immunizing alpacas or Balb / c mice in a conventional manner, and then developing and obtaining it by using known conventional nanobody or murine monoclonal antibody preparation technical schemes;

[0043] The polyclonal antibody of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi can be obtained by the following method: using AFT-YJFZ01 as an immunizing antigen, immunizing experimental rabbits such as New Zealand white rabbits in a conventional manner, and then developing and obtaining the rabbit-derived polyclonal antibody of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi by using known conventional polyclonal antibody preparation technical schemes;

[0044] The horseradish peroxidase-labeled antibody that binds to the polyclonal antibody of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 is a horseradish peroxidase-labeled goat anti-rabbit antibody, which can be directly purchased as a commodity.

[0045] The aflatoxin-producing ability of Aspergillus flavus strains is an index to measure the ability of strains to produce aflatoxins. The stronger the aflatoxin-producing ability of the strain, the more aflatoxins the strain can produce under the same time and culture conditions. The present invention provides an aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi. By using the characteristic that the content of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi is positively correlated with the aflatoxin-producing ability of the strain, the uses of identifying the aflatoxin-producing ability of Aspergillus flavus aflatoxin-producing strains and identifying whether there are aflatoxin-producing strains with strong aflatoxin-producing ability in farmland, agricultural products and feeds are further provided. It is practical and easy to promote, and provides a key starting point and scientific basis for timely discovering the risk of aflatoxin pollution and early prevention and control, and has important significance for promoting the high-quality development of the agricultural industry and ensuring food safety.

[0046] The beneficial effects of the present invention are as follows:

[0047] 1. It can be used to identify the aflatoxin-producing ability of Aspergillus flavus aflatoxin-producing strains;

[0048] 2. It can be used to identify whether there are aflatoxin-producing strains with strong aflatoxin-producing ability in farmland, agricultural products and feeds;

[0049] 3. It is easy to operate, has strong practicability, and is easy to promote and apply;

[0050] It is of great significance to promote the high-quality development of the agricultural industry and ensure food safety. Specific implementation manners

[0051] Example 1 Preparation of the aflatoxin-producing fungus toxin-producing ability indicator molecule AFT-YJFZ01

[0052] Prepare Czapek medium according to the following formula: 3% (w / v) sucrose, 0.3% (w / v) NaNO3, 0.1% (w / v) K2HPO4, 0.05% (w / v) MgSO4·7H2O, 0.05% (w / v) KCl, 0.001% (w / v) FeSO4, pH 6.5, and obtain Czapek medium. Randomly select 10 toxin-producing strains published in the public literature "Research on the Distribution, Toxin-Producing Ability and Infection of Aspergillus flavus in Typical Peanut Producing Areas of China" - Master's thesis of the Chinese Academy of Agricultural Sciences, author Zhang Xing, page 33 - HLJ-1, HeNZY-2, HuBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-122-2, JSnt-1, HuNdx-7, HBHA-8-17, etc., and inoculate them into the above Czapek medium respectively. After culturing at 28°C and 20 rpm / min for 5 days, homogenize and break cells by conventional methods, and use conventional protein purification systems, protein electrophoresis, immunoaffinity and other methods to purify the aflatoxin-producing fungus toxin-producing ability indicator molecule AFT-YJFZ01. The test results show that AFT-YJFZ01 can be prepared from the above toxin-producing strain cultures. Under the same culture conditions, the amount of AFT-YJFZ01 prepared by HBHA-8-17 is the largest, while the amount of AFT-YJFZ01 prepared by HLJ-1 is the smallest.

[0053] The initial acquisition of the aflatoxin-producing fungus toxin-producing ability indicator molecule AFT-YJFZ01 uses the discovery method:

[0054] The method for discovering the aflatoxin-producing strain toxin-producing ability indicator molecule AFT-YJFZ01 is as follows:

[0055] (1) Take aflatoxin high-toxin-producing strains, culture to obtain strain cultures and extracellular secreted protein mixtures; then break the strain culture cells to obtain intracellular protein mixtures; combine the above extracellular secreted protein mixtures and intracellular protein mixtures, and add carbodiimide for coupling to obtain aflatoxin antigens;

[0056] (2) Immunize experimental animals with the above aflatoxin antigens to obtain a nanobody library or a monoclonal antibody library;

[0057] (3) Obtain the protein combined solution of Aspergillus flavus strains with different toxin-producing abilities, and use the antibodies in the antibody library obtained in step (2) to detect the proteins of Aspergillus flavus strains with different toxin-producing abilities, so as to obtain a series of detection signals;

[0058] (4) Identify the nanobodies whose detection signals are positively correlated with the toxin-producing ability of the above-mentioned Aspergillus flavus strains, that is, the antibody of the Aspergillus flavus strain toxin-producing ability indicator molecule, and the protein corresponding to the antibody of the Aspergillus flavus strain toxin-producing ability indicator molecule is the discovered Aspergillus flavus strain toxin-producing ability indicator molecule.

[0059] In the above solution, the Aspergillus flavus strain with strong toxin-producing ability in step (1) is isolated and identified from nature by conventional methods, or obtained by artificial modification, and its toxin-producing ability is identified by the standard method of NY / T 2311—2013 and the result is not less than 10 μg / kg.

[0060] In step (3), there are no less than 3 Aspergillus flavus strains with different toxin-producing abilities, and their toxin-producing abilities are identified by the standard method of NY / T 2311—2013 and presented in at least 3 levels of high, medium and low.

[0061] The medium used in the cultivation of the Aspergillus flavus strain with strong toxin-producing ability is Czapek medium or other nutrients that can support the normal growth of Aspergillus flavus. The cultivation time is not less than 12 h, and the cultivation environmental temperature is 15-35 °C.

[0062] The cell disruption of the strain culture is carried out by conventional methods such as liquid nitrogen grinding or cell disruptor.

[0063] The dosage of the carbodiimide is 0.005-0.1 g of carbodiimide added to every 1.0 mL of the combined extracellular secreted protein mixture and intracellular protein mixture.

[0064] The coupling reaction refers to reacting at 15-37 °C for 2-6 h and reacting overnight at 4-10 °C.

[0065] The immunization is a conventional immunization method, inoculating with Aspergillus flavus antigen. The test animals refer to mice or alpacas or other test animals with similar effects.

[0066] According to the above solution, the antibody preparation process refers to the conventional nanobody preparation technical process or the conventional cell fusion-based hybridoma monoclonal antibody preparation technical process.

[0067] According to the above solution, the detection of the proteins of Aspergillus flavus strains with different toxin-producing abilities refers to using the conventional Western Blot technical process, that is, transferring the proteins of Aspergillus flavus strains with different toxin-producing abilities onto a nitrocellulose membrane, and then using the antibodies in the above antibody library to detect by direct method or indirect method, or using other technical processes with similar effects.

[0068] According to the above solution, the above direct method refers to conjugating the antibodies in the above antibody library with a signal material by a conventional method, and then performing an immunobinding reaction with the corresponding protein transferred onto the nitrocellulose membrane.

[0069] According to the above solution, the above indirect method refers to first performing an immunobinding reaction between the antibodies in the above antibody library and the corresponding protein transferred onto the nitrocellulose membrane, and then using a second antibody-conjugated signal material to perform an immunobinding reaction with the antibody bound to the nitrocellulose membrane.

[0070] In the above detection, the signal material is horseradish peroxidase or colloidal gold or fluorescent material or other materials with similar functions. The detection signal is a color reaction signal or a spot signal or a fluorescence signal.

[0071] Example 2 Preparation of Nanobody of Aflatoxin-Producing Bacteria Virulence-Indicating Molecule AFT-YJFZ01

[0072] Using AFT-YJFZ01 as an immunizing antigen, immunize alpacas or Balb / c mice in a conventional manner, and then it can be developed and obtained by using known conventional nanobody or murine monoclonal antibody preparation technical solutions.

[0073] Dissolve the prepared AFT-YJFZ01 above in a conventional PBS buffer or physiological saline to a concentration not lower than 0.1 mg / mL, and then mix it with Freund's complete adjuvant in equal volume and emulsify it. Immunize alpacas by multi-point subcutaneous or intradermal injection on the back. After that, strengthen the immunization once every 2 - 4 weeks. When strengthening the immunization, replace Freund's complete adjuvant with Freund's incomplete adjuvant. Monitor the immunization effect using a conventional ELISA procedure. After the alpaca serum titer no longer rises, then collect blood from the immunized alpaca's vein, extract total RNA, synthesize cDNA, amplify the VHH gene, recover the VHH gene fragment, ligate the VHH gene with the pCANTAB 5E(his) vector treated with double digestion, electrotransform the ligation product, construct a nanobody gene library, and rescue the nanobody gene library, etc. These operations are completed according to the method of patent document CN103866401A, and finally a rescued nanobody gene library is obtained.

[0074] The AFT-YJFZ01 obtained by the above preparation was immobilized on a solid-phase carrier such as a 96-well enzyme-labeled plate at gradients of 8 μg / well, 2 μg / well, 0.5 μg / well, and 0.1 μg / well. The rescued nanobody gene library was subjected to 2-4 rounds of panning according to the method of patent document CN103866401A. Then, the antibodies produced by each phage clone were identified using AFT-YJFZ01 and indirect non-competitive ELISA. The phage corresponding to the positive result was the phage positive clone. This positive clone was then used to prepare nanobodies by the conventional method for preparing nanobodies, which were the nanobodies of AFT-YJFZ01 and used for further application research work. Preferably, the nanobodies were characterized by the ELISA method and had strong specificity and high affinity.

[0075] Example 3 Preparation of Monoclonal Antibodies Against the Aflatoxin-producing Fungus Virulence Indicator Molecule AFT-YJFZ01

[0076] Using AFT-YJFZ01 as the immunizing antigen, immunize alpacas or Balb / c mice in a conventional manner, and then the monoclonal antibodies can be developed using known conventional nanobody or murine monoclonal antibody preparation technical solutions.

[0077] Dissolve the AFT-YJFZ01 obtained by the above preparation in a conventional PBS buffer or physiological saline to a concentration not lower than 0.1 mg / mL, and then mix it with an equal volume of Freund's complete adjuvant and emulsify it. Inject BALB / c mice by multiple subcutaneous or intradermal injections in the back. Then, strengthen the immunization once every 2-4 weeks. When strengthening the immunization, replace Freund's complete adjuvant with Freund's incomplete adjuvant. Monitor the immunization effect using the conventional ELISA procedure. After the serum titer of BALB / c mice no longer increases, then isolate the immune mouse spleen cells, fuse the spleen cells with murine myeloma cells SP2 / 0, and the selective culture of hybridoma cells in semi-solid medium was completed according to the method of patent document CN103849604A. After white pinhead spots appeared on the semi-solid medium, pick the white spots into 96-well culture plates containing conventional hybridoma culture medium respectively, so as to obtain a monoclonal hybridoma resource library.

[0078] Obtain the above monoclonal hybridoma culture supernatant, that is, monoclonal antibodies, according to the method of patent document CN103849604A. Immobilize the AFT-YJFZ01 obtained by the above preparation on a solid-phase carrier such as a 96-well enzyme-labeled plate at gradients of 8 μg / well, 2 μg / well, 0.5 μg / well, and 0.1 μg / well. Then, identify each monoclonal antibody using the indirect non-competitive ELISA procedure, pick the positive clones, obtain the AFT-YJFZ01 monoclonal antibodies, and use them for further application research work. Preferably, the AFT-YJFZ01 monoclonal antibodies are detected to have strong specificity and high affinity.

[0079] Example 4 Preparation of rabbit polyclonal antibodies against the toxicity indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria

[0080] AFT-YJFZ01 is used as the immune antigen, New Zealand white rabbits and other experimental rabbits are immunized in a conventional manner, and then the antibody is developed using the known conventional rabbit polyclonal antibody preparation technology.

[0081] AFT-YJFZ01, an indicator of the toxicity of aflatoxin-producing bacteria, was used directly as an antigen. A solution with a concentration of no less than 0.1 mg / mL was mixed with an equal volume of Freund's complete adjuvant and emulsified. The solution was then injected subcutaneously or intradermally into New Zealand white rabbits at multiple points on the back. Booster immunizations were then administered every two to four weeks, replacing Freund's complete adjuvant with incomplete adjuvant. The efficacy of the immunization was monitored using a conventional ELISA procedure. Once the titer of the immunized animal serum ceased to rise, the serum was prepared using conventional methods to generate a rabbit polyclonal antibody against AFT-YJFZ01, an indicator of the toxicity of aflatoxin-producing bacteria.

[0082] Example 5 Identification of aflatoxin-producing strains using the aflatoxin-producing strain toxicity indicator molecule AFT-YJFZ01

[0083] The first step is to prepare the test solution of the strain to be identified: according to the strength of the toxin production ability of the strain, 10 toxin-producing strains HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSnt-1, HuNdx-7, GDZJ-108-19, and HBHA-8-17 published in the public document "Research on the Distribution, Toxicity and Infection of Aflatoxin in Typical Flower Production Areas in China" - a master's thesis of the Chinese Academy of Agricultural Sciences, author Zhang Xing, page 33 - were selected. The strain to be identified was cultured in conventional Czapek medium or other culture medium suitable for the growth of toxin-producing aflatoxin. The culture environment temperature was 15-35°C. After the culture time was not less than 12 hours, the mixture of the culture medium and the culture was fully homogenized and then diluted 5 times with sterile water to obtain the test solution of the strain to be identified.

[0084] Step 2, determination of the test solution of the strain to be identified: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 above in a conventional ELISA coating buffer to form a coating solution with a concentration of 2 μg / mL, and then add 200 μL per well to a 96-well ELISA plate. After placing it at 4°C overnight or at 37°C for at least 2 hours, remove the coating solution in the ELISA plate, and then wash the ELISA plate with a conventional ELISA washing solution; then, use skimmed milk powder with a concentration not less than 1% as the blocking solution, add 300 μL per well, place it at room temperature or 37°C for at least 1 hour for blocking, then discard the blocking solution, and then wash the ELISA plate with a conventional ELISA washing solution; then appropriately dilute the above test liquid with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well, or add 200 μL of the solution of the toxigenicity-indicating molecule AFT-YJFZ01 of the present invention with a series of concentrations per well. After placing it at room temperature or 37°C for at least 1 hour for blocking, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, appropriately dilute the rabbit polyclonal antibody of AFT-YJFZ01 above with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37°C for at least 1 hour for blocking, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody according to the instructions with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37°C for at least 1 hour for blocking, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, add the conventional chromogenic solution and stop solution of ELISA in sequence, and finally read and calculate the content of AFT-YJFZ01 through an ELISA reader.

[0085] According to the results of the above literature "Research on the Distribution, Toxigenicity and Infection of Aspergillus flavus in Typical Peanut Production Areas in China", the toxigenicity of 10 strains such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSnt-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 is as follows: 0 μg / L, 0 μg / L, 3.8 μg / L, 4.9 μg / L, 67.2 μg / L, 81.7 μg / L, 192.0 μg / L, 204.4 μg / L, 297.4 μg / L, 1027.5 μg / L, indicating that HBZHX-21 and HBXY-36 are non-toxigenic strains, HBHA-1-4 and GDZJ-6 are weakly toxigenic strains, and HBHA-8-17 and GDZJ-108-19 are strongly toxigenic strains.

[0086] Step 3, Judgment of the identification result: The higher the content of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 in the strain to be identified, the stronger the aflatoxin-producing ability (i.e., the toxin-producing ability) of the identified strain. According to the above technical solution, the results of measuring the content of AFT-YJFZ01 in 10 strains such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSnt-1, HuNdx-7, GDZJ-108-19, and HBHA-8-17 are as follows: the amount of AFT-YJFZ01 contained in each milliliter of the culture medium is 0 ng, 0 ng, 8 ng, 12 ng, 49 ng, 51 ng, 104 ng, 115 ng, 175 ng, and 536 ng in sequence. The results also show that HBZHX-21 and HBXY-36 are non-toxin-producing strains, HBHA-1-4 and GDZJ-6 are weak toxin-producing strains, and HBHA-8-17 and GDZJ-108-19 are strong toxin-producing strains. The order of the toxin-producing ability of the strains determined by this result is consistent with the order of the toxin-producing ability of the strains published in the above-mentioned public literature "Research on the Distribution, Toxin-Producing Ability and Infection of Aspergillus flavus in Typical Peanut Production Areas in China", and the identification results of the strong toxin-producing strains and weak toxin-producing strains are consistent with the literature, which proves that the technical solution provided by the present invention can be used to identify the toxin-producing ability of strains producing aflatoxin, and the method is simple, easy to operate, and has strong practicability.

[0087] Example 6 Identification of Strong Toxin-Producing Strains Producing Aflatoxin in Farmland by Using the Aflatoxin-Producing Ability Indicator Molecule AFT-YJFZ01

[0088] Step 1, Preparation of the test solution of the sample to be identified: Select 4 peanut rhizosphere soil samples during the flowering period from Jilin, Liaoning, Jiangxi, Fujian, etc., and name them soil sample - 1, soil sample - 2, soil sample - 3, and soil sample - 4 in sequence. Weigh the farmland soil samples to be tested in sequence, crush them and transfer them to sterile water, with a concentration of 0.5 g / mL, shake at room temperature until uniform, and make a uniformly dispersed solution of the sample to be tested. Take 50 μL of the soil dilution and add it to 30 mL of a conventional Sabouraud liquid medium, place it in a shaker at 28 °C with a speed of 200 rpm for cultivation, and take samples after 24 hours of cultivation to form the test solution of the sample to be identified.

[0089] Step 2, determination of the test solution of the sample to be identified: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 in a conventional ELISA coating buffer to form a coating solution with a concentration of 0.2-8.0 μg / mL, and then add 200 μL per well to a 96-well ELISA plate. After placing it at 4°C overnight or at 37°C for at least 2 hours, remove the coating solution in the ELISA plate, and then wash the ELISA plate with a conventional ELISA washing solution; then, use skim milk powder with a concentration not lower than 1% as the blocking solution, add 300 μL per well, place it at room temperature or 37°C for at least 1 hour, then discard the blocking solution, and then wash the ELISA plate with a conventional ELISA washing solution; then, appropriately dilute the above-mentioned test liquid with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well, or add 200 μL of a solution of the virulence-indicating molecule AFT-YJFZ01 of the present invention with a series of concentrations (concentrations of 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300 ng / mL, for obtaining a standard curve) per well. After placing it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, appropriately dilute the rabbit polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody according to the instructions with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37°C for at least 1 hour, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, add the conventional chromogenic solution and stop solution of ELISA in sequence, and finally read through an ELISA reader and calculate the content result of AFT-YJFZ01 based on the standard curve.

[0090] Step 3, judgment of the identification result: If the content of the virulence-indicating molecule AFT-YJFZ01 of the aflatoxin-producing fungus in the sample to be identified is high, it indicates that the sample to be identified contains strong virulence strains of aflatoxin. According to the above technical solution, the content results of AFT-YJFZ01 in soil sample -1, soil sample -2, soil sample -3, and soil sample -4 were measured - the amounts of AFT-YJFZ01 contained in each milliliter of the culture solution were: 0.3 ng, 0.2 ng, 16 ng, and 19 ng in sequence. This result shows that the amounts of AFT-YJFZ01 contained in each milliliter of the culture solution in soil sample -1 and soil sample -2 are both below 1.0 ng, and they do not contain strong virulence strains of aflatoxin, and the post-harvest pollution risk of peanuts in the corresponding farmland is very low; the amounts of AFT-YJFZ01 contained in each milliliter of the culture solution in soil sample -1 and soil sample -2 are both above 10 ng, and they contain strong virulence strains of aflatoxin, and the post-harvest pollution risk of peanuts in the corresponding farmland is relatively high.

[0091] Example 7: Using the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing fungi to identify whether strongly aflatoxin-producing strains are contained in agricultural products

[0092] First step: Preparation of the test solution of the sample to be identified: Select 4 agricultural product samples such as peanuts, corn, rice, and wheat. Weigh the test farmland soil samples in sequence, crush them and transfer them to sterile water at a concentration of 0.5 g / mL, shake at room temperature until uniform, and prepare a uniformly dispersed solution of the test sample. Take 100 μL of the uniformly dispersed sample solution and add it to 50 mL of a conventional Sabouraud liquid medium, place it in a shaker at 28 °C and 200 rpm for cultivation. After 6 hours of cultivation, take a sample to form the test solution of the sample to be identified.

[0093] Second step: Determination of the test solution of the sample to be identified above: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 above in a conventional ELISA coating buffer to form a coating solution with a concentration of 0.2 - 8.0 μg / mL, and then add 200 μL per well to a 96-well ELISA plate. After placing it at 4 °C overnight or at 37 °C for at least 2 hours, remove the coating solution in the ELISA plate, and then wash the ELISA plate with a conventional ELISA washing solution; then, use skim milk powder with a concentration not lower than 1% as the blocking solution, add 300 μL per well, place it at room temperature or 37 °C for at least 1 hour of blocking, then discard the blocking solution, and then wash the ELISA plate with a conventional ELISA washing solution; then appropriately dilute the above test liquid with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well, or add 200 μL of a solution of the aflatoxin-producing ability indicator molecule AFT-YJFZ01 of the present invention with a series of concentrations (concentrations are 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300 ng / mL, for obtaining a standard curve) per well (for obtaining a standard curve). After placing it at room temperature or 37 °C for at least 1 hour of blocking, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, appropriately dilute the rabbit polyclonal antibody of AFT-YJFZ01 above with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37 °C for at least 1 hour of blocking, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody according to the instructions with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37 °C for at least 1 hour of blocking, discard the liquid, and then wash the ELISA plate with a conventional ELISA washing solution; then, add the conventional color-developing solution and terminating solution of ELISA in sequence, and finally read with an ELISA reader, and calculate the content result of AFT-YJFZ01 based on the standard curve.

[0094] Step 3, judgment of the identification result: If the content of the aflatoxin-producing strain virulence indicator molecule AFT-YJFZ01 in the sample to be identified is high, it indicates that the sample to be identified contains strong aflatoxin-producing strains. The results of the AFT-YJFZ01 content in peanut, corn, rice, and wheat samples measured according to the above technical solution - the amount of AFT-YJFZ01 contained in each milliliter of the culture medium is 12 ng, 13 ng, 0.7 ng, and 0 ng in sequence. This result shows that the amount of AFT-YJFZ01 contained in each milliliter of the culture medium in peanut and corn samples is above 10 ng, and they contain strong aflatoxin-producing strains with a relatively high pollution risk; the amount of AFT-YJFZ01 contained in each milliliter of the culture medium in the rice sample is below 1.0, and it does not contain strong aflatoxin-producing strains with a very low pollution risk; the amount of AFT-YJFZ01 contained in each milliliter of the culture medium in the measured wheat sample is 0, and it does not contain aflatoxin-producing strains with basically no aflatoxin pollution risk.

[0095] Example 8 Use of the aflatoxin-producing strain virulence indicator molecule AFT-YJFZ01 to identify whether a feed contains strong aflatoxin-producing strains

[0096] Step 1, preparation of the test solution of the sample to be identified: Select a total of 4 test feed samples from the market, named Feed-1, Feed-2, Feed-3, and Feed-4 in sequence. Weigh the test feed samples in sequence, crush them and transfer them to sterile water with a concentration of 0.5 g / mL, shake at room temperature until uniform, and make a uniformly dispersed test sample solution. Take 50 μL of the uniformly dispersed sample solution and add it to 30 mL of a conventional Sabouraud liquid medium, place it in a shaker at 28 °C and 200 rpm for incubation. After incubation for 24 h, take a sample to form the test solution of the sample to be identified.

[0097] Step 2. Determination of the test solution of the sample to be identified: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 in a conventional ELISA coating buffer to form a coating solution with a concentration of 0.2-8.0 μg / mL. Then add 200 μL per well to a 96-well ELISA plate. After placing it at 4°C overnight or at 37°C for at least 2 h, remove the coating solution in the ELISA plate, and then wash the ELISA plate with a conventional ELISA washing solution. Then, use skim milk powder with a concentration not less than 1% as the blocking solution, add 300 μL per well, place it at room temperature or 37°C for at least 1 h for blocking, then discard the blocking solution, and wash the ELISA plate with a conventional ELISA washing solution. Then, appropriately dilute the above-mentioned test liquid with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well, or add 200 μL of the solution of the virulence-indicating molecule AFT-YJFZ01 of the present invention with a series of concentrations (concentrations are 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300 ng / mL, for obtaining a standard curve) per well. After placing it at room temperature or 37°C for at least 1 h for blocking, discard the liquid, and wash the ELISA plate with a conventional ELISA washing solution. Then, appropriately dilute the rabbit polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37°C for at least 1 h for blocking, discard the liquid, and wash the ELISA plate with a conventional ELISA washing solution. Then, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody according to the instructions with a conventional phosphate buffer with a pH close to neutral, and then add 200 μL per well. After placing it at room temperature or 37°C for at least 1 h for blocking, discard the liquid, and wash the ELISA plate with a conventional ELISA washing solution. Then, add the conventional chromogenic solution and termination solution of ELISA in sequence, and finally read and calculate the content result of AFT-YJFZ01 through an ELISA reader.

[0098] Step 3. Judgment of the identification result: If the content of the virulence-indicating molecule AFT-YJFZ01 of the aflatoxin-producing fungus in the sample to be identified is high, it indicates that the sample to be identified contains strong virulence strains of aflatoxin. According to the above technical scheme, the content results of AFT-YJFZ01 in Feed-1, Feed-2, Feed-3, and Feed-4 are as follows: the amount of AFT-YJFZ01 contained in each milliliter of the culture solution is 0.6 ng, 22 ng, 13 ng, and 69 ng in sequence. This result shows that the amount of AFT-YJFZ01 contained in each milliliter of the culture solution in the Feed-1 sample is below 1.0 ng, and it does not contain strong virulence strains of aflatoxin, and its pollution risk is very low; taking the example of uniformly dispersing 50 μL of the sample dispersion liquid in 30 mL of the culture medium, the amount of AFT-YJFZ01 contained in each milliliter of the culture solution in Feed-2, Feed-3, and Feed-4 is above 10 ng, and they contain strong virulence strains of aflatoxin. <110> Oil Crops Research Institute, Chinese Academy of Agricultural Sciences <120> An indicator molecule for the toxin-producing ability of aflatoxin-producing fungi and its application <160> 1 <210> 1 <211> 33172 <212> PRT <213> Aspergillus flavus <400> 1 AIGVEEPEAD PTYYHNAIGV EEPEADPTYY HNNKAIGVEE PEADPTYYHN NKTTASMVWE 60 EAQQVSGKAS VETPASIEAA SELSKAVSPS FEDVWSQPRD GAGQMFIPLN PNAYSPNTLN 120 KDGAGQMFIP LNPNAYSPNT LNKGSPKDGV YVDKSVTSGF VDGIKDGLRD VGGPIEDQNS 180 LQVGDRDVHG FATRFEQLPI NQPRFGKPVG AVGSAAFGKP VGAVGSAATA LKGDNEIPQA 240 ATAHDSAWDF FSQQPSGDNE IPQAATAHDS AWDFFSQQPS SLHGPNFEQL PINQPRGPTL 300 LEDFIFRGVD FTEDPLLQGR GVGAHGVFTS YHGGPNFEQL PINQPRHVDG FGIHTFRLAS 360 VETPASIEAA SELSKLFSYL DTQLNRLFYN SLTPAEQQFV VDAIRNAGIQ TSRNAGIQTS 420 RDGVYVDKNS LTPAEQQFVV DAIRNYFAET EQVMFQPGHN YFAETEQVMF QPGHIVRPEE 480 YVPITKPLQI VIDGFRPNAY SPNTLNKPVG AVGSAATALK QDLFEAIEAG RQLSEDGVDV 540 VVVAERSEDG VDVVVVAERS LTPAEQQFVV DAIRSMVWEE AQQVSGKSPS FEDVWSQPRS 600 VETPASIEAA SELSKSVTSG FVDGIKDGLR TTDVGTFGQK VDFTEDPLLQ GRVETPASIE 660 AASELSKVGF LASVETPASI EAASELSKYP EWELGVQIMD EEDQLKFDHE RVPERFGFDL 720 FDPTKFDLFD PTKFGKPVGA VGSAATNPDF MRQDLFEAIE AGRFVTDNGD SKLVKFVTDN 780 GDSKSVTSGF VDGIKGFDLF DPTKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKIVPEEY 840 VPITKDGVYV DKSVTSGFVD GIKFENSNVK SSVVRVGGPI EDQNSLQVGD RGDNEIPQAA 900 TAHDSVTSGF VDGIKDTTDV GTFGQKLKGV GAHGVFTGDN EIPQAATAHF VVDAIRTLLE 960 DFIFRFTEDP LLQGRPDLIH AVKPRVDGFG IHTFRGVGAH GVFYLDTQLN RNVIIQLNRK 1020 PVGAVGSAAT ALKDGVYVDK NNVIIQLNRI VPEEYVPITK LGKTPAEQQF VVDAIRFENS 1080 NVKEQVMFQP GHIVRGVDFT EDPLLQGRAI GVEEPEADPT YYHNAIGVEE PEADPTYYHN 1140 ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKASMVWEEA QQVSGKASMV 1200 WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKASMVWEEA 1260 QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS MVWEEAQQVS 1320 GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE EAQQVSGKAS 1380 MVWEEAQQVS GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ VSGKASMVWE 1440 EAQQVSGKAS MVWEEAQQVS GKASMVWEEA QQVSGKASMV WEEAQQVSGK ASMVWEEAQQ 1500 VSGKASMVWE EAQQVSGKAS MVWEEAQQVS GKAVSPSFED VWSQPRAVSP SFEDVWSQPR 1560 AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED VWSQPRAVSP 1620 SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED 1680 VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV SPSFEDVWSQ 1740 PRAVSPSFED VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF EDVWSQPRAV 1800 SPSFEDVWSQ PRAVSPSFED VWSQPRAVSP SFEDVWSQPR AVSPSFEDVW SQPRAVSPSF 1860 EDVWSQPRAV SPSFEDVWSQ PRAVSPSFED VWSQPRDGAG QMFIPLNPNA YSPNTLNKDG 1920 AGQMFIPLNP NAYSPNTLNK DGAGQMFIPL NPNAYSPNTL NKDGAGQMFI PLNPNAYSPN 1980 TLNKDGAGQM FIPLNPNAYS PNTLNKDGAG QMFIPLNPNA YSPNTLNKDG AGQMFIPLNP 2040 NAYSPNTLNK GSPKDGAGQM FIPLNPNAYS PNTLNKGSPK DGVYVDKDGV YVDKDGVYVD 2100 KSVTSGFVDG IKDGVYVDKS VTSGFVDGIK FENSNVKSSV VRFGFDLFDP TKFGFDLFDP 2160 TKFGKPVGAV GSAATALKFG KPVGAVGSAA TALKFGKPVG AVGSAATALK FGKPVGAVGS 2220 AATALKFGKP VGAVGSAATA LKFGKPVGAV GSAATALKFG KPVGAVGSAA TALKFGKPVG 2280 AVGSAATALK FGKPVGAVGS AATALKFGKP VGAVGSAATA LKFGKPVGAV GSAATALKFG 2340 KPVGAVGSAA TALKFGKPVG AVGSAATALK FVTDNGDSKF VTDNGDSKLV KFVTDNGDSK 2400 LVKFVTDNGD SKLVKGPTLL EDFIFRGVDF TEDPLLQGRG VDFTEDPLLQ GRGVDFTEDP 2460 LLQGRGVDFT EDPLLQGRGV DFTEDPLLQG RGVDFTEDPL LQGRHGGPNF EQLPINQPRH 2520 GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2580 GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2640 GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2700 GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2760 GGPNFEQLPI NQPRHGGPNF EQLPINQPRH GGPNFEQLPI NQPRHGGPNF EQLPINQPRH 2820 GGPNFEQLPI NQPRHGGPNF EQLPINQPRH VDGFGIHTFR HVDGFGIHTF RHVDGFGIHT 2880 FRHVDGFGIH TFRHVDGFGI HTFRHVDGFG IHTFRHVDGF GIHTFRHVDG FGIHTFRHVD 2940 GFGIHTFRHV DGFGIHTFRH VDGFGIHTFR HVDGFGIHTF RHVDGFGIHT FRHVDGFGIH 3000 TFRHVDGFGI HTFRHVDGFG IHTFRHVDGF GIHTFRHVDG FGIHTFRHVD GFGIHTFRHV 3060 DGFGIHTFRI VPEEYVPITK IVPEEYVPIT KIVPEEYVPI TKLFSYLDTQ LNRLFSYLDT 3120 QLNRLFSYLD TQLNRLFSYL DTQLNRLFYN SLTPAEQQFV VDAIRLFYNS LTPAEQQFVV 3180 DAIRNAGIQT SRNAGIQTSR DGVYVDKNNV IIQLNRNYFA ETEQVMFQPG HNYFAETEQV 3240 MFQPGHIVRN YFAETEQVMF QPGHIVRNYF AETEQVMFQP GHIVRNYFAE TEQVMFQPGH 3300 IVRNYFAETE QVMFQPGHIV RNYFAETEQV MFQPGHIVRN YFAETEQVMF QPGHIVRNYF 3360 AETEQVMFQP GHIVRNYFAE TEQVMFQPGH IVRNYFAETE QVMFQPGHIV RNYFAETEQV 3420 MFQPGHIVRN YFAETEQVMF QPGHIVRNYF AETEQVMFQP GHIVRNYFAE TEQVMFQPGH 3480 IVRNYFAETE QVMFQPGHIV RQDLFEAIEA GRQDLFEAIE AGRQLSEDGV DVVVVAERQL 3540 SEDGVDVVVV AERQLSEDGV DVVVVAERQL SEDGVDVVVV AERSLQGKAS MVWEEAQQVS 3600 GKSLQGKASM VWEEAQQVSG KSPSFEDVWS QPRSVTSGFV DGIKSVTSGF VDGIKDGLRS 3660 VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS 3720 VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS VTSGFVDGIK DGLRSVTSGF VDGIKDGLRS 3780 VTSGFVDGIK DGLRTTDVGT FGQKTTDVGT FGQKTTDVGT FGQKTTDVGT FGQKTTDVGT 3840 FGQKLKTTDV GTFGQKLKVG FLASVETPAS IEAASELSKV GFLASVETPA SIEAASELSK 3900 VGFLASVETP ASIEAASELS KVGFLASVET PASIEAASEL SKVGFLASVE TPASIEAASE 3960 LSKVGFLASV ETPASIEAAS ELSKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKVPVHNN 4020 NRDGAGQMFI PLNPNAYSPN TLNKVPVHNN NRDGAGQMFI PLNPNAYSPN TLNKYPEWEL 4080 GVQIMDEEDQ LKYPEWELGV QIMDEEDQLK ALFNRDIATG KANNYCSNQV EGPYSLYSGR 4140 DIATGKVSIA KDYACPWNGG EEVSLKDYAC PWNGGEEVSL KVEYSDAAKE GDPEMYGNNE 4200 TVNKVCAKAN NYCSNQVEGP YSLYSGREPG ICETTPGVKE QTASVVNGTA VIKGFSATGD 4260 YPRGGPGSSS MIGLMQENGP CRGYLEDIAY VLDSGIKGYY DISHFDPDIG LMQENGPCRL 4320 AAEGDPEMYG NNETVNKNAP LSIWMNGGPG SSSMIGLMQE NGPCRNQVEG PYSLYSGRNY 4380 CSNQVEGPYS LYSGRPGGCK DQIIECRQVE GPYSLYSGRQ YGNFSFTRSN QVEGPYSLYS 4440 GRTNASYVGG LVRTVYDMAM EAWSKTVYDM AMEAWSKPGG TVYDMAMEAW SKPGGCKVAL 4500 VYGDRDYACP WNGGEEVSLK VFEAGHEVPA YQPETAYEIF VFEAGHEVPA YQPETAYEIF 4560 HRVSIWTESY GGRYCSNQVE GPYSLYSGRY GPSFTAFFQE QNEKVALVYG DRVEYSDAAK 4620 FRISYKEPGI CETTPGVKFT AFFQEQNEKS IWTESYGGRN YIVVDADSSF WFFESRISYK 4680 EPGIHDDRVS IWTESYGGRY GPSFTAFFQE QNEKVALVYG DRDYAANNYC SNQVEGPYSL 4740 YSGRANNYCS NQVEGPYSLY SGRANNYCSN QVEGPYSLYS GRANNYCSNQ VEGPYSLYSG 4800 RANNYCSNQV EGPYSLYSGR ANNYCSNQVE GPYSLYSGRA NNYCSNQVEG PYSLYSGRAN 4860 NYCSNQVEGP YSLYSGRANN YCSNQVEGPY SLYSGRANNY CSNQVEGPYS LYSGRANNYC 4920 SNQVEGPYSL YSGRANNYCS NQVEGPYSLY SGRANNYCSN QVEGPYSLYS GRANNYCSNQ 4980 VEGPYSLYSG RANNYCSNQV EGPYSLYSGR ANNYCSNQVE GPYSLYSGRA NNYCSNQVEG 5040 PYSLYSGRAN NYCSNQVEGP YSLYSGRANN YCSNQVEGPY SLYSGRDIAT GKVSIAKDYA 5100 CPWNGGEEVS LKDYACPWNG GEEVSLKGDP EMYGNNETVN KVCAKANNYC SNQVEGPYSL 5160 YSGRGGPGSS SMIGLMQENG PCRIGLMQEN GPCRIGLMQE NGPCRISYKE PGICETTPGV 5220 KISYKEPGIC ETTPGVKISY KEPGICETTP GVKISYKEPG ICETTPGVKI SYKEPGICET 5280 TPGVKISYKE PGICETTPGV KISYKEPGIC ETTPGVKNAP LSIWMNGGPG SSSMIGLMQE 5340 NGPCRNAPLS IWMNGGPGSS SMIGLMQENG PCRNAPLSIW MNGGPGSSSM IGLMQENGPC 5400 RNAPLSIWMN GGPGSSSMIG LMQENGPCRN APLSIWMNGG PGSSSMIGLM QENGPCRNAP 5460 LSIWMNGGPG SSSMIGLMQE NGPCRNAPLS IWMNGGPGSS SMIGLMQENG PCRNAPLSIW 5520 MNGGPGSSSM IGLMQENGPC RNAPLSIWMN GGPGSSSMIG LMQENGPCRN APLSIWMNGG 5580 PGSSSMIGLM QENGPCRNAP LSIWMNGGPG SSSMIGLMQE NGPCRPGGCK DQIIECRTVY 5640 DMAMEAWSKT VYDMAMEAWS KTVYDMAMEA WSKTVYDMAM EAWSKTVYDM AMEAWSKTVY 5700 DMAMEAWSKT VYDMAMEAWS KTVYDMAMEA WSKPGGTVYD MAMEAWSKPG GTVYDMAMEA 5760 WSKPGGCKTV YDMAMEAWSK PGGCKTVYDM AMEAWSKPGG CKTVYDMAME AWSKPGGCKT 5820 VYDMAMEAWS KPGGCKTVYD MAMEAWSKPG GCKTVYDMAM EAWSKPGGCK TVYDMAMEAW 5880 SKPGGCKVAL VYGDRDYACP WNGGEEVSLK VALVYGDRDY ACPWNGGEEV SLKVALVYGD 5940 RDYACPWNGG EEVSLKVALV YGDRDYACPW NGGEEVSLKV ALVYGDRDYA CPWNGGEEVS 6000 LKVALVYGDR DYACPWNGGE EVSLKVALVY GDRDYACPWN GGEEVSLKVA LVYGDRDYAC 6060 PWNGGEEVSL KVALVYGDRD YACPWNGGEE VSLKVALVYG DRDYACPWNG GEEVSLKVFE 6120 AGHEVPAYQP ETAYEIFHRV FEAGHEVPAY QPETAYEIFH RVFEAGHEVP AYQPETAYEI 6180 FHRVFEAGHE VPAYQPETAY EIFHRVFEAG HEVPAYQPET AYEIFHRVFE AGHEVPAYQP 6240 ETAYEIFHRV FEAGHEVPAY QPETAYEIFH RVFEAGHEVP AYQPETAYEI FHRVFEAGHE 6300 VPAYQPETAY EIFHRVSIWT ESYGGRVSIW TESYGGRVSI WTESYGGRVS IWTESYGGRV 6360 SIWTESYGGR VSIWTESYGG RVSIWTESYG GRVSIWTESY GGRVSIWTES YGGRVSIWTE 6420 SYGGRVSIWT ESYGGRVSIW TESYGGRYGP SFTAFFQEQN EKYGPSFTAF FQEQNEKYGP 6480 SFTAFFQEQN EKAAWLFEDS QAKADEINQI FDAISYMKAD VPSGSTNITH GRAFPCFDEP 6540 ALKAGMIADA GALASSGYQS TSGLLSLLKA VEQSLDAIRD GHILQQFKFA AGETSAIHPN 6600 IRGFDNEAEF IVWNEIVARI VDVLLDEKIV DVLLDEKNSG ASRLNADHSA IYRLTFTGIL 6660 NDNMAGFYRN GGEKEYNVVY DRNQDIYMPL GGLRNVGFPV VTVAEDAASS SIKSSHPIEV 6720 PVKSSHPIEV PVKRTGDVRP EEDTTLYPVM LGLRTHEIGW EFSEKTKQGL DENTMLTERT 6780 LGLALSDEVK VYATPDQDIE HGRYLASTQM EPTDARYLGE DVFIQGVRQG LLTVEDRGSV 6840 FSIVLKAAQE MFQRQGLDEN TMLTERTDVE SWLKAGMIAD AGALASSGYQ STSGLLSLLK 6900 FAAGETSAIH PNIRLTFTGI LNDNMAGFYR NGGEKEYNVV YDRNGGEKEY NVVYDRNGGE 6960 KEYNVVYDRN GGEKEYNVVY DRNQDIYMPL GGLRVYATPD QDIEHGRYLA STQMEPTDAR 7020 AWYENGITNC VGDNTRCCDS GVEQLVSFSD VSDFKDADAC NGGGIEYDSP ADTPLEFKDN 7080 TCNAPIPVSF PVAPTDTKDP YMFHQANLRD QCNYSLQYTI GNKFAANGNY GSETTAAVIN 7140 NFNGRFTTSA SDGFDGMQVN PRGNGVIEAA AGKGNVAGNI LVIAKITTAD MDGISSWLPT 7200 INGKIYYNCD TPACTVQEWI DTSAGSGDFS NLLATEKKPL GTGTDLWPKL DDLFVWWTTP 7260 ANRLVDWPIV TITHQEMSAN MNAGSSYFVE VGHNMNAGSS YFVEVGHNGN GVIEAAAGKQ 7320 AWVETMVQEF VRSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 7380 VNPRVIANGN VAGNILVIAK YFTSNGIIPP AITGLHNGDA LRQITGVTLS AKSASDGFDG 7440 MQVNPRDPYM FHQANSDVSD FKEAGLKGII PPAITGLHNG DALRYFTSNG IIPPAITGLH 7500 NEISFNQAWL RLVDWPIVTI THQEMSANFL DRKPLGTGTD LANGNVAGNI LVIAKHQEMS 7560 ANFLDRYNRD QCNYSLQYTI GNKAWYENGI TNCVGDNTRA WYENGITNCV GDNTRAWYEN 7620 GITNCVGDNT RAWYENGITN CVGDNTRAWY ENGITNCVGD NTRAWYENGI TNCVGDNTRA 7680 WYENGITNCV GDNTRAWYEN GITNCVGDNT RDADACNGGG IEYDSPADTP LEFKDADACN 7740 GGGIEYDSPA DTPLEFKDAD ACNGGGIEYD SPADTPLEFK DNTCNAPIPV SFPVAPTDTK 7800 DNTCNAPIPV SFPVAPTDTK DPYMFHQAND PYMFHQANLR DPYMFHQANL RDPYMFHQAN 7860 LRDPYMFHQA NLRDQCNYSL QYTIGNKEIS FNQAWLREIS FNQAWLREIS FNQAWLREIS 7920 FNQAWLREIS FNQAWLREIS FNQAWLREIS FNQAWLRFAA NGNYGSETTA AVINNFNGRF 7980 AANGNYGSET TAAVINNFNG RFAANGNYGS ETTAAVINNF NGRFAANGNY GSETTAAVIN 8040 NFNGRFAANG NYGSETTAAV INNFNGRFAA NGNYGSETTA AVINNFNGRF AANGNYGSET 8100 TAAVINNFNG RFAANGNYGS ETTAAVINNF NGRFAANGNY GSETTAAVIN NFNGRITTAD 8160 MDGISSWLPT INGKITTADM DGISSWLPTI NGKITTADMD GISSWLPTIN GKITTADMDG 8220 ISSWLPTING KITTADMDGI SSWLPTINGK KPLGTGTDLW PKKPLGTGTD LWPKKPLGTG 8280 TDLWPKKPLG TGTDLWPKKP LGTGTDLWPK KPLGTGTDLW PKKPLGTGTD LWPKKPLGTG 8340 TDLWPKKPLG TGTDLWPKKP LGTGTDLWPK KPLGTGTDLW PKKPLGTGTD LWPKLDDLFV 8400 WWTTPANRLD DLFVWWTTPA NRLDDLFVWW TTPANRLDDL FVWWTTPANR LVDWPIVTIT 8460 HQEMSANLVD WPIVTITHQE MSANFLDRLV DWPIVTITHQ EMSANFLDRM NAGSSYFVEV 8520 GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA 8580 AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS 8640 YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN 8700 GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKTPLLNQ QNSMWPYFTT SASDGFDGMQ 8760 VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8820 VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8880 VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8940 VNPRVIANGN VAGNILVIAK VIANGNVAGN ILVIAKYFTS NGIIPPAITG LHNGDALRAS 9000 HTVDKNGIWS SEVKATDNYI ANAAAAVAKD LLQDIVTWDD KEAGIYLIAR GPLNEGGLYA 9060 ERGTNYVALS LWALESDGAK HTDYSSQEST SYKLGSFELS YTTPVLTGYG NVESPEQPKL 9120 SGQDASAITW KLTGNLGGED YQDKLTGNLG GEDYQDKVRN SAYNYWVPEL PTEGTSPGFS 9180 TSKPGIGFYT AQFDLDLPKQ GFHQPQPPSE SWESGSPLEG LSKSPGSFFV VRSSYDDSAW 9240 VSADLPKTSY DYGSPITETR YPDADYMQYV MDQARNGIWS SEVKVLVLYG GPKASPSYLT 9300 ATPRYLDTLP EIKTLHLEQS PSTPYAQLYV NGYQYGKKAD IVVPFPWGGP GFEKAQLYVN 9360 GYQYGKAQLY VNGYQYGKAT DNYIANAAAA VAKLSGQDAS AITWKLTGNL GGEDYQDKVR 9420 NGIWSSEVKQ GFHQPQPPSE SWESGSPLEG LSKSSYDDSA WVSADLPKYP DADYMQYVMD 9480 QARYPDADYM QYVMDQARAL TNGAGAIKEA IADVLEHLGE NDEDIAVYAP NPFYKGFAPL 9540 EYLGSNFENG ELPKGFDNAG FVMGTSSSLF NQFILRGKMP MPILVADGRH FQLINTAAYW 9600 KIPNVAIAVS GGGYRLNGTD IPNFLKLNLS SFDASGYIDR LPDICNTCFK MPMPILVADG 9660 RNSILEGPDV KSAAALSTSE KDWLQVRSSS LFNQFILRTF LNLGLNKTNT KLPDICNTCF 9720 KTSLTDYWGR SSFDASGYID RSTSEKDWLQ VRGTDIPNFL KLNLGLNKGF DNAGFVMGTS 9780 SSLFNQFEYL GSNFENGELP KMPILVADGR DLYDAVKINT AAYWKSIALG DDFKKALTNG 9840 AGAIKALTNG AGAIKALTNG AGAIKALTNG AGAIKGKMPM PILVADGRGK MPMPILVADG 9900 RGKMPMPILV ADGRGKMPMP ILVADGRGKM PMPILVADGR GKMPMPILVA DGRHFQLINT 9960 AAYWKHFQLI NTAAYWKLPD ICNTCFKLPD ICNTCFKMPI LVADGRMPMP ILVADGRMPM 10020 PILVADGRMP MPILVADGRM PMPILVADGR MPMPILVADG RMPMPILVADGR MPMPILVA 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 [[ID=I4]]VSLVANHIDT VGKTAATVNT WTGGWSDSKA IGTYQNSGLS QYTVRYSEGV HFPARTVANW 22800 LVREVAGDVD NAVNPAWRLG AGVQGFEAYE AANAQGLRLD GGVIEDFAQK NPDLSSTSDT 22860 TDVIRGPDEP YSGQYDEERQ FISVTNPTGA EPVPKFLSEL LEDEYFTKFV NIWLENTDYE 22920 SAANDPHLSK LLEYDIASGT PVYRNNNQGL EALTISPDGK VYAISDQDDT GPWIRDAYQN 22980 DFAARVDPST AVDYNHYSDA ADRSGDVQTL QFAWALQHLS ERTGNQTGQL GTYFNKNINM 23040 LLYGTDDCSG KGMVFSIDAQ GEKNINMLLY GTDDCSGKNP NPDQAFLQVR FWVATGDSSK 23100 AALDALQQSI YLQPKFSDGN GLFYQYERVA VAGYDDTTGG VGPLLAQKAI AIALQSSHRQ 23160 TGSVDGYAYT DANKNDLITY LKSVVENNND GLTAAYRVAP NSGAYLNEAD FRSLPLIVGN 23220 SDQEGKANEQ PTWVYRSDYQ ECADAPGQKF GATGDEYREP SNDPNPPETY SKVALLFSER 23280 LADGSIPTRS ISSDEDSAET EQSDSSDPKV AIIDGLADPW RTMGVGYATN DDSTIRDVPI 23340 MQELNTNTIR DVDPIVIKVE TGVIKPGMVV TFAPANVTTE VKIVVIGHVD SGKDDADLQE 23400 LGAKSSAFAF RKDPSDAIPS IPSIPISYKE AIPFLKEAAE IDSHWERDDY MPFIEVPRDD 23460 YMPFIEVPRA LQGVDELVEK DALHPQFDNF YQEQPKASYG AGVTIQDRDL SVFFTRINDL 23520 LPVYVELLQK AALTNMVNAA KGEITPEQYE KIINEPTAAA IAYGLDKQLT SEEIWQAEEK 23580 LDKPAGDYTI RDYSNNWESG ALKLAPNQMT GSLDATYLKL APNQMTGSLD ATYLKCASDG 23640 SAETCSWEGH CKCASDGSAE TCSWEGHCKC ASDGSAETCS WEGHCKCASD GSAETCSWEG 23700 HCKCASDGSA ETCSWEGHCK LAAISVEPGK ASELGCSSGD LDCLCKVGEC AQMCISNMNA 23760 KGTLLSSNQG SQAADVKNDD DFLNYGISSK APSVITYDEA TKYEAAGITV HKDDYLPFID 23820 MPSEVTQIDA KIPMALDDWP TLSNMIFSGK DNFDTSSVTH RGYQINFLSN SAKDNIQGIT 23880 KPAIRDNIQG ITKPAIGPLG LSPKLQLWDT AGQERAEDYL LNPSPKNFGI GQDIQPKIDA 23940 TTNPGMRQLG LAMLGNKLVI DGLKEVTEIP ATADASRVNV DYTEVPRLAV NMVPFPRYCD 24000 LILGEWRDQI KDVLRIDYIG GGDLFRGSSP NVLYKSANWT PPEGIVRAAS TGSMAEQYTK 24060 AATGTYASST TVYKAENQAV AVGRALVEGS TFAKALYSSA ATGTYASSTT VYKATGTYAS 24120 STTVYKATTV YGESIKAYAD GYVQIVQTYA ASTGSMAEQY TKDLTWSYAA LLTANNRFNV 24180 DETAFTGAWG RGQSAQGASP GVVIASPSKI GADGQSAQGA SPGVVIASPS KIGSLAITDV 24240 SLPFFKIQGI SNPSGALSSG GLGEPKKYTV PSTCGVKPSG ALSSGGLGEP KQAILNNIGA 24300 DGQSAQGASP GVVIASPSKS DPDYFYTWTR SIYAINSGRT GTYASSTTVY KTVGSSCPYC 24360 DSQAPQVRVQ NGAVTWESDP NRVQNGAVTW ESDPNRKPDY FYTWTRYTVP STCGVKSSAA 24420 TGTYASSTTV YKGAVTWESD PNRIVGSISQ LGSWNPSSAT ALSAVQSDVW RDINTVLGSI 24480 HTFDPQNIGA DGQSAQGASP GVVIASPSKA ASTGSMAEQY TKAENQAVAV GRAENQAVAV 24540 GRALVEGSTF AKALVEGSTF AKALYSSAAT GTYASSTTVY KAYADGYVQI VQTYAASTGS 24600 MAEQYTKDLT WSYAALLTAN NRDLTWSYAA LLTANNRIQG ISNPSGALSS GGLGEPKIQG 24660 ISNPSGALSS GGLGEPKNIG ADGQSAQGAS PGVVIASPSK PDYFYTWTRQ AILNNIGADG 24720 QSAQGASPGV VIASPSKQAI LNNIGADGQS AQGASPGVVI ASPSKQAILN NIGADGQSAQ 24780 GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS KQAILNNIGA DGQSAQGASP 24840 GVVIASPSKQ AILNNIGADG QSAQGASPGV VIASPSKQAI LNNIGADGQS AQGASPGVVI 24900 ASPSKQAILN NIGADGQSAQ GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS 24960 KQAILNNIGA DGQSAQGASP GVVIASPSKQ AILNNIGADG QSAQGASPGV VIASPSKQAI 25020 LNNIGADGQS AQGASPGVVI ASPSKQAILN NIGADGQSAQ GASPGVVIAS PSKSAVQSDV 25080 WRSAVQSDVW RSAVQSDVWR SAVQSDVWRS AVQSDVWRSD PDYFYTWTRS DPDYFYTWTR 25140 SDPDYFYTWT RSDPDYFYTW TRSDPDYFYT WTRSDPDYFY TWTRSIYAIN SGRSIYAINS 25200 GRSIYAINSG RTVGSSCPYC DSQAPQVRTV GSSCPYCDSQ APQVRVQNGA VTWESDPNRV 25260 QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN RVQNGAVTWE SDPNRVQNGA 25320 VTWESDPNRV QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN RVQNGAVTWE 25380 SDPNRVQNGA VTWESDPNRV QNGAVTWESD PNRVQNGAVT WESDPNRVQN GAVTWESDPN 25440 RKVQNGAVTW ESDPNRKVQN GAVTWESDPN RKVQNGAVTW ESDPNRKASM VWEEAQQVSG 25500 KAVSPSFEDV WSQPRDGAGQ MFIPLNPNAY SPNTLNKDVG GPIEDQNSLQ VGDRFGFDLF 25560 DPTKGPTLLE DFIFRHGGPN FEQLPINQPR HVDGFGIHLF SYLDTQLNRL FYNSLTPAEQ 25620 QFVVDAIRNN VIIQLNRQDL FEAIEAGRSL TPAEQQFVVD AIRSVTSGFV DGIKVGFLAS 25680 VETPASIEAA SELSKTTDVG TFGQKDVHGF ATRIVPEEYV PITKFVTDNG DSKASMVWEE 25740 AQQVSGKASM VWEEAQQVSG KASMVWEEAQ QVSGKASMVW EEAQQVSGKA SMVWEEAQQV 25800 SGKASMVWEE AQQVSGKAVS PSFEDVWSQP RAVSPSFEDV WSQPRAVSPS FEDVWSQPRA 25860 VSPSFEDVWS QPRAVSPSFE DVWSQPRDGA GQMFIPLNPN AYSPNTLNKD GAGQMFIPLN 25920 PNAYSPNTLN KHGGPNFEQL PINQPRHGGP NFEQLPINQP RHGGPNFEQL PINQPRHGGP 25980 NFEQLPINQP RLFSYLDTQL NRNNVIIQLN RNNVIIQLNR NNVIIQLNRS VTSGFVDGIK 26040 SVTSGFVDGI KAAALAELVW SGNRAELVWS GNRASNSLQY VNVQVKDAYS PHEIYSREYL 26100 VANGVQAQAL VPKGIMLDTG RGVQAQALVP KHIVGATAPL WGEQVDDINV SHIVGATAPL 26160 WGEQVDDINV SSMIFEQLEG MSLSKVIPEI DMPSHSSSGW KYNVMANPDA NTPNFNYGGN 26220 GGSWCAPYKT YEVVGNVYKD IEADLQHAET VWGALHAFLV MWEDIALSAD NAHDVPKAAA 26280 LAELVWSGNR AAALAELVWS GNRAAALAEL VWSGNRAAAL AELVWSGNRA SNSLQYVNVQ 26340 VKASNSLQYV NVQVKASNSL QYVNVQVKAS NSLQYVNVQV KDAYSPHEIY SRDAYSPHEI 26400 YSRDAYSPHE IYSRDAYSPH EIYSRDAYSP HEIYSRDAYS PHEIYSRDAY SPHEIYSREY 26460 LVANGVQAQA LVPKEYLVAN GVQAQALVPK EYLVANGVQA QALVPKGIML DTGRIFEQLE 26520 GMSLSKIFEQ LEGMSLSKIF EQLEGMSLSK IFEQLEGMSL SKTYEVVGNV YKYNVMANPD 26580 ANTPNFNYGG NGGSWCAPYK YNVMANPDAN TPNFNYGGNG GSWCAPYKYN VMANPDANTP 26640 NFNYGGNGGS WCAPYKYNVM ANPDANTPNF NYGGNGGSWC APYKATSGGT SAAAPVFAGL 26700 VGMLNDARAW YQESAVSKDF TDITAGSSIG CDGVNPQTGK GFPDVAAHSL TPRPDVAAHS 26760 LTPRPNSALP QVLSNSYGDE EQTVPEYYAK SALPQVLSNS YGDEEQTVPE YYAKSNSYGD 26820 EEQTVPEYYA KSYGDEEQTV PEYYAKYLDQ QITAETKAWY QESAVSKAWY QESAVSKAWY 26880 QESAVSKAWY QESAVSKGFP DVAAHSLTPR GFPDVAAHSL TPRGFPDVAA HSLTPRGFPD 26940 VAAHSLTPRG FPDVAAHSLT PRPDVAAHSL TPRSALPQVL SNSYGDEEQT VPEYYAKYLD 27000 QQITAETKYL DQQITAETKA NEQPTWVYRF FCPTDYLIDV RGTPSVLTEQ GLVKLGVPGN 27060 ELAIEIGSTG DYNARQGTPS VLTEQGLVKS LPLIVGNSDQ EGKTFQGTPS VLTEQGLVKY 27120 PVVQNPVTLA ESSCQSVFNP NIPKNPSAGP GWDQAKPTNG PLAKFQGTPS VLTEQGLVKA 27180 NEQPTWVYRA NEQPTWVYRG TPSVLTEQGL VKNPSAGPGW DQAKPTNGPL AKSLPLIVGN 27240 SDQEGKSLPL IVGNSDQEGK SLPLIVGNSD QEGKARNHGT STVAPQVQAS VYRASVDISN 27300 VDTYSSTEVA NDDSFQQVGK ATFLVWDQQR KASVDISNVD TYSSTEVAND DSFQQVGKNH 27360 GTSTVAPQVQ ASVYRSSTEV ANDDSFQQVG KTLYLTDTDA GVPMIDPRTV YAFDVSEDGS 27420 YLKVASNGYV ITGAGKARNH GTSTVAPQVQ ASVYRARNHG TSTVAPQVQA SVYRATFLVW 27480 DQQRATFLVW DQQRATFLVW DQQRKASVDI SNVDTYSSTE VANDDSFQQV GKTVYAFDVS 27540 EDGSYLKVAS NGYVITGAGK VASNGYVITG AGKIENQSDA DGYSSCSTLK LTGLTTLTTL 27600 SFAALTKSAS SLNSIGDTFK SDKLNVIDFP KSLNSIGDTF KVGSIEFTAL PQLQSLDFTK 27660 TVNGGFQIAR LNVIDFPKGQ LGFWGNKGTY SGDLQLNGVK LNVIDFPKSD KLNVIDFPKS 27720 DKLNVIDFPK TVNGGFQIAR TVNGGFQIAR AVGSDEWTVR LGITYTTYSK MTNDYISALT 27780 KSEMLAEQDK MTNDYISALT KSTITTPWKS VVENNNDGLT AAYRVAPNSG AYLNEADFRY 27840 FYGDNYATLR SGAYLNEADF RTAVGSDEWT VRLGITYTTY SKMTNDYISA LTKMTNDYIS 27900 ALTKMTNDYI SALTKSEMLA EQDKMTNDYI SALTKSEMLA EQDKMTNDYI SALTKSEMLA 27960 EQDKMTNDYI SALTKSVVEN NNDGLTAAYR SVVENNNDGL TAAYRSVVEN NNDGLTAAYR 28020 SVVENNNDGL TAAYRETTMF VLQGFGASMA RLNALQGGRL VQNDFNTLLR QDILGGMVDS 28080 YTDPKTGETT QIHARYGLEA AVPLMYESTG LGLGDMTKVS VADVKIDYIG GGDLFRLVQN 28140 DFNTLLRQDI LGGMVDSYTD PKYGLEAAVP LMYESTGLGL GDMTKALEAY KVNGKAVDFS 28200 GHDEFQGKEP SNDPNPPETY SKFGATGDEY RSDYQECADA PGQKYIARPD IMKSTLPDLS 28260 EVIKVNGKEV GQFKSVDNFH LLTVYAVDFS GHDEFQGKFG ATGDEYRYIA RPDIMKFLDE 28320 ALTYPPPKGI QINDPSINDD SVMIYAPAVR IASAMLDEED EKYFNVKNGD QSPPSALGPL 28380 PSVIERPSIN DDSVMIYAPA VRTDYSVCGE TTIFKTVDDE EGVAAQFKIA SAMLDEEDEK 28440 FLDEALTYPP PKFLDEALTY PPPKGIQIND PSINDDSVMI YAPAVRGIQI NDPSINDDSV 28500 MIYAPAVRIA SAMLDEEDEK IASAMLDEED EKYFNVKIAS AMLDEEDEKY FNVKNGDQSP 28560 PSALGPLPSV IERNGDQSPP SALGPLPSVI ERAGAVAAVV YNNEKHGIPG GGIATGAEGI 28620 KLVLGDAVPE SAAPMGLTPP TKSDKELVSS SAFQSHVKSF EGFPKRLVAH SVATYARIAD 28680 LGKEEYNHPT RAGAVAAVVY NNEKHGIPGG GIATGAEGIK HGIPGGGIAT GAEGIKLVLG 28740 DAVPESAAPM GLTPPTKLVL GDAVPESAAP MGLTPPTKLV LGDAVPESAA PMGLTPPTKS 28800 DKELVSSSAF QSHVKFTDTP VLYGPKKFTD TPVLYGPKML DDAGIYLITD LSSPSESINR 28860 QQLSFVMNQW YEKYGAYSVC SPKSDGQCSD LLKWDVDLYS RLITDLSSPS ESINRDLSSP 28920 SESINRKFTD TPVLYGPKKF TDTPVLYGPK QQLSFVMNQW YEKQQLSFVM NQWYEKCDVA 28980 TTDVYYSGKG GTPDFGPGRS KYTAEGYEAA TKVATIGSAT FARYCASAQE DNATLQALLR 29040 YTAEGYEAAT KYVDAGGFEP SIKSKYTAEG YEAATKVATI GSATFARVAT IGSATFARYV 29100 DAGGFEPSIK DADACNGGGI EYDSPADTPL EFKDNTCNAP IPVSFPVAPT DTKEISFNQA 29160 WLRFAANGNY GSETTAAVIN NFNGRITTAD MDGISSWLPT INGKVIANGN VAGNILVIAK 29220 SDVSDFKEAG LKDADACNGG GIEYDSPADT PLEFKDADAC NGGGIEYDSP ADTPLEFKDA 29280 DACNGGGIEY DSPADTPLEF KFAANGNYGS ETTAAVINNF NGRANNYCSN QVEGPYSLYS 29340 GRVSIWTESY GGRYGPSFTA FFQEQNEKTV YDMAMEAWSK ISYKEPGICE TTPGVKSAGY 29400 APLKPGGCKD QIIECRANNY CSNQVEGPYS LYSGRANNYC SNQVEGPYSL YSGRANNYCS 29460 NQVEGPYSLY SGRISYKEPG ICETTPGVKV SIWTESYGGR AIMGAEEAAK TGGAMWPYRT 29520 LIPDVVGIFA GTPKFVTNMQ AALLKALSEM ILQSEKDYYA SMLQQPKANF EVETPRGITG 29580 TSIARANFEV ETPRDYYASM LQQPKFVTNM QAALLKFVTN MQAALLKTGG AMWPYRTGGA 29640 MWPYRAEDYL LNPSPKLSDL TGDTEYAQLS QKTDDQVSLF ETTIRTDSGF AGLTNVNAAN 29700 GGGRYDNQES FLFAEVLKIT GQEIYRAEDY LLNPSPKTDD QVSLFETTIR AGFAGDDAPR 29760 QEYDESGPSI VHRSYELPDG QVITIGNERV APEEHPVLLT EAPINPKDLT DYLMKIIAPP 29820 ERSYELPDGQ VITIGNERVA PEEHPVLLTE APINPKALLF GAAGSAEDPV VVKNVGFPVV 29880 TVAEDAASSS IKVYATPDQD IEHGRAVEQS LDAIRQGLLT VEDRGPLNEG GLYAERLSGQ 29940 DASAITWKLT GNLGGEDYQD KASPSYLTAT PRALMNGAGA IKDAGYETSI TDYWGRPTVY 30000 GFVPLEYVGS KTAFSDILAK TNTQVPDACT QCFQKMPMPL LVADGRTAFS DILAKAFPDV 30060 AAQGMNFAVY DKELYNIGDY QADANSGSKI AFASYLEEYA RLETIGDTFK QGLQDITLGA 30120 SIGCTGRAFP DVAAQGMNFA VYDKELYNIG DYQADANSGS KQGLQDITLG ASIGCTGRLV 30180 EGAAAGIVVA SPSKQGVLNN IGADGKSSSA YESLTSAVKA SALIAYGNSL ISSDKSVYGI 30240 NNGRPDYFYT WTRPDYFYTW TRSNPDYFYT WTRSNPDYFY TWTRSNPDYF YTWTRSNPDY 30300 FYTWTRSNPD YFYTWTRSNP DYFYTWTRFS VAEILPGAKN VVLDTTALSA NTKVGTIITG 30360 DPLDPPVLKY PAEVFLPGGT YQLGKADKET DIGSAIEKAS AIQLDGIIYR QLSGHVGPLT 30420 SSSSKETDIG SAIEKADKET DIGSAIEKQL SGHVGPLTSS SSKQLSGHVG PLTSSSSKIY 30480 SFFVGGAVPE NLRQTSSEQN PSLEEIQAAQ ATVLPHSPVS NVKSAGEYNT FSPEWPVPLT 30540 KTFDENDTYE IGNKSIDQFF NDAKVPTVLM SPWVGKDGQE ATFHFDRVDW SPSFRAAEVI 30600 NYYTPDHVPV FNAMSIDQFF NDAKNAFITN YPSEQRYDTA TFIDKRIDAT TNPGMRQLGL 30660 AMLGNKNMHD VIGNDGTVPS EFRIDATTNP GMRIDATTNP GMRNAFITNY PSEQRNAFIT 30720 NYPSEQRQLG LAMLGNKAQI TAVNLEARGD GGGGPTFEHL EKSMDNDNTS LLVFGKMGES 30780 VDDFFARAQI TAVNLEARMG ESVDDFFARG IVSGEGEESS DPVKVDNVVA SFKYMQQLLD 30840 QTKVVWQDSV RAQNDPNAFG VVAARDPNAF GVVAARNDPN AFGVVAARAA TYCPENIEKA 30900 QNDPNAFGVV AARAQNDPNA FGVVAARTIF GWDIAEGQKS AGYTPLKVNG VEYGETRSAG 30960 YTPLKVNGVE YGETRSAGYT PLKVNGVEYG ETRGGSILPM QEVALTTRWA SVIDATKKAT 31020 GDVLFNTKHT ADGAWAKGGS ILPMQEVALT TRGGSILPMQ EVALTTRWAS VIDATKDGLE 31080 GSFKWDNLDS AALNTKVEDG NLILTMPKVE DGNLILTMPK VEDGNLILTM PKWDNLDSAA 31140 LNTKWDNLDS AALNTKSAIS QYGDSFAKSQ SDFESEFSTA KVNAGIGIGP DDLVSFIKSA 31200 ISQYGDSFAK SQSDFESEFS TAKGFNIVVA PGLDGRHVDV PLTGEDEITI LAIHDEVSPV 31260 GDTDALLERA PVVQYALNRY LVDQLNPEGK AIHDEVSPVG DTDALLERAI HDEVSPVGDT 31320 DALLERAPVV QYALNRAPVV QYALNRDAEL TDAGVKQAQV AHDFWQKLNT GAVIPVLVRL 31380 GDLSANPIER VLPQVIEATN RIYVTGQSYA GRLGDLSANP IERVLPQVIE ATNRVLPQVI 31440 EATNRNDPVA VFDGSVIPKE AGLVPFQVSP TTKTLGIDIA RGQTPLPILV ADGRTSTTLP 31500 EVCSKNNILE GPDVKGQTPL PILVADGRIN TAAYWKEAIA DVLEHLGEND EDIAVYAPNP 31560 FYKNSILEGP DVKMPMPILV ADGRMPMPIL VADGRNDDDF LNYGISSKSP VTSEYTSVRS 31620 IFEAANEKAI NDYIDSQLDK YLTNSQALAD LPYFAEKGVQ ISTNIPKGVQ ISTNIPKEII 31680 STYSIDGLRS VYQTMTDRYN TDAELYKGGS ELGFRSAADG LASAITSKSG DDISTTDALA 31740 LPEPVQALTK AGSSPTDIIS GISDKTDALD SAIKYDYENV DSDGANKYNL SNGAPAPETV 31800 TNKSMPTSGA VDLVAKCSSH DDCSDELACT DGVCACTADS AVTCSWEGHC AGAKGDNPSI 31860 LGLRCTADSA VTCSWEGHCA GAKDSPNWDV DSDALPAIPE GAKTLADFDA LKTLADFDAL 31920 KINPGPLARL YPDDNLAFIQ AGISDEKLYT GEVFKTDEGK GQEPPAAIVE VQKVAIIDGL 31980 ADPWRSVNIV NYTPSDSYTY SDNSGSWQSV KVTTGGQGAE FTLAKDQTTW SVDGNVVRVT 32040 TGGQGAEFTL AKVTTGGQGA EFTLAKAGQF PISANDGATS TKEAASAALA AGYKQTYTSC 32100 NPLKKTLNYA DALDGENYPQ TPSRVAVAGY DDTTGGVGPL LAQKINPSSG LLEPQTPLAV 32160 SPGSGPRVAV AGYDDTTGGV GPLLAQKTTG AFDESGPPLS QKNALQTMYD TQDKAALDAL 32220 QQSIYLQPKF SDGNGLFYQY ERLGAEVVTA GRIYAVADTQ ERLINQVELS EDKAVITDIV 32280 NQQRAVITDI VNQQRGENIL SAPLITYAPA GPELDEKELG FTAVGGEGKD IQYLENYQGQ 32340 GYSGPAVKIL QYAQGRDYSN NWESGALKDD YMPFIEVPRE AAEIDSHWER TIPIDNDVDY 32400 VVTGYRVYWV DSGPRTGDGV NDAPSLKDGQ EQEILARADA IVAAIRSALI AFEKNINMLL 32460 YGTDDCSGKG MVFSIDAQGE KTGTDQASVG YYKDAVYALD AIYGIDARDN ILPENLDDGL 32520 PSQFVYEKIL VENLQDQTAK ILVENLQDQT AKAEPYVTGS SAASGSNFVA DFAEAGTDGK 32580 QISYWAFTTP AVKFEPPAVY NDELKDAEEE PYDWSNEGRL SDELEDDNAP IGFETTKDQE 32640 MAVAAFRTSD DFASQMDGRC MGCDSTSIDV SRCMGCDSTS IDVSRDASGG DQITEWQDIY 32700 LPPITKGIQD AGVIATAKGV GSDAWTVSES GRGIDGDKGL VVKDLYGNIV MSGGSTLYPG 32760 IADRAGFAGD DAPRQEYDES GPSIVHRSYE LPDGQVITIG NERSYELPDG QVITIGNERL 32820 SGGVAVIKTT AVLFDEGKIG GSAGTELQSD KDLALVDPGL ELSYNTKLVG GSDFGEDEAK 32880 TLSTNEEGYE TSAVRASYGA GVTIQDRSAY VVYDLSNNEI SLANTKVPYL IGANTDEGTS 32940 FAIRLPVEAF QALASSTSET KDAGNAATND PLFPFSRAAL PGTEVLFADS VAKVTSAQYY 33000 VNPKIQAFVI YPENFDKVGA GVNVGELYAF ADKALTQYSV KTTYNVVAQT KTYANLPQAL 33060 VNSGAIKVIP LQGCDADEYG RDNIQGITKP AIRLANAYTW EGGRYQGASQ CPFRTMGVGY 33120 ATNDDSTIRC ASDGSAETCS WEGHCKCASD GSAETCSWEG HCKLILPGEL AK 33172

Claims

1. A molecule indicating the toxicity of aflatoxin-producing fungi, AFT-YJFZ01, characterized by: The amino acid sequence of the aflatoxin-producing fungus toxicity indicator molecule AFT-YJFZ01 is shown in SEQ ID NO.

1.

2. A method for identifying the toxicity of aflatoxin-producing fungi in the system is based on the content detection of the toxicity indicator molecule AFT-YJFZ01 for aflatoxin-producing fungi. The amino acid sequence of the toxicity indicator molecule AFT-YJFZ01 for aflatoxin-producing fungi is shown in SEQ ID NO.

1.

3. An application for identifying the aflatoxin-producing ability of aflatoxin-producing strains of the genus Aspergillus, characterized in that: Specific application methods include: (1) Provide nanobodies or monoclonal antibodies against the toxicity indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria: (2) Provide polyclonal antibodies against AFT-YJFZ01, a toxicity indicator molecule for aflatoxin-producing fungi; (3) Preparation of the test solution of the strain to be identified: culturing and diluting the strain to be identified to obtain the test solution of the strain to be identified; (4) Determination of the aflatoxin-producing ability of the strain to be identified: An indirect non-competitive double antibody sandwich method is used to identify the aflatoxin-producing ability of aflatoxin-producing strains of the genus Aspergillus, comprising the following steps: coating an AFT-YJFZ01 nanoantibody or monoclonal antibody in an ELISA plate, washing the plate; adding a blocking solution for blocking, washing the plate; adding a test solution for reaction, washing the plate; adding an AFT-YJFZ01 polyclonal antibody for reaction, washing the plate; adding a horseradish peroxidase-labeled antibody that reacts with a specific polyclonal antibody for the toxicity indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria, reacting, washing the plate; adding a color development solution for reaction; adding a stop solution, and reading and calculating the results with an ELISA reader; The amino acid sequence of the aflatoxin-producing fungus toxicity indicator molecule AFT-YJFZ01 is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that: The step (3) is as follows: the strain to be identified is cultured in a conventional Czapek medium or other medium suitable for the growth of the strain, the culture environment temperature is 15 ~ 35 ° C, and the culture time is not less than 12 hours. The mixture of the culture medium and the culture is fully homogenized and then diluted 1-10 times with sterile water to obtain a test solution of the strain to be identified.

5. The use according to claim 3, characterized in that: The step (4) is: a. Prepare a 0.2-8.0 μg / mL coating solution of the AFT-YJFZ01 nanobody or monoclonal antibody with ELISA coating buffer, then add it to the ELISA plate. Place it at 4°C overnight or at 37°C for at least 2 hours, then remove the coating solution from the ELISA plate and wash the plate with a conventional ELISA washing solution. Then, use skim milk powder with a concentration of not less than 1% as a blocking solution, place it at room temperature or at 37°C for at least 1 hour, then discard the blocking solution and wash the plate with a conventional ELISA washing solution. b. Then, dilute the test solution appropriately with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells, place it at room temperature or 37°C to block for at least 1 hour, discard the liquid, and then wash the ELISA plate with conventional ELISA washing solution; c Then, dilute the AFT-YJFZ01 polyclonal antibody appropriately with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells, place it at room temperature or 37°C for blocking for at least 1 hour, discard the liquid, and then wash the ELISA plate with conventional ELISA washing solution; d Then, dilute the commercial horseradish peroxidase-labeled antibody as needed with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells, and block it at room temperature or 37°C for at least 1 hour. Discard the liquid and wash the ELISA plate with a conventional ELISA wash solution. Then, conventional ELISA colorimetric solution and stop solution were added successively, and finally the AFT-YJFZ01 content was read and calculated using a microplate reader.

6. The application of identifying whether there are strong aflatoxin-producing strains in the sample is as follows: (1) Provide nanobodies or monoclonal antibodies against the toxicity indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria: (2) Provide polyclonal antibodies against AFT-YJFZ01, a toxicity indicator molecule for aflatoxin-producing fungi; (3) Preparation of samples to be identified: culturing and diluting the samples to be identified to obtain the test solution of the samples to be identified; (4) To identify whether there are strong aflatoxin-producing strains in the sample: the indirect non-competitive double antibody sandwich method is used to identify the sample, and the steps include: The ELISA plate is coated with the nanoantibody or monoclonal antibody of AFT-YJFZ01, and the plate is washed; a blocking solution is added to block the plate, and the plate is washed; Add the sample solution to be identified for reaction and wash the plate; add the AFT-YJFZ01 polyclonal antibody for reaction and wash the plate; add the horseradish peroxidase-labeled antibody that reacts with the polyclonal antibody of the aflatoxin-producing fungus toxicity indicator molecule AFT-YJFZ01, react and wash the plate; add the color development solution for reaction; add the stop solution, and read and calculate the results with a microplate reader; (5) Evaluation of identification results: The higher the content of AFT-YJFZ01, an indicator molecule for the toxicity of aflatoxin-producing bacteria, in the strain to be identified, the stronger the aflatoxin-producing ability, i.e., the toxicity, of the identified strain. Based on the calculation results of the above-mentioned microplate reader, the content of AFT-YJFZ01, an indicator molecule for the toxicity of aflatoxin-producing bacteria, per unit volume of the sample solution to be tested is obtained to determine whether the sample to be identified contains a strong aflatoxin-producing strain. The amino acid sequence of the aflatoxin-producing fungus toxicity indicator molecule AFT-YJFZ01 is shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that: The step (3) is as follows: weigh the sample to be identified, transfer it into sterile water, shake it at room temperature until it is uniform, and prepare a uniform dispersion of the sample to be identified; take 10-1000 μL of the uniform dispersion of the sample and add it to 6-600 mL of conventional Czapek medium or other culture medium suitable for the growth of toxin-producing aflatoxin; place it at 15-35°C and shake it at 200±50 rpm; take a sample after culturing for 6-24 hours to form a test solution of the sample to be identified; the sample to be identified is soil, Chinese medicinal materials, agricultural products or feed.

8. The use according to claim 6, characterized in that: The specific steps of step (4) are: a. Prepare a 0.2-8.0 μg / mL coating solution of the AFT-YJFZ01 nanobody or monoclonal antibody with ELISA coating buffer, then add it to the ELISA plate. Place it at 4°C overnight or at 37°C for at least 2 hours, then remove the coating solution from the ELISA plate and wash the plate with a conventional ELISA washing solution. Then, use skim milk powder with a concentration of not less than 1% as a blocking solution, place it at room temperature or at 37°C for at least 1 hour, then discard the blocking solution and wash the plate with a conventional ELISA washing solution. b. Then, dilute the sample solution to be identified appropriately with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the ELISA plate, place it at room temperature or 37°C to block for at least 1 hour, discard the liquid, and then wash the ELISA plate with conventional ELISA washing solution; c Then, dilute the AFT-YJFZ01 polyclonal antibody appropriately with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells, place it at room temperature or 37°C for blocking for at least 1 hour, discard the liquid, and then wash the ELISA plate with conventional ELISA washing solution; d Then, dilute the commercial horseradish peroxidase-labeled antibody as needed with a conventional phosphate buffer with a pH close to neutral, add it to the ELISA plate wells, and block it at room temperature or 37°C for at least 1 hour. Discard the liquid and wash the ELISA plate with a conventional ELISA wash solution. Then, conventional ELISA colorimetric solution and stop solution were added successively, and finally the AFT-YJFZ01 content was read and calculated using a microplate reader.

9. The use according to claim 3 or 6, characterized in that: The animal sources of the polyclonal antibody against the aflatoxin-producing fungus toxicity indicator molecule AFT-YJFZ01 and the nanoantibody or monoclonal antibody against the aflatoxin-producing fungus toxicity indicator molecule AFT-YJFZ01 are different.

10. The use according to claim 5 or 8, characterized in that: In step (b), a series of concentration gradients of the toxicity indicator molecule AFT-YJFZ01 solution are used to replace the test solution or the sample solution to be identified to prepare a standard curve.