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

Abstract

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

Description

Molecular early warning method of aflatoxin in agricultural products

technical field

The invention relates to an early warning method for aflatoxin molecules in agricultural products.

Background technique

Aflatoxins are highly toxic and harmful, and are the most common pollutants that contaminate food. In recent years, there has been an overall trend of increasing pollution, seriously threatening food safety and people's health. Aflatoxin is the most toxic mycotoxin in nature, among which aflatoxin B1 is a Class I carcinogen identified by the International Agency for Research on Cancer (IARC), which has caused many poisoning incidents of human and animal groups , has become one of the main reasons for the high incidence of liver cancer cases. According to the statistics retrieved by Web of Science in the past 5 years, there are more than 110 types of food and raw materials contaminated by aflatoxin, ranking first among pollutants. However, so far, there is still no research example of molecular early warning before the contamination of microbial toxins such as aflatoxin at home and abroad, which is difficult to meet the urgent needs of early warning.

Aflatoxins are mainly produced by toxin-producing fungi such as Aspergillus flavus. Take peanuts as an example. Peanuts have already carried aflatoxin-producing fungal strains in the field. After harvesting, these strains enter packaging bags, transport vehicles, warehouses, processing lines, etc. together with peanuts. Once the conditions are suitable, they can produce A large amount of aflatoxins threaten the consumption safety of peanuts and their products and the safety of people's lives and health. Internationally renowned research institutions such as the Agricultural Research Service of the United States Department of Agriculture and the International Semi-Arid Research Institute of the Food and Agriculture Organization of the United Nations (FAO) have listed aflatoxin-producing fungi as soil-borne pathogens of peanuts and other crops for research and prevention.

Studies have shown that the virulence of different strains of aflatoxin-producing fungi varies greatly, ranging from non-toxigenic fungal strains to highly virulent strains. Taking the Aspergillus flavus strains isolated from peanuts as an example, a considerable number of Aspergillus flavus strains have lost toxin-producing genes or key regulatory genes for toxin production, and they do not have the ability to produce aflatoxin; less than 20% of Aspergillus flavus strains are Strong virulence-producing strains. How to assess the early warning risk of aflatoxin contamination? This has always been a worldwide problem in this field.

In response to the above problems, after more than ten years of systematic research, the inventor team discovered the toxin-producing indicator molecule of aflatoxin-producing fungi, and for the first time clarified that it has the function of early warning of aflatoxin pollution, so it can be used as an indicator of aflatoxin pollution. Warning elements. Our research found that there is a significant positive correlation between the occurrence level of the early warning molecule and the level of aflatoxin contamination. Based on this correlation, we further invented the early warning method and application of aflatoxin molecules in agricultural products, which can be used for agricultural products aspergillus flavus The early warning of the risk of toxins provides a scientific basis for the prevention and control of the source of aflatoxins in agricultural products and early prevention and control.

Contents of the invention

Aiming at the deficiencies in the prior art, the present invention provides an early warning method for aflatoxin molecules in agricultural products and its application. The method is used for early warning of the risk of agricultural products being contaminated by aflatoxin, has high speed, strong timeliness and practicability, and is easy to popularize and apply.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

Aflatoxin molecular early warning method for agricultural products, measuring the concentration of aflatoxin early warning molecules in agricultural product samples: when the measured concentration of aflatoxin early warning molecules is less than 0.2 μg/g, the level of aflatoxin pollution is below 0.2 μg/g′, which is low Risk; when the measured concentration of aflatoxin early warning molecules is greater than 1.2 μg/g, the level of aflatoxin pollution is above 10 μg/kg, which is high risk; when the measured concentration of aflatoxin early warning molecules is between 0.2 μg/g and 1.2 In μg/g, the risk is medium. The aflatoxin early warning molecule is AFT-YJFZ01 peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

In the above scheme, the quantitative detection of the concentration of aflatoxin early-warning molecule can be prepared by using the amino acid sequence of the toxin-producing early-warning molecule AFT-YJFZ01 of aflatoxin-producing bacteria or a partial sequence thereof, and through a conventional antibody preparation process to prepare a The antibody corresponding to the protein, and realize the quantitative detection of the early warning molecular protein, and also realize the quantitative detection of these aflatoxin early warning molecular proteins with a one-to-one correspondence relationship through other detection techniques, so as to achieve the above purposes, partial sequence It refers to a part of the whole sequence that has a one-to-one correspondence with the aflatoxin early warning molecule protein.

Specifically, the aflatoxin early warning molecule AFT-YJFZ01 detection method as described above measures the concentration of AFT-YJFZ01 in agricultural product samples, and the aflatoxin early warning molecule AFT-YJFZ01 immune rapid detection method can be used to quantitatively detect the concentration of AFT-YJFZ01 in agricultural product samples , the method steps are:

Prepare the test solution of the agricultural product sample to be tested, add it to the microplate well of the nanobody or monoclonal antibody coated with the aflatoxin early warning molecule AFT-YJFZ01 at the bottom of the well, react, and wash the plate;

Add aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody reaction, wash the plate;

Add a horseradish peroxidase-labeled antibody that reacts with the aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody, react, and wash the plate;

Add chromogenic solution to react; add stop solution, read and calculate the concentration of AFT-YJFZ01 in the sample to be tested with a microplate reader.

The specific steps are: add 100-200 μL per well to the microplate wells of the nanobody or monoclonal antibody coated with the aflatoxin early warning molecule AFT-YJFZ01 at the bottom of the well, and place it at room temperature or 37°C for no less than 1 hour. , Discard the liquid after reaction, wash the ELISA plate, then add AFT-YJFZ01 rabbit polyclonal antibody, and then add 100-200 μL to each well, let it stand at room temperature or 37°C for no less than 1 hour, discard the liquid, and wash again Then add 100-200 μL of horseradish peroxidase-labeled goat anti-rabbit antibody to each well of the enzyme-labeled plate, place it at room temperature or 37°C for no less than 1 hour, discard the liquid, wash the enzyme-labeled plate, and then add ELISA to display Color solution, stop solution, and finally read and calculate the concentration of AFT-YJFZ01 in the sample to be tested by a microplate reader. The determination of the concentration of the above-mentioned AFT-YJFZ01 can also use other detection methods of the aflatoxin early warning molecule AFT-YJFZ01 with equivalent effects.

According to the above scheme, replace the solution to be tested with a series of concentrations of the pure product solution of the early warning molecule AFT-YJFZ01 (100-200 μL per well) that acts as a standard substance, and use it to make a standard curve, and calculate the concentration of AFT-YJFZ01 in the sample to be tested. concentration.

According to the above scheme, the method steps of preparing the test solution of the agricultural product sample to be tested are as follows: weigh the agricultural product sample to be tested, pulverize it and transfer it to the sample diluent, leave it to stand after homogenizing in a conventional method, and take the supernatant. It can be made into a sample to be tested and a solution to be tested.

The sample diluent can be a 0.01mol/L phosphate buffer solution containing 0.1% sorbitol and cotton sugar, and the preparation method is: 0.5 g each of sorbitol and cotton sugar, 4.0 g of NaCl, Na ₂ HPO ₄ ·12H ₂ O 1.45g, KCl 0.1g, KH ₂ PO ₄ 0.1g, add deionized water to 500mL.

The microtiter plate of the nanobody or monoclonal antibody of the aflatoxin warning molecule AFT-YJFZ01 is coated on the bottom of the well, and its preparation method is: dissolving the nanobody or monoclonal antibody of AFT-YJFZ01 in the ELISA coating buffer Form a coating solution of 0.2-8.0 μg/mL in the solution, then add it to the microplate (100-200 μL/well), place it at 4°C overnight or at 37°C for no less than 2 hours, then remove the above plate Add coating solution, and then wash the microplate with ELISA conventional washing solution; then add ELISA conventional blocking solution (200-300 μL per well), leave it at room temperature or 37°C for blocking for no less than 1 hour, then discard the blocking solution, and then Wash the microtiter plate with ELISA routine washing solution.

According to the above scheme, the polyclonal antibody of AFT-YJFZ01 is different from the animal source of the nanobody or monoclonal antibody of AFT-YJFZ01, and the toxin-producing early warning molecule of aflatoxin-producing bacteria can be directly used as an antigen to prepare a nanobody or a monoclonal antibody. Clonal antibodies, and polyclonal antibodies derived from rabbits, specifically:

The nanobody or monoclonal antibody of aflatoxin early warning molecule AFT-YJFZ01 can be obtained by the following method: use AFT-YJFZ01 as the immune antigen, immunize alpaca or Balb/c mice in a conventional way, and then use known conventional nanobody Or mouse monoclonal antibody preparation technical scheme can be developed;

The following method can be used to obtain the polyclonal antibody of the aflatoxin early warning molecule AFT-YJFZ01: use AFT-YJFZ01 as the immune antigen, immunize New Zealand white rabbits and other experimental rabbits in a conventional way, and then use the known conventional polyclonal antibody preparation technical scheme The rabbit-derived polyclonal antibody of the aflatoxin early warning molecule AFT-YJFZ01 can be developed;

The horseradish peroxidase-labeled antibody that reacts with the polyclonal antibody of the aflatoxin-producing bacteria toxin-producing early warning molecule AFT-YJFZ01 is a horseradish peroxidase-labeled goat anti-rabbit antibody, which can be directly Obtained by purchasing goods.

The ELISA coating buffer refers to a conventional carbonate buffer, and the preparation method is as follows: Weigh 1.465g of NaHCO ₃ and 0.795g of Na ₂ CO ₃ , add deionized water to dilute to 500mL.

The ELISA color developing solution refers to the conventional hydrogen peroxide and TMB color developing solution of ELISA.

The terminator solution refers to the conventional chromogenic terminator solution of ELISA 2mol/L sulfuric acid aqueous solution, the preparation method is: take 44mL concentrated sulfuric acid and add it to 300mL deionized water, stir until it cools down, and finally set the volume to 400mL.

The present invention sets the aflatoxin concentration as low risk when it is 1.0 μg/kg and high risk when it is above 10 μg/kg by combining the European Union, the United States, China and other domestic and foreign aflatoxin limit regulations. When the aflatoxin concentration is below 1.0 μg/kg, the AFT-YJFZ01 concentration is less than 0.2 μg/g; when the aflatoxin content is above 10 μg/kg, the AFT-YJFZ01 concentration is greater than 1.2 μg/g, which confirms the aflatoxin in agricultural products Molecular early warning method: measure the concentration of aflatoxin early warning molecules in agricultural product samples. When the measured concentration of aflatoxin early warning molecules is less than 0.2 μg/g, the level of aflatoxin contamination is below 0.2 μg/g, which is low risk; When the concentration of aflatoxin early warning molecules is greater than 1.2 μg/g, the level of aflatoxin pollution is above 10 μg/kg, which is high risk; when the measured concentration of aflatoxin early warning molecules is between 0.2 μg/g and 1.2 μg/g, for medium risk.

Further, 293 samples of agricultural products were used to verify the results: when the measured AFT-YJFZ01 concentration was less than 0.2 μg/g, the aflatoxin contamination level of 86% of the samples was below 1.0 μg/kg, which was low risk; when the measured AFT-YJFZ01 - When the concentration of YJFZ01 is greater than 1.2 μg/g, the aflatoxin contamination level of 76% of the samples is above 10 μg/kg, which is a high risk. It shows that the accuracy of the method for predicting the low risk of aflatoxin in agricultural products is more than 80%, and the accuracy of predicting the high risk can be more than 75%.

To sum up, the present invention evaluates the ex-ante risk of aflatoxin pollution in agricultural products by detecting the concentration of aflatoxin early-warning molecules in agricultural products, and based on the significant correlation between the concentration of aflatoxin early-warning molecules and the level of aflatoxin pollution, so as to realize The early warning of aflatoxin in agricultural products provides a scientific basis for the source prevention and control of aflatoxin in agricultural products and early prevention and control.

The beneficial effects of the present invention are:

1. The early warning of aflatoxin in agricultural products is fast - it can be completed within half a day, accurate and easy to operate. 2. It can be applied to a wide range of agricultural products, has strong practicability, and is easy to promote and apply. 3. Promote the high-quality development and protection of agricultural products Food safety is of great significance.

Description of drawings

Figure 1 is the standard curve of the rapid immunological detection method for aflatoxin early warning molecule AFT-YJFZ01.

Detailed ways

Embodiment 1 Preparation of aflatoxin early warning molecule AFT-YJFZ01 that can act as a standard substance

According to the following formula configuration Cha's medium: 3% (w/v) sucrose, 0.3% (w/v) NaNO3, 0.1% (w/v) K2HPO4, 0.05% (w/v) MgSO4 7H2O, 0.05% ( w/v) KCl, 0.001% (w/v) FeSO4, pH6.5, configured to obtain Chapei's medium. Random selection of public literature "Distribution, Toxicity and Infection of Aspergillus flavus in Typical Peanut Production Areas in China" - Chinese Academy of Agricultural Sciences master's degree thesis, author Zhang Xing, page 33 - published toxin-producing fungal strain HLJ-1 , HeNZY-2, HuBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-108-19, JSnt-1, HuNdx-7, HBHA-8-17 and other 10 strains were inoculated into the above-mentioned After culturing at 28°C and 200rpm/min for 5 days in Cha's medium, fully homogenize and break the cells by conventional methods, and then use conventional protein purification systems, protein electrophoresis, immunoaffinity and other methods to purify and obtain aflatoxin early warning Molecule AFT-YJFZ01. The test results show that AFT-YJFZ01 can be prepared from the cultures of the above-mentioned toxin-producing fungal strains. Under the same culture conditions, the amount of AFT-YJFZ01 produced by HBHA-8-17 is the largest, and the amount of AFT-YJFZ01 produced by HLJ-1 Minimum amount.

The immunoaffinity method is to use the nanobody or monoclonal antibody of the aflatoxin early warning molecule AFT-YJFZ01 to prepare an immunoaffinity column by a conventional method, and then use the immunoaffinity method to extract the aflatoxin from the aflatoxin-producing fungal cells It is enriched and purified in the crushing solution, and obtained by dissolving in deionized water. Specifically, the sample liquid can be used to dilute the aflatoxin-producing fungal cell disruption solution, and after filtering through conventional filter paper, it is continuously added to the above-mentioned immunoaffinity column. Finally, use pH 2.2 glycine buffer solution or 70% methanol aqueous solution to elute. After collecting the eluate, remove the solution in time by conventional ultrafiltration centrifugation method, and then use sterile water to dissolve it from the ultrafiltration centrifuge tube and leave it in the ultrafiltration centrifuge tube. Filter the protein in the centrifuge tube to obtain the aqueous solution of aflatoxin early warning molecule AFT-YJFZ01.

The excavation method for the initial acquisition of the virulence warning molecule AFT-YJFZ01 of aflatoxin-producing bacteria:

(1) Get the strong virulence-producing strain of Aspergillus flavus, cultivate and obtain the strain culture and the extracellular secreted protein mixture; then the cells of the bacterial strain culture are broken to obtain the intracellular protein mixture; the above-mentioned extracellular secreted protein mixture and the intracellular protein mixture are combined, Add carbodiimide coupling to obtain Aspergillus flavus antigen;

(2) Immunizing the experimental animals with the above-mentioned Aspergillus flavus antigen to obtain a nanobody library or a monoclonal antibody library;

(3) Obtain the protein combination solution of the Aspergillus flavus strains of different virulence, utilize the antibody in the antibody library that step (2) obtains, detect the protein of the Aspergillus flavus strains of different virulence, obtain a series of detection signals;

(4) Find out the nano-antibody whose detection signal is positively correlated with the virulence produced by the above-mentioned Aspergillus flavus strain, that is, the early warning molecular antibody of the virulence produced by the Aspergillus flavus strain, and the protein corresponding to the early warning molecular antibody of the virulence produced by the Aspergillus flavus strain is excavated Aspergillus flavus strains produce virulence warning molecules.

In the above scheme, the strong virulence-producing strain of Aspergillus flavus in step (1) is isolated and identified from nature by conventional methods, or obtained through artificial transformation, and its virulence is not less than 10 μg after being identified by the NY/T 2311-2013 standard method /kg.

There are not less than 3 strains of Aspergillus flavus with different virulence in step (3), and their virulence is identified by the NY/T 2311-2013 standard method as at least 3 levels of high, medium and low.

The culture medium used in the cultivation of the strong virulence-producing strain of Aspergillus flavus is Chapei's medium or other nutrients for the normal growth of Aspergillus flavus, the cultivation time is not less than 12 hours, and the cultivation environment temperature is 15-35°C.

The cells of the strain culture are disrupted by conventional methods such as liquid nitrogen grinding or a cell disruptor.

The dosage of the carbodiimide is 0.005-0.1 g for every 1.0 mL of combined extracellular secreted protein mixture and intracellular protein mixture.

The coupling reaction refers to the reaction at 15-37°C for 2-6 hours, and the reaction at 4-10°C overnight.

The immunization is a routine immunization method, inoculating the Aspergillus flavus antigen. The test animals refer to mice or alpacas or other test animals with similar effects.

According to the above scheme, the antibody preparation process refers to the conventional nanobody preparation technology process or the conventional cell fusion-based hybridoma monoclonal antibody preparation technology process.

According to the above scheme, the detection of proteins of different virulence-producing Aspergillus flavus strains refers to the use of conventional Western Blot technical process, that is, the proteins of different virulence-producing Aspergillus flavus strains are moved to nitrocellulose membranes, and then the above-mentioned Antibodies in the antibody library are detected by direct methods or indirect methods, or other technical processes with similar efficacy are adopted.

According to the above-mentioned scheme, the above-mentioned direct method refers to coupling the antibody in the above-mentioned antibody library with the signal material by conventional methods, and then reacts with the above-mentioned corresponding protein transferred to the nitrocellulose membrane for immune binding reaction.

According to the above-mentioned scheme, the above-mentioned indirect method refers to that the antibody in the above-mentioned antibody library first reacts with the above-mentioned corresponding protein transferred to the nitrocellulose membrane, and then uses the second antibody and the signal material conjugate to bind to the above-mentioned nitrocellulose membrane. Antibodies on the cellulose membrane undergo an immunological binding reaction.

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

The above-mentioned aflatoxin early warning molecule AFT-YJFZ01 antibody can also be prepared by using all or part of the peptides of the aflatoxin early warning molecule AFT-YJFZ01 sequence through conventional antibody preparation technology processes.

Example 2 Nanobody Preparation of Aflatoxin Early Warning Molecule AFT-YJFZ01

Using AFT-YJFZ01 as the immune antigen, immunizing alpaca or Balb/c mice in a conventional way, and then using the known conventional nanobody or mouse monoclonal antibody preparation technology, it can be developed and obtained.

Dissolve the AFT-YJFZ01 prepared above in conventional PBS buffer or normal saline to a concentration of not less than 0.1mg/mL, then mix and emulsify with Freund's complete adjuvant in equal volume, and inject it subcutaneously or intradermally at multiple points on the back Immunize the alpaca, and then boost the immunization once every 2-4 weeks, and replace the Freund's complete adjuvant with Freund's incomplete adjuvant during the booster immunization. Use conventional ELISA process to monitor the immune effect, and after the alpaca serum titer no longer rises, then take blood from the vein of the immunized alpaca, extract total RNA, synthesize cDNA, amplify VHH gene, recover VHH gene fragments, and VHH gene The connection with the pCANTAB 5E(his) carrier treated with double restriction enzymes, the electrotransformation of the ligation product, the construction of the nanobody gene library and the rescue of the nanobody gene library were completed with reference to the method of the patent document CN103866401A, and finally the rescued nanobody gene was obtained library.

The AFT-YJFZ01 prepared above was immobilized on a solid phase carrier such as a 96-well microplate plate according to a gradient of 8 μg/well, 2 μg/well, 0.5 μg/well, and 0.1 μg/well. The final nanobody gene library was panned for 2-4 times, and then AFT-YJFZ01 and indirect non-competitive ELISA were used to identify the antibody produced by each phage clone. The phage corresponding to the positive result was a phage positive clone, and then the positive clone was passed through nano The conventional method of antibody preparation prepares the nanobody, namely the nanobody of AFT-YJFZ01, which is used for further application research work, preferably the nanobody with strong specificity and high affinity characterized by ELISA method.

Example 3 Monoclonal Antibody Preparation of Aflatoxin Early Warning Molecule AFT-YJFZ01

Using AFT-YJFZ01 as the immune antigen, immunizing alpaca or Balb/c mice in a conventional way, and then using the known conventional nanobody or mouse monoclonal antibody preparation technology, it can be developed and obtained.

Dissolve the AFT-YJFZ01 prepared above in conventional PBS buffer or normal saline to a concentration of not less than 0.1mg/mL, then mix and emulsify with Freund's complete adjuvant in equal volume, and inject it subcutaneously or intradermally at multiple points on the back BALB/c mice were then boosted every 2-4 weeks, and Freund's complete adjuvant was replaced with Freund's incomplete adjuvant during the booster immunization. Use conventional ELISA process to monitor the immune effect, and after the serum titer of BALB/c mice no longer rises, then isolate the splenocytes of immunized mice, splenocytes are fused with mouse myeloma cells SP2/0, and semi-solid medium is used for hybridoma cells The selective cultivation operation of the method is completed by referring to the method of the patent document CN103849604A. After the white spots on the needle tip grow on the semi-solid medium, the white spots are respectively picked into the 96-well culture plate with built-in hybridoma conventional medium to obtain monoclonal hybridization. tumor resource bank.

Refer to the method of patent document CN103849604A to obtain the above-mentioned monoclonal hybridoma culture supernatant, that is, the monoclonal antibody, and fix the AFT-YJFZ01 prepared above at the gradient of 8 μg/well, 2 μg/well, 0.5 μg/well, and 0.1 μg/well Use the indirect non-competitive ELISA program to identify each monoclonal antibody on a solid phase carrier such as a 96-well microtiter plate, pick positive clones, and obtain the AFT-YJFZ01 monoclonal antibody for further application research, preferably with specificity after testing AFT-YJFZ01 monoclonal antibody with strong sex and high affinity.

Example 4 Preparation of Rabbit-derived Polyclonal Antibody of Aflatoxin Early Warning Molecule AFT-YJFZ01

Using AFT-YJFZ01 as the immune antigen, the New Zealand white rabbit and other test rabbits are immunized in a conventional way, and then the known conventional rabbit polyclonal antibody preparation technical scheme can be used to develop and obtain it.

The AFT-YJFZ01 prepared above was directly used as an antigen, mixed and emulsified with an equal volume of Freund's complete adjuvant with a solution of not less than 0.1 mg/mL, and injected subcutaneously or intradermally into New Zealand white rabbits at multiple points on the back, and then Boost immunization once every 2-4 weeks, and replace complete Freund's adjuvant with Freund's incomplete adjuvant during booster immunization. The immunization effect was monitored by conventional ELISA process, and after the titer of immunized animal serum no longer increased, the serum of immunized animals was prepared by conventional methods, which was the rabbit-derived polyclonal antibody of aflatoxin early warning molecule AFT-YJFZ01.

Example 5 Establishment of a Rapid Immunological Detection Method for Aflatoxin Early Warning Molecule AFT-YJFZ01

AFT-YJFZ01 immunological rapid detection method, that is, the basic operation procedure of the double-antibody sandwich indirect non-competitive ELISA method: the nanobody coated with AFT-YJFZ01 in the microtiter plate, wash the plate; add blocking solution to block, wash the plate; add AFT-YJFZ01 Or react with the test solution, wash the plate; add AFT-YJFZ01 rabbit polyclonal antibody to react, wash the plate; add horseradish peroxidase-labeled goat anti-rabbit antibody to react, wash the plate; add chromogenic solution to react; add stop solution , microplate reader toxin and calculate the result. The following work is done using this basic procedure.

Determination of the optimal concentration of the antibody: multiple parallel experiments were carried out simultaneously using the design checkerboard titration method, and the above-mentioned nanobody was coated with different concentrations, and the above-mentioned rabbit-derived polyclonal antibody was set at different concentrations, and finally the nanobody and rabbit were determined according to the results. The optimal working concentration of the source polyclonal antibody is to select the concentration of the above two antibodies corresponding to the point where the OD450nm value is approximately 1.0 under the principle of saving the amount of antibody. Concentration can also be detected, the optimal coating concentration of the above-mentioned nanobody is 2.0 μg/mL, and the optimal concentration of the above-mentioned rabbit-derived polyclonal antibody is 3.0 μg/mL.

Determination of the best antibody coating conditions: Coating is the first step in the research of ELISA methods, and the quality of the coating effect has a very critical impact on the results of ELISA. In order to determine the influence of different coating conditions on the detection results, three different conditions were selected to coat the wells under 4°C overnight, 37°C constant temperature for 2 hours, and 37°C constant temperature for 1 hour. The test results showed that the studied All three coating methods can achieve coating, and overnight coating at 4°C is the best coating condition.

Determination of the best blocking agent: After the antibody is coated, in order to avoid the unoccupied positions of the microplate wells from interfering with the subsequent steps of the ELISA, it is necessary to use some inert proteins, namely blocking agents, to occupy these positions. A specific blocking agent will bind to the secondary antibody non-specifically, resulting in false positives. In this study, 3% BSA/PBST, 3% skimmed milk powder/PBST, and 5% skimmed milk powder/PBST were used for sealing. The blocking effect was best with % skim milk powder/PBST.

Determination of the sealing time: the study set three different sealing times of 1h, 2h, and 3h at 37°C for sealing, and the test results showed that the positive hole OD450nm value/negative hole OD450nm value was the largest under the setting of 37°C and 2h blocking , Positive sample OD450nm value > 1, 37 ℃ constant temperature blocking 2h is the best blocking time.

Determination of the reaction time of the above-mentioned rabbit-derived polyclonal antibody: the research adopts three reaction times of 30min, 50min, and 1h at a constant temperature of 37°C for the reaction. Reaction time.

ELISA standard curve drawing of aflatoxin early warning molecule AFT-YJFZ01: Dilute AFT-YJFZ01 molecules to 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300ng/mL, inject 200μL into each well, and use the above-mentioned best The ELISA standard curve of AFT-YJFZ01 was drawn according to the conditions, and the results are shown in Figure 1. The correlation coefficient of the double-antibody sandwich enzyme-linked immunosorbent assay method established under the above optimal conditions is 0.9980, and the detection limit of AFT-YJFZ01 molecule can reach 0.1ng/mL, indicating that the detection method has good detection sensitivity and accuracy .

Evaluation of method specificity: In order to evaluate the specificity of the above-mentioned immunoassay method for aflatoxin early warning molecule AFT-YJFZ01, several strains of fungi Fusarium oxysporum, Aspergillus niger and Ochrae sp. with certain homology to Aspergillus flavus were selected for the study , Fusarium moniliforme, etc., by detecting the cell disruption liquid of these fungal cultures, the results are shown in Table 1. The above methods have no obvious cross-reaction to the proteins of fungi that have homology with Aspergillus flavus, indicating that the established flavus The aspergillus toxin early warning molecule AFT-YJFZ01 immunological rapid detection method has good specificity.

Table 1. Specificity determination results of aflatoxin early warning molecular immunological rapid detection method

Method repeatability evaluation: In order to evaluate the repeatability of the aflatoxin early-warning molecule AFT-YJFZ01 immunological rapid detection method established above, four aflatoxin-producing fungal strains were randomly selected—positive 1, positive 2, positive 3, positive 4 and 1 aflatoxin non-toxigenic fungal strain—negative 5 studies, analyzed the data variation within and between plates of the assay results. The above research results are shown in Tables 2 and 3. The calculated coefficient of variation within the plate is 0.5%-3.5%, and the coefficient of variation between plates is 0.9%-5.7%, both of which are below 7%. Therefore, the above method has good repeatability.

Table 2. In-plate repeatability determination results of early warning molecule AFT-YJFZ01 immunological rapid detection method

Table 3 Reproducibility determination results between plates of early warning molecule AFT-YJFZ01 immunological rapid detection method

Method accuracy evaluation: In order to evaluate the detection accuracy of the above method and also to investigate the practicability of the method, peanut and corn samples were selected as examples for research and evaluation, and aflatoxin early warning molecule AFT-YJFZ01 was added to corn and peanut samples. The closer the recovery rate of the detection result is to 100%, the more accurate and practical the method is. The results of this study are shown in Table 4. The results show that the above established aflatoxin early warning molecule immune rapid detection method is used to detect the early warning molecule AFT-YJFZ01 in peanuts and corn, and the recovery rate reaches 82.5%-109.5%, indicating that the method is accurate. The accuracy is high, and it can be applied to the actual sample detection, and has good practicability.

Table 4. Addition recovery test results of aflatoxin early warning molecular immune rapid detection method

Example 6 Correlation between the concentration of aflatoxin early warning molecules in agricultural products and the level of aflatoxin pollution in agricultural products

A total of 293 samples of agricultural products such as peanuts, corn, rice, wheat, walnuts, and pistachios were randomly selected, and the method of Example 5 was used to measure the concentration of the aflatoxin early warning molecule AFT-YJFZ01 in the above samples, and "GB 5009.22-2016 food Determination of Aflatoxin Group B and Group G in Safe National Standards Food "High Performance Liquid Chromatography-Mass Spectrometry Method for Determination of Aflatoxin Levels. The measurement results show that: when the measured AFT-YJFZ01 concentration is less than 0.2 μg/g, the aflatoxin contamination level of 86% of the samples is below 1.0 μg/kg, which is low risk; when the measured AFT-YJFZ01 concentration is greater than 1.2 μg/g When the aflatoxin contamination level of 76% of the samples was above 10 μg/kg, it was considered as high risk; for the remaining AFT-YJFZ01 concentrations measured between 0.2 μg/g and 1.2 μg/g, it was considered as medium risk.

Further, 6 aflatoxin-producing toxin-producing strains from different origins were randomly cultured using the conventional Chapei culture medium method, and the above-mentioned early warning molecular detection method and aflatoxin detection method were used to track and monitor AFT-YJFZ01 and aflatoxin during the cultivation process. According to the law of growth and decline of toxins, AFT-YJFZ01 can be detected after 16 hours of culture, and aflatoxin is almost zero at this time. This result shows that the early warning molecule AFT-YJFZ01 is produced before the production of aflatoxin.

Therefore, based on the aflatoxin early warning molecule AFT-YJFZ01, we established an early warning method for aflatoxin molecules in agricultural products: that is, when the concentration of AFT-YJFZ01 in agricultural products is less than 0.2 μg/g, it is low risk. In guiding production, it shows that the determination The agricultural products can be safely eaten or stored; when the measured concentration of AFT-YJFZ01 is greater than 1.2 μg/g, it is considered a high risk, and in guiding production, it indicates that prevention and control or even reduction measures need to be taken to prevent and control the hazards of aflatoxin pollution; when When the measured concentration of AFT-YJFZ01 is between 0.2μg/g and 1.2μg/g, the risk is medium. In terms of guiding production, it indicates that the measured agricultural products need continuous tracking and monitoring. The method was more than 80% accurate in predicting low risk of aflatoxin in agricultural products, and more than 75% accurate in predicting high risk.

Example 7 Application of the Aflatoxin Molecular Early Warning Method in the Above-mentioned Agricultural Products

The above molecular early warning method was used to predict peanuts from 10 county-level peanut production areas, corn from 3 county-level production areas, rice from 3 county-level production areas, wheat from 3 county-level production areas, 1 walnut, 1 Agricultural products such as pistachios. The prediction results showed that: 1) AFT-YJFZ01 concentration of peanuts in one county-level production area was greater than 1.63 μg/g, which belonged to high risk, and effective aflatoxin prevention and control or reduction measures should be taken for the products. 2) The concentrations of AFT-YJFZ01 in 2 county-level production areas for peanuts, 1 county-level production area for corn, and 1 production area for walnuts were in the range of 0.27 μg/g to 0.83 μg/g, followed by 0.58 μg/g and 0.30 μg/g μg/g, 0.83μg/g, and 0.27μg/g belong to medium risk, and the pollution risk needs to be continuously tracked and monitored.

Based on the above specific examples,

The technical scheme of the present invention can be used for early warning of the risk of aflatoxin in agricultural products, and timely guide production. 1. The early warning of aflatoxin in agricultural products is fast—it can be completed within half a day, accurate and easy to operate. 2. It can be applied to a wide range of agricultural products , Strong practicability, easy to promote and apply, 3. It is of great significance to promote the high-quality development of agricultural products and ensure food safety.

＜110＞ Institute of Oil Crops, Chinese Academy of Agricultural Sciences

＜120＞ Molecular early warning method of aflatoxin in agricultural products

<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

TAHD SVTSGF 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

NAYSP NTLNK 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 KVKAKANNYC 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 GGEKEYNVVYDRNQDIYMPL 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 NRLDDLFVWWTTPANRLDDL FVWWTTPANR LVDWPIVTIT 8460

HQEMSANLVD WPIVTITHQE MSANFLDRLV DWPIVTITHQ EMSANFLDRM NAGSSYFVEV 8520

GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA 8580

AAGKMNAGSS YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS 8640

YFVEVGHNGN GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKMNAGSS YFVEVGHNGN 8700

GVIEAAAGKM NAGSSYFVEV GHNGNGVIEA AAGKTPLLNQ QNSMWPYFTT SASDGFDGMQ 8760

VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8820

VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8880

VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ VNPRTPLLNQ QNSMWPYFTT SASDGFDGMQ 8940

VNPRVIANGN VAGNILVIAK VIANGNVAGN ILVIAKYFTS NGIIPPAITG LHNGDALRAS 9000

HTVDKNGIWS SEVKATDNYI ANAAAAVAKD LLQDIVTWDD KEAGIYLIAR GPLNEGGLYA 9060

ERGTNYVALS LWALESDGAK HTDYSSQEST SYKLGSFELS YTTPVLTGYG NVESPEQPKL 9120

SGQDASAITW KLTGNLGGED YQDKLTGNLG GEDYQDKVRN SAYNYWVPEL PTEGTSPGFS 9180

TSKPGIGFYT AQFDLDLPKQ GFHQPQPPSE SWESGSPLEG LSKSPGSFFV VRSSYDDSAW 9240

VSADLPKTSY DYGSPITETR YPDADYMQYV MDQARNGIWS SEVKVLVLYG GPKASPSYLT 9300

ATPRYLDTLP EIKTLHLEQS PSTPYAQLYV NGYQYGKKAD IVVPFPWGGP GFEKAQLYVN 9360

GYQYGKAQLY VNGYQYGKAT DNYIANAAAA VAKLSGQDAS AITWKLTGNL GGEDYQDKVR 9420

NGIWSSEVKQ GFHQPQPPSE SWESGSPLEG LSKSSYDDSA WVSADLPKYP DADYMQYVMD 9480

QARYPDADYM QYVMDQARAL TNGAGAIKEA IADVLEHLGE NDEDIAVYAP NPFYKGFAPL 9540

EYLGSNFENG ELPKGFDNAG FVMGTSSSLF NQFILRGKMP MPILVADGRH FQLINTAAYW 9600

KIPNVAIAVS GGGYRLNGTD IPNFLKLNLS SFDASGYIDR LPDICNTCFK MPMPILVADG 9660

RNSILEGPDV KSAAALSTSE KDWLQVRSSS LFNQFILRTF LNLGLNKTNT KLPDICNTCF 9720

KTSLTDYWGR SSFDASGYID RSTSEKDWLQ VRGTDIPNFL KLNLGLNKGF DNAGFVMGTS 9780

SSLFNQFEYL GSNFENGELP KMPILVADGR DLYDAVKINT AAYWKSIALG DDFKKALTNG 9840

AGAIKALTNG AGAIKALTNG AGAIKALTNG AGAIKGKMPM PILVADGRGK MPMPILVADG 9900

RGKMPMPILV ADGRGKMPMP ILVADGRGKM PMPILVADGR GKMPMPILVA DGRHFQLINT 9960

AAYWKHFQLI NTAAYWKLPD ICNTCFKLPD ICNTCFKMPI LVADGRMPMP ILVADGRMPM 10020

PILVADGRMP MPILVADGRM PMPILVADGR MPMPILVADG RMPMPILVAD GRMPMPILVA 10080

DGRMPMPILV ADGRMPMPIL VADGRNSILE GPDVKNSILE GPDVKNSILE GPDVKSAAAL 10140

STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS 10200

AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL 10260

QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE 10320

KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL 10380

STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL QVRSAAALST SEKDWLQVRS 10440

AAALSTSEKD WLQVRSAAAL STSEKDWLQV RSAAALSTSE KDWLQVRSAA ALSTSEKDWL 10500

QVRSAAALST SEKDWLQVRS AAALSTSEKD WLQVRSAAAL STSEKDWLQV RTNTKLPDIC 10560

NTCFKTNTKL PDICNTCFKT NTKLPDICNT CFKAASLPAS FSGFKALVSH DGTFVADAKE 10620

NSLVWHQQVL GWLNKFNAVF SGTLKGDAGS PVFSPDSKGD AGSPVFSPDS KKIAYWQMAD 10680

ESYEADHRIQ AFVIYPENFD KLFSIPADAG DDYKPKLNPE GLISAPRLPV SEGLSLFNIL 10740

QERMINWIQG SDLGRNAESP YPPFGGASDY DLSPDGKRSE AIPNPSGDVA VFSQSQYSFK 10800

SEAIPNPSGD VAVFSQSQYS FKTAVPINGP DSPGTPEGVK TAVPINGPDS PGTPEGVKGD 10860

AGSPVFSPDS KTLATANKID PELKTLYVYT VGSEETIPSL AADWDRTTSQ WNVLDLKVVT 10920

TDSGDVRWIQ GSDLGRALVS HDGTFVADAK ALVSHDGTFV ADAKGDAGSP VFSPDSKKIA 10980

YWQMADESYE ADHRIAYWQM ADESYEADHR IAYWQMADES YEADHRLFSI PADAGDDYKP 11040

KLFSIPADAG DDYKPKLNPE GLISAPRRSE AIPNPSGDVA VFSQSQYSFK RSEAIPNPSG 11100

DVAVFSQSQY SFKTAVPING PDSPGTPEGV KTAVPINGPD SPGTPEGVKT AVPINGPDSP 11160

GTPEGVKGDA GSPVFSPDSK TTSQWNVLDL KTTSQWNVLD LKAASNFDGD TLVLGYDSGN 11220

GNPETSFMTL MRAGSTIAVT DVQITGGAVG IKASGTCSGP IQSAPTSYWL ADQDHSGDAR 11280

DAGSPKPVVQ IGHEGDVGVA EIQNMRFSVA EILPGAKGDG STDDSASLNA ILANNAANCK 11340

GTCSGPIQSA PTSYWLADQD HSGDARIVGE AWAVITGAGD AFKNSQILIQ NLSHDDNNAI 11400

AVDSKNSQIL IQNLSHDDNSN 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 FSVPGTKSS 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 GHEPYYQPI 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 IASAMLDEEED 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 FRNDPAVVFD 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 GRQADMMPL 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 LNTGAVIVPVL 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 APPTPPDFSLG YIQSKIEQDG 21180

SESLLTNEYA PLKGYAFIWN MPAQGRTSGW GGNPGGYRYQ LASYLRDYLD EYLVFPPAGV 21240

QPQKIYVTGE SYAGRVDHLP DVPFDVGEMY SGLVPIDKDD KNFQELFGIK KTPLDDFRAD 21300

DVLEVNPLAD PEVVSYFRLD ADAITAQYFG NDAPWYRSDY ASFLYGPYRL VFAPQEEKAT 21360

WDGVDADKIR FQYPGDLFDQ GTTIRFTGDA ATVNSIAARP DSDEIYFGGQ FEKNLEVLSL 21420

TKHVAAYSFG SKAWTALGGG VNGPVHKNLA LLDGKDLTDY LMKSYELPDG QVITIGNERV 21480

APEEHPVLLT EAPINPKQEY DESGPSIVHR QEYDESGPSI VHRVAPEEHP VLLTEAPINP 21540

KVAPEEHPVL LTEAPINPKV APEEHPVLLT EAPINPKAEL PEGYPESSAN PAFRDIQYLE 21600

NYQGQGYSGP AVKFVTVTEE TDPDLFWALR LSLGDSGACK DAEVEPANWG VEGRLGGAQL 21660

FTSRTVSLVE DGSNTPATLS AGTFARSLVD IYRTVSLVED GSNTPATLSA GTFARAYVEM 21720

MQCTDEKEPL VRLEKDLPGT TLSSKYYGYG GGNPLGPAQG IGFANELIAR LEKDLPGTTL 21780

SSKLSEAGHS VLLIEKTPIE SDATSYLNDR GGPMGTYLVS ASERVILSAG TFGTPKAVIT 21840

DIVNQQRLIN QVELSEDKTI ARGGSNNFGI VTRLLLSDAM WYTRERPSAQ LLSGKFDTLG 21900

TSGPTAKLAN AYTWEGGRAV ADRIPLAIHD EVSPVGDTDA LLERAPVVQY ALNRDGYMGY 21960

HGVPQIPIYA YKAIHDEVSP VGDTDALLER AIHDEVSPVG DTDALLERAI HDEVSPVGDT 22020

DALLERDGYM GYHGVPQIPI YAYKLAEESA ALGVKVNPII LTGDMWRVSD NAGLGDWVPN 22080

PDRFPDGLTP LVEDVTKALG GTSTINGMAY TRSQLSDYAA ATVKAEDVQI DVWQKALGGT 22140

STINGMAYTR EIGFTVQEDN TDGKIDFGSA PNIVNAIAED RNSPVWPDGI QQTYEYPNRA 22200

STNMITSNSG AGLVPQDENR IGGSAGTELQ SDKITGEVWP VLMAYGQKSA ISQYGDSFAK 22260

SQSDFESEFS TAKVNAGIGI GPDDLVSFIK GNAMDHFSSI MERYGPTYAA YFLDQNEKAI 22320

TETQYEEAKD LTLDLPDIGL QYAGTVKTVA ALINWRNIWD GTTAQNVKKE DLETSSYSFS 22380

GDGKVDFASL QSAATQSTTY SNAPAVKTCS LVFLFPKKED LETSSYSFSG DGKFDGILGL 22440

GFDTISVNKY GSGSLSGFVS QDTLKWYSVY DLGNGAVGLA KDAYSPHEIY SRIFEQLEGM 22500

SLSKTYEVVG NVYKDQVLKD VALIKAVPGT AQQAAAIKDA GEFDVERGTV VTNDQCGGAS 22560

SVRLHLVTAE EADIKIIYDM PDGSSCKGID VAKPTGRVNG VWTVTHSPFE GLSALKSAMT 22620

LPRVNGVWTV THSPFEGLSA LKVPTVLMSP WVGKSIDQFF NDAKNVAPDD PDHSITGGNQ 22680

QVYSTYHPNA KESTLHLVLR IQDKEGIPPD QQRIQDKEGI PPDQQRIQDK EGIPPDQQRD 22740

VSLVANHIDT VGKTAATVNT WTGGWSDSKA IGTYQNSGLS QYTVRYSEGV HFPARTVANW 22800

LVREVAGDVD NAVNPAWRLG AGVQGFEAYE AANAQGLRLD GGVIEDFAQK NPDLSSTSDT 22860

TDVIRGPDEP YSGQYDEERQ FISVTNPTGA EPVPKFLSEL LEDEYFTKFV NIWLENTDYE 22920

SAANDPHLSK LLEYDIASGT PVYRNNNQGL EALTISPDGK VYAISDQDDT GPWIRDAYQN 22980

DFAARVDPST AVDYNHYSDA ADRSGDVQTL QFAWALQHLS ERTGNQTGQL GTYFNKNINM 23040

LLYGTDDCSG KGMVFSIDAQ GEKNINMLLY GTDDCSGKNP NPDQAFLQVR FWVATGDSSK 23100

AALDALQQSI YLQPKFSDGN GLFYQYERVA VAGYDDTTGG VGPLLAQKAI AIALQSSHRQ 23160

TGSVDGYAYT DANKNDLITY LKSVVENNND GLTAAYRVAP NSGAYLNEAD FRSLPLIVGN 23220

SDQEGKANEQ PTWVYRSDYQ ECADAPGQKF GATGDEYREP SNDPNPPETY SKVALLFSER 23280

LADGSIPTRS ISSDEDSAET EQSDSSDPKV AIIDGLADPW RTMGVGYATN DDSTIRDVPI 23340

MQELNTNTIR DVDPIVIKVE TGVIKPGMVV TFAPANVTTE VKIVVIGHVD SGKDDADLQE 23400

LGAKSSAFAF RKDPSDAIPS IPSIPISYKE AIPFLKEAAE IDSHWERDDY MPFIEVPRDD 23460

YMPFIEVPRA LQGVDELVEK DALHPQFDNF YQEQPKASYG AGVTIQDRDL SVFFTRINDL 23520

LPVYVELLQK AALTNMVNAA KGEITPEQYE KIINEPTAAA IAYGLDKQLT SEEIWQAEEK 23580

LDKPAGDYTI RDYSNNWESG ALKLAPNQMT GSLDATYLKL APNQMTGSLD ATYLKCASDG 23640

SAETCSWEGH CKCASDGSAE TCSWEGHCKC ASDGSAETCS WEGHCKCASD GSAETCSWEG 23700

HCKCASDGSA ETCSWEGHCK LAAISVEPGK ASELGCSSGD LDCLCKVGEC AQMCISNMNA 23760

KGTLLSSNQG SQAADVKNDD DFLNYGISSK APSVITYDEA TKYEAAGITV HKDDYLPFID 23820

MPSEVTQIDA KIPMALDDWP TLSNMIFSGK DNFDTSSVTH RGYQINFLSN SAKDNIQGIT 23880

KPAIRDNIQG ITKPAIGPLG LSPKLQLWDT AGQERAEDYL LNPSPKNFGI GQDIQPKIDA 23940

TTNPGMRQLG LAMLGNKLVI DGLKEVTEIP ATADASRVNV DYTEVPRLAV NMVPFPRYCD 24000

LILGEWRDQI KDVLRIDYIG GGDLFRGSSP NVLYKSANWT PPEGIVRAAS TGSMAEQYTK 24060

AATGTYASST TVYKAENQAV AVGRALVEGS TFAKALYSSA ATGTYASSTT VYKATGTYAS 24120

STTVYKATTV YGESIKAYAD GYVQIVQTYA ASTGSMAEQY TKDLTWSYAA LLTANNRFNV 24180

DETAFTGAWG RGQSAQGASP GVVIASSPSKI GADGQSAQGA SPGVVIASPS KIGSLAITDV 24240

SLPFFKIQGI SNPSGALSSG GLGEPKKYTV PSTCGVKPSG ALSSGGLGEP KQAILNNNIGA 24300

DGQSAQGASP GVVIASSPSKS DPDYFYTWTR SIYAINSGRT GTYASSTTVY KTVGSSCPYC 24360

DSQAPQVRVQNGAVTWESDPNRVQNGAVTWESDPNRKPDYFYTWTRYTVPSTCGVKSSAA 24420

TGTYASSTTV YKGAVTWESD PNRIVGSISQ LGSWNPSSAT ALSAVQSDVW RDINTVLGSI 24480

HTFDPQNIGA DGQSAQGASP GVVIASSPSKA ASTGSMAEQY TKAENQAVAV GRAENQAVAV 24540

GRALVEGSTF AKALVEGSTF AKALYSSAAT GTYASSTTVY KAYADGYVQI VQTYAASTGS 24600

MAEQYTKDLT WSYAALLTAN NRDLTWSYAA LLTANNRIQG ISNPSGALSS GGLGEPKIQG 24660

ISNPSGALSS GGLGEPKNIG ADGQSAQGAS PGVVIASSPSK PDYFYTWTRQ AILNNIGADG 24720

QSAQGASPGV VIASPSKQAI LNNIGADGQS AQGASPGVVI ASPSKQAILN NIGADGQSAQ 24780

GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS KQAILNNIGA DGQSAQGASP 24840

GVVIASSPSKQ AILNNIGADG QSAQGASPGVVIASPSKQAI LNNIGADGQS AQGASPGVVI 24900

ASPSKQAILN NIGADGQSAQ GASPGVVIAS PSKQAILNNI GADGQSAQGA SPGVVIASPS 24960

KQAILNNIGA DGQSAQGASP GVVIASPSKQ AILNNIGADG QSAQGASPGVVIASPSKQAI 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 ESDPNRRKVQN 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

VKANSLQYV 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 IMKSTLPPDLS 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 GAVIVLVRL 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 TIPIDNDDVDY 32400

VVTGYRVYWV DSGPRTGDGV NDAPSLKDGQ EQEILARADA IVAAIRSALI AFEKNINMLL 32460

YGTDDCSGKG MVFSIDAQGE KTGTDQASVG YYKDAVYALD AIYGIDARDN ILPENLDDGL 32520

PSQFVYEKIL VENLQDQTAK ILVENLQDQT AKAEPYVTGS SAASGSNFVA DFAEAGTDGK 32580

QISYWAFTTP AVKFEPPAVY NDELKDAEEE PYDWSNEGRL SDELEDDNAP IGFETTKDQE 32640

MAVAAFRTSD DFASQMDGRC MGCDSTSIDV SRCMGCDSTS IDVSRDASGG DQITEWQDIY 32700

LPPITKGIQD AGVIATAKGV GSDAWTVSES GRGIDGDKGL VVKDLYGNIV MSGGSTLYPG 32760

IADRAGFAGD DAPRQEYDES GPSIVHRSYE LPDGQVITIG NERSYELPDG QVITIGNERL 32820

SGGVAVIKTT AVLFDEGKIG GSAGTELQSD KDLALVDPGL ELSYNTKLVG GSDFGEDEAK 32880

TLSTNEEGYE TSAVRASYGA GVTIQDRSAY VVYDLSNNEI SLANTKVPYL IGANTDEGTS 32940

FAIRLPVEAF QALASSTSET KDAGNAATND PLFPFSRAAL PGTEVLFADS VAKVTSAQYY 33000

VNPKIQAFVI YPENFDKVGA GVNVGELYAF ADKALTQYSV KTTYNVVAQT KTYANLPQAL 33060

VNSGAIKVIP LQGCDADEYG RDNIQGITKP AIRLANAYTW EGGRYQGASQ CPFRTMGVGY 33120

ATNDDSTIRC ASDGSAETCS WEGHCKCASD GSAETCSWEG HCKLILPGEL AK 33172

Claims (9)

1. An agricultural product aflatoxin molecule early warning method is characterized in that: determining the concentration of aflatoxin early warning molecules in an agricultural product sample: when the concentration of the detected aflatoxin early-warning molecules is less than 0.2 mug/g, the aflatoxin pollution level is below 1.0 mug/kg, and the risk is low; when the concentration of the detected aflatoxin early-warning molecules is more than 1.2 mug/g, the aflatoxin pollution level is more than 10 mug/kg, and the risk is high; when the concentration of the detected aflatoxin early-warning molecule is 0.2-1.2 mug/g, the aflatoxin early-warning molecule is a AFT-YJFZ01 peptide, and the amino acid sequence is shown as SEQ ID NO. 1.

2. The method of claim 1, wherein: the method for quantitatively detecting the concentration of AFT-YJFZ01 in an agricultural product sample by adopting an aflatoxin early warning molecule AFT-YJFZ01 immune rapid detection method comprises the following steps:

preparing an agricultural product sample to be detected, namely adding the agricultural product sample to be detected into an enzyme-labeled plate hole of which the bottom is coated with a nano antibody or a monoclonal antibody of aflatoxin early-warning molecule AFT-YJFZ01, reacting, and washing the plate;

adding aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody for reaction, and washing the plate;

adding a horseradish peroxidase labeled antibody which is combined with an aflatoxin early warning molecule AFT-YJFZ01 polyclonal antibody, reacting, and washing a plate;

adding a color development liquid for reaction;

and adding a stop solution, and reading and calculating the concentration of AFT-YJFZ01 in the sample to be detected by using an enzyme-labeled instrument.

3. The method of claim 2, wherein: 100-200 mu L of each hole is added into an enzyme-labeled plate hole of a nano antibody or a monoclonal antibody of which the bottom is coated with aflatoxin early-warning molecules AFT-YJFZ01, the solution after reaction is removed after the reaction is placed at room temperature or 37 ℃ for no less than 1h, the enzyme-labeled plate is washed, then the AFT-YJFZ01 rabbit-derived polyclonal antibody is added, then 100-200 mu L of each hole is added, the solution is removed after the reaction is placed at room temperature or 37 ℃ for no less than 1h, the enzyme-labeled plate is washed, then 100-200 mu L of horseradish peroxidase-labeled goat anti-rabbit antibody is added into each hole, the solution is removed after the reaction is placed at room temperature or 37 ℃ for no less than 1h, the enzyme-labeled plate is washed, then the chromogenic solution and the stop solution are sequentially added, and finally the concentration of the AFT-YJFZ01 in a sample to be detected is read and calculated through an enzyme-labeled instrument.

4. The method of claim 2, wherein: and replacing the solution to be measured with a serial concentration of an early warning molecule AFT-YJFZ01 pure product solution serving as a standard substance, and preparing a standard curve, so as to calculate and obtain the concentration of AFT-YJFZ01 in the sample to be measured.

5. The method of claim 2, wherein: the method for preparing the agricultural product sample to be measured liquid comprises the following steps: weighing an agricultural product sample to be measured, crushing, transferring to a sample diluent, homogenizing by a conventional method, standing, and taking the supernatant to prepare a sample to be measured.

6. The method of claim 5, wherein: the sample diluent is 0.01mol/L phosphate buffer solution containing 0.1% sorbitol and soft sugar, and the preparation method comprises the following steps: sorbitol and soft sugar 0.5. 0.5g each, naCl 4.0. 4.0g, na ₂ HPO ₄ ·12H ₂ O 1.45 g、KCl 0.1g、KH ₂ PO ₄ 0.1 And g, adding deionized water to a volume of 500mL.

7. The method of claim 2, wherein: the hole bottom is coated with a nano antibody or a monoclonal antibody of aflatoxin early-warning molecule AFT-YJFZ01, and the preparation method comprises the following steps: dissolving the nanometer antibody or monoclonal antibody of AFT-YJFZ01 in ELISA coating buffer solution to form coating solution of 0.2-8.0 mug/mL, adding the coating solution into an ELISA plate, standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2h, removing the coating solution in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; then adding ELISA routine blocking solution, standing at room temperature or 37 ℃ to block at least 1h, discarding the blocking solution, and washing the ELISA routine washing solution.

8. The method of claim 2, wherein: the color developing solution refers to conventional hydrogen peroxide and TMB color developing solution for ELISA.

9. The method of claim 2, wherein: the stop solution is a conventional chromogenic stop solution of ELISA, namely 2mol/L sulfuric acid aqueous solution.

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