CN113820491B

CN113820491B - Aflatoxin pollution early risk early warning intelligent perception card and application thereof

Info

Publication number: CN113820491B
Application number: CN202111041457.3A
Authority: CN
Inventors: 李培武; 张奇; 姜俊; 唐晓倩
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2023-06-27
Anticipated expiration: 2041-09-07
Also published as: CN113820491A

Abstract

The invention discloses an aflatoxin pollution early risk early warning intelligent perception card and application thereof. The aflatoxin early-warning molecule comprises an aflatoxin early-warning molecule nano antibody or an aflatoxin early-warning molecule monoclonal antibody, a signal material marked aflatoxin early-warning molecule rabbit polyclonal antibody, a sample pad, a nitrocellulose membrane, a water absorption pad, a bottom plate and a sheep anti-rabbit antibody, wherein the aflatoxin early-warning molecule is AFT-YJFZP008 peptide, and the amino acid sequence of the aflatoxin early-warning molecule is shown as SEQ ID NO. 1. The method is used for rapidly sensing the aflatoxin production capacity of the aspergillus strain producing the toxins, identifying whether the aflatoxin producing strains with strong toxicity exist in products such as farmlands, agricultural products, chinese medicinal materials and feeds, and intelligently sensing the aflatoxin pollution risk level in the agricultural products, and is easy to popularize and apply.

Description

Aflatoxin pollution early risk early warning intelligent perception card and application thereof

Technical Field

The invention relates to an aflatoxin pollution early risk early warning intelligent perception card and application thereof.

Background

The aflatoxin has strong toxicity and great harm, is the pollutant with the largest variety of polluted foods, is one of the most serious aflatoxin pollution countries in the world in China, generally presents a pollution aggravating trend in recent years, and seriously threatens the food safety and the health of people. Aflatoxin is the most toxic mycotoxin in nature, wherein aflatoxin B1 is a class I carcinogen identified by International cancer research organization (International Agency for Research on Cancer, IARC), and has caused excessive poisoning events of human and animal populations, and becomes one of the main causes of high incidence of liver cancer cases. Statistics of data retrieved according to the last 5 years Web of Science: the aflatoxin pollutes food and raw materials more than 110 kinds, and the high-concentration pollutants are first.

Aflatoxins are mainly produced by toxic fungi such as aspergillus flavus. Taking peanuts as an example, the peanuts are provided with toxin-producing fungus strains of aflatoxin in the field, and after the peanut is harvested, the strains enter packaging bags, transport vehicles, warehouses, processing lines and the like along with the peanuts, and once the conditions are proper, a large amount of aflatoxin can be produced, so that the consumption safety and the life health safety of people of the peanuts and products thereof are threatened. The agricultural research bureau of the United states department of agriculture, the International semi-arid research bureau of the United nations grain and agriculture organization (FAO) and other internationally known research institutions list the toxic fungi of aflatoxins as soil-borne pathogens of crops such as peanuts for research, prevention and control.

Studies have shown that the virulence of different strains of aflatoxin are very different from strains of non-virulent fungi to high-virulence strains. Taking Aspergillus flavus strains separated from peanuts as examples, a considerable part of Aspergillus flavus strains lack toxigenic genes or toxigenic key regulatory genes, and do not have the capability of producing aflatoxin; less than about 20% of the Aspergillus flavus strains are virulent strains. How does the early warning risk assess in advance of aflatoxin contamination? This has been a worldwide problem in the art.

According to the technical scheme, the inventor team discovers the indicator molecule of the toxicity-producing fungi of the aflatoxin through more than ten years of systematic researches, and the indicator molecule has the early warning function of aflatoxin pollution for the first time, so that the indicator molecule becomes an aflatoxin early warning molecule, the research discovers that obvious positive correlation exists between the occurrence level of the early warning molecule and the aflatoxin pollution level, and the intelligent perception card for early risk early warning of aflatoxin pollution is further invented by utilizing the correlation, is used for early risk early warning of the aflatoxin of agricultural products, and provides scientific basis for the prevention and control of the aflatoxin source and early prevention and control of the agricultural products.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides an early risk early warning intelligent perception card for aflatoxin pollution, application and application thereof, which are used for rapidly perceiving the aflatoxin production capability of aspergillus strain producing toxins, identifying whether strong virulence aflatoxin producing strains exist in products such as farmlands, agricultural products, chinese herbal medicines, feeds and the like, intelligently perceiving the risk level of aflatoxin pollution in the agricultural products, and are easy to popularize and apply.

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

The utility model provides an early risk early warning intelligent perception card of aflatoxin pollution, including aflatoxin early warning molecule nanobody or aflatoxin early warning molecule monoclonal antibody, signal material marked aflatoxin early warning molecule rabbit polyclonal antibody, sample pad, nitrocellulose membrane, absorbent pad, bottom plate, sheep anti-rabbit antibody, the one side of bottom plate from top to bottom pastes absorbent pad, detection pad and sample pad in proper order, and adjacent each pad overlaps the junction and connects, the detection pad regards nitrocellulose membrane as the base pad, and the top-down sets up horizontal matter control line and detection line on the nitrocellulose membrane, matter control line on be coated with sheep anti-rabbit antibody, the detection line on be coated with aflatoxin early warning molecule nanobody or aflatoxin early warning molecule monoclonal antibody, signal material marked aflatoxin early warning molecule polyclonal antibody load on sample pad, or independently load, aflatoxin molecule refer to AFT-YJFZP008 peptide, its amino acid sequence is as shown in SEQ ID NO. 1.

According to the scheme, the signal material refers to conventional europium latex microspheres, gold nanoparticles and other marking materials capable of achieving similar effects, and the signal material can be obtained through commercial purchase.

According to the scheme, the aflatoxin early-warning molecular rabbit polyclonal antibody marked by the signal material refers to a product obtained after the signal material is coupled with the aflatoxin early-warning molecular rabbit polyclonal antibody by a conventional marking method.

The sample pad, the nitrocellulose membrane, the water absorption pad, the bottom plate and the goat anti-rabbit antibody are conventional materials of an immune test strip and can be obtained through commercial purchase.

The application of the aflatoxin pollution early risk early warning intelligent perception card is provided, which comprises the following steps: the method is used for rapidly sensing the aflatoxin production capacity of the aspergillus strain, or identifying whether the aflatoxin production strain with strong toxicity exists in products such as farmlands, agricultural products, chinese medicinal materials, feeds and the like, or intelligently sensing the aflatoxin pollution risk level in the agricultural products.

The application method for rapidly sensing the aflatoxin production capacity of the aspergillus strain and identifying whether the aflatoxin production strain with strong toxicity exists in products such as farmlands, agricultural products, chinese herbal medicines, feeds and the like comprises the following steps: culturing and preparing a strain to be identified or a sample to be identified to be tested liquid by using a conventional Chlamydia cell culture medium, and then determining the content of aflatoxin early warning molecules AFT-YJFZP008 by using the aflatoxin pollution early risk early warning intelligent perception card, wherein the specific method comprises the following steps: when the sample pad is loaded with the aflatoxin early-warning molecular rabbit polyclonal antibody marked by the signal material, the strain to be authenticated or the liquid to be tested of the sample to be authenticated or the liquid to be tested of the agricultural product sample is added to the sample pad of the aflatoxin pollution early-warning intelligent perception card, and the reaction is carried out for a period of time, and the analysis result is read;

The sample pad is not loaded with the aflatoxin early warning molecular rabbit polyclonal antibody marked by the signal material, the strain to be authenticated or the sample to be authenticated of the agricultural product is added into the aflatoxin early warning molecular rabbit polyclonal antibody marked by the signal material, and then the aflatoxin pollution early risk early warning intelligent perception card is inserted into the strain to be authenticated or the sample to be authenticated to react for a period of time, and the analysis result is read.

According to the scheme, the application method further comprises the following steps: judging the detection result: according to the determination result of the aflatoxin pollution early risk early warning intelligent perception card, the content of aflatoxin early warning molecule AFT-YJFZP008 in a unit volume of strain to be detected liquid is obtained, and the aflatoxin toxigenic power of the strain to be identified is analyzed. The higher the concentration of aflatoxin early warning molecules AFT-YJFZP008 in the strain to be identified, the stronger the aflatoxin production capacity, i.e. the virulence of the identified strain;

according to the determination result of the early risk early warning intelligent perception card of aflatoxin pollution, the content of aflatoxin early warning molecule AFT-YJFZP008 in a unit volume of sample liquid to be identified is obtained, and whether the sample to be identified contains a strong virulence strain of aflatoxin is determined. And if the concentration of the aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified is high, the strong virulence strain containing aflatoxin in the sample to be identified is indicated.

According to the determination result of the early-risk early-warning intelligent perception card for aflatoxin pollution, the content of aflatoxin early-warning molecules AFT-YJFZP008 in a unit volume or a to-be-detected liquid of an agricultural product sample is obtained, and the risk of aflatoxin pollution in the agricultural product is predicted.

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

preparing the sample liquid to be identified: culturing a sample to be identified to obtain a sample to be identified to be tested; the method comprises the following steps: weighing a sample to be identified, transferring the sample to be identified into sterile water, vibrating the sample to be identified to be uniform at room temperature, preparing uniform dispersion liquid of the sample to be identified, adding 10-1000 mu L of the uniform dispersion liquid of the sample to be identified into a conventional Saussurea liquid culture medium containing 6-600-mL, placing the culture medium to be subjected to shake culture at 200+/-50 rpm at 15-35 ℃, and sampling after culturing 6-24 h to form the sample to be identified.

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

In the scheme, the aflatoxin early warning molecular nano-antibody can be prepared by using AFT-YJFZP008 as an immune antigen, immunizing alpaca in a conventional mode and then using a known conventional nano-antibody preparation technical scheme.

In the scheme, the aflatoxin early-warning molecular monoclonal antibody can be prepared by using AFT-YJFZP008 as an immune antigen, immunizing Balb/c mice in a conventional mode, and then using a known conventional murine monoclonal antibody preparation technical scheme.

The aflatoxin early warning molecular rabbit polyclonal antibody can be prepared by using AFT-YJFZP008 as an immune antigen, immunizing test rabbits such as New Zealand white rabbits in a conventional manner, and then using a known conventional rabbit polyclonal antibody preparation technical scheme.

The early risk early warning intelligent perception card for aflatoxin pollution can be used for identifying the aflatoxin production capability of an aspergillus strain, and the higher the content of aflatoxin early warning molecules in the strain to be identified is, the stronger the aflatoxin production capability, namely the virulence of the identified strain is; the early risk early warning intelligent perception card for aflatoxin pollution can be used for identifying whether a strong virulent aflatoxin strain exists in products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like, and if the content of the aflatoxin early warning molecules in a sample to be identified is high, the strong virulent strain containing aflatoxin in the sample to be identified is indicated;

The virulence of the aspergillus flavus strain is an index for measuring the capability of the strain to produce aflatoxin, and the stronger the virulence of the strain is, the more the amount of the aflatoxin the strain can produce under the same time and culture condition is shown. The invention utilizes the aflatoxin early warning molecules obtained by research, combines the nano-antibody or monoclonal antibody of the aflatoxin toxigenic early warning molecules and the rabbit-source polyclonal antibody (the aflatoxin early warning molecules can be directly used as antigens to prepare the nano-antibody or monoclonal antibody and the rabbit-source polyclonal antibody), constructs a sandwich immunodetection method by the antibodies, and finally assembles the intelligent perception card for early risk early warning of aflatoxin pollution; in the application of the aflatoxin pollution early-stage risk early-warning intelligent perception card, (1) the characteristic that the aflatoxin early-warning molecule is used for determining the characteristic that the concentration is positively correlated with the bacterial strain virulence is utilized, so that the aflatoxin early-warning intelligent perception card is used for identifying the aflatoxin producing capability of aspergillus strains and the application of the aflatoxin virulence bacterial strain with strong virulence in farmlands, agricultural products and feeds, and (2) the characteristic that the aflatoxin early-warning molecule is utilized for determining the characteristic that the concentration is positively correlated with the aflatoxin pollution risk in the agricultural products is utilized, so that the aflatoxin pollution risk level in the agricultural products is intelligently perceived, and the aflatoxin early-warning intelligent perception card is practical and easy to popularize and provides key grippers and scientific basis for timely finding the aflatoxin pollution risk and early prevention and early control, and has important significance for promoting the high-quality development of agricultural industry and guaranteeing the food safety.

The invention has the beneficial effects that:

1. the early risk early warning intelligent perception card for aflatoxin pollution, which can be used for quick detection of aflatoxin early warning molecular immunity, can be used for quick quantitative detection of aflatoxin early warning molecular AFT-YJFZP008, can be used for identifying and comparing aflatoxin production capacity of aflatoxin production strains of aspergillus, can be used for identifying whether aflatoxin production strains with strong toxicity exist in farmlands, agricultural products and feeds, can be used for quick intelligent perception of aflatoxin pollution risk level in the agricultural products, is easy to operate, has strong practicability, is easy to popularize and apply, and has important significance for promoting high-quality development of agricultural industry and guaranteeing food safety.

Detailed Description

Example 1 preparation of Aft-YJFZP008

The preparation of the culture medium was carried out as follows: 3% (w/v) sucrose, 0.3% (w/v) NaNO3,0.1% (w/v) K2HPO4,0.05% (w/v) MgSO4.7H2O, 0.05% (w/v) KCl,0.001% (w/v) FeSO4, pH6.5 were prepared to obtain a Chlamydomonas medium. Randomly selecting 10 strains of published open literature (national institute of peanut production area Aspergillus flavus distribution, virulence and infection) namely national institute of agriculture, namely national institute of Chinese agricultural, namely, shuoshi institute of science, author Zhang Xing, page 33, namely published toxigenic strains HLJ-1, heNZY-2, huBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-122-2, jcnt-1, huNdx-7, HBHA-8-17 and the like, respectively inoculating the 10 strains into the Chlamys culture medium, culturing for 5 days at 28 ℃ at 200 rpm/min, fully homogenizing and crushing cells by a conventional method, and purifying to obtain aflatoxin early-warning molecules AFT-YJFZP008 by using a conventional protein purification system, protein electrophoresis, immunoaffinity and other methods. Test results show that AFT-YJFZP008 can be prepared in the culture of the toxigenic strain, under the same culture conditions, the quantity of AFT-YJFZP008 prepared by HBHA-8-17 is the largest, and the quantity of AFT-YJFZP008 prepared by HLJ-1 is the smallest.

The immunoaffinity method is to prepare an immunoaffinity column by using a nanometer antibody or a monoclonal antibody of aflatoxin early warning molecule AFT-YJFZP008 through a conventional method, and then to enrich and purify from aflatoxin toxigenic fungi cell disruption liquid by using the immunoaffinity method, and dissolve with deionized water to obtain the product. The immunoaffinity column is prepared by using aflatoxin early warning molecule AFT-YJFZP008 antibody through a conventional affinity column preparation scheme. Specifically, the aflatoxin toxigenic bacteria cell disruption solution can be diluted by using a sample solution, filtered by using filter paper, continuously added into the immunoaffinity column, and after the aflatoxin toxigenic bacteria cell disruption solution is basically drained, the immunoaffinity column is washed by using a conventional eluent, and finally the column is eluted by using a glycine buffer solution with the pH of 2.2 or a 70% methanol aqueous solution, the solution is timely removed by a conventional ultrafiltration centrifugation method after the eluent is collected, and then the protein remained in the ultrafiltration centrifuge tube is dissolved by using sterile water from the ultrafiltration centrifuge tube, so that the aflatoxin early-warning molecule AFT-YJFZP008 aqueous solution can be obtained.

The initial acquisition of aflatoxin toxigenic bacteria toxigenic early warning molecule AFT-YJFZP008 is carried out by a mining method:

the method for exploring the toxicity early-warning molecules of the aspergillus flavus strain is as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The aflatoxin early-warning molecule AFT-YJFZP008 antibody can also be prepared by using all peptide fragments or part of peptide fragments of the aflatoxin early-warning molecule AFT-YJFZP008 after knowing the whole sequence of the aflatoxin early-warning molecule AFT-YJFZP 008.

Example 2 preparation of Aft-YJFZP008 nanobody as Aflatoxin early warning molecule

AFT-YJFZP008 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then the preparation technical scheme of known conventional nano antibodies or mouse monoclonal antibodies is utilized to develop and obtain the alpaca or Balb/c mice.

Dissolving AFT-YJFZP008 obtained by the preparation method in a conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1 mg/mL, mixing and emulsifying with Freund's complete adjuvant in an equal volume, immunizing alpaca by subcutaneous or intradermal multipoint injection at back, and enhancing immunity for 1 time every 2-4 weeks, wherein Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during enhancing immunity. The immune effect is monitored by adopting a conventional ELISA flow until serum titer of alpaca is not increased any more, then the operations of venous blood collection, total RNA extraction, cDNA synthesis, VHH gene amplification, VHH gene fragment recovery, connection of the VHH gene and a double enzyme digestion pCANTAB 5E (his) carrier, electric conversion of a connection product, construction of a nanobody gene library, rescue of the nanobody gene library and the like of the alpaca are completed according to the method of a patent document CN103866401A, and finally the rescued nanobody gene library is obtained.

Fixing AFT-YJFZP008 obtained by the preparation on solid-phase carriers such as 96-hole ELISA plates according to gradients of 8 mug/hole, 2 mug/hole, 0.5 mug/hole and 0.1 mug/hole, panning the saved nanobody gene library for 2-4 times according to a method of patent document CN103866401A, identifying antibodies generated by each phage clone by using AFT-YJFZP008 and indirect non-competitive ELISA, identifying phage corresponding to positive results as phage positive clones, preparing the nanobody by the positive clones in a conventional mode of nanobody preparation, namely the nanobody of AFT-YJFZP008, for further application research work, preferably characterizing the nanobody with strong specificity and high affinity by ELISA method.

Example 3 preparation of Aft-YJFZP008 monoclonal antibody as an Aflatoxin early warning molecule

The AFT-YJFZP008 obtained by the preparation is dissolved in a conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1 mg/mL, and is mixed and emulsified with Freund's complete adjuvant in an equal volume, BALB/c mice are subjected to subcutaneous or intradermal multipoint injection mode at the back, and then are subjected to boosting for 1 time every 2-4 weeks, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during boosting. And (3) monitoring the immune effect by adopting a conventional ELISA flow, after the serum titer of the BALB/c mice is no longer increased, then separating immune mouse spleen cells, fusing the spleen cells with mouse myeloma cells SP2/0, completing the selective culture operation of a semisolid culture medium on hybridoma cells by using a method of reference patent document CN103849604A, and after a needle point white spot grows on the semisolid culture medium, respectively picking the white spots into 96-hole culture plates with the built-in hybridoma conventional culture medium, thereby obtaining a monoclonal hybridoma resource library.

The monoclonal antibody obtained by the culture supernatant of the monoclonal hybridoma is obtained by the method of the reference CN103849604A, AFT-YJFZP008 obtained by the preparation is fixed on a solid-phase carrier such as a 96-well ELISA plate according to the gradient of 8 mug/well, 2 mug/well, 0.5 mug/well and 0.1 mug/well, each monoclonal antibody is identified by an indirect non-competitive ELISA program, positive clones are picked up, and the AFT-YJFZP008 monoclonal antibody is obtained and used for further application research work, and the AFT-YJFZP008 monoclonal antibody with the characteristics of strong specificity and high affinity is preferably detected.

Example 4 preparation of Aft-YJFZP008 Rabbit polyclonal antibody as African aflatoxin Pre-alarm molecule

AFT-YJFZP008 is used as an immune antigen, test rabbits such as New Zealand white rabbits are immunized by a conventional mode, and a known conventional rabbit polyclonal antibody preparation technical scheme is utilized to develop and obtain the antibody.

The AFT-YJFZP008 obtained by the preparation is directly used as an antigen, the solution with the concentration not lower than 0.1mg/mL is mixed and emulsified with Freund's complete adjuvant in an equal volume, new Zealand white rabbits are subjected to subcutaneous or intradermal multipoint injection at the back, then the immunization is enhanced for 1 time every 2-4 weeks, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during the enhancement. And monitoring the immune effect by adopting a conventional ELISA flow, and preparing and obtaining serum of the immune animal by a conventional method after the serum titer of the immune animal is not increased, namely the rabbit-derived polyclonal antibody of aflatoxin early-warning molecule AFT-YJFZP 008.

Example 5 preparation of Aflatoxin pollution early risk early warning smart sensor card

The aflatoxin pollution early risk early warning intelligent perception card is formed by assembling an aflatoxin early warning molecular nano antibody or an aflatoxin early warning molecular monoclonal antibody, a signal material marked aflatoxin early warning molecular rabbit polyclonal antibody, a sample pad, a nitrocellulose membrane, a water absorbing pad, a bottom plate and a goat anti-rabbit antibody through a conventional immune test strip construction mode, namely, the water absorbing pad, a detection pad and the sample pad are sequentially stuck on one surface of the bottom plate from top to bottom, adjacent pads are overlapped and connected at a connecting part, the detection pad takes the nitrocellulose membrane as a base pad, a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with the goat anti-rabbit antibody, the signal material marked aflatoxin early warning molecular rabbit polyclonal antibody is loaded on the sample pad or independently loaded, and the difference is that a core reagent is loaded:

(1) The detection line of the nitrocellulose membrane is fixed with an aflatoxin early-warning molecule nano antibody or an aflatoxin early-warning molecule monoclonal antibody, and the fixing method comprises the following steps: spraying by adopting a conventional spot film instrument, wherein the concentration of the aflatoxin early-warning molecule nano antibody or the aflatoxin early-warning molecule monoclonal antibody is 1 mg/mL, and the spraying speed is 0.6 mu L/cm; the quality control line is fixed with goat anti-rabbit antibody, and the fixing method comprises the following steps: spraying by a conventional spot film instrument, wherein the concentration of the anti-rabbit antibody of the sprayed sheep is 0.5 mg/mL, and the spraying speed is 0.6 mL/cm.

(2) The aflatoxin early-warning molecular rabbit polyclonal antibody marked by the signal material is loaded on a sample pad or is independently loaded in a vessel such as a conventional small hole, a small bottle and the like, and is subjected to conventional freeze drying treatment.

The preparation method of the signal material marked aflatoxin early-warning molecule rabbit polyclonal antibody is illustrated by taking a conventional europium latex microsphere as an example as follows: taking 100 mu L of europium latex microspheres, uniformly mixing with 900 mu L of MES solution with pH of 6.0 by vortex, placing in an ultrasonic cell disruption instrument, centrifuging for 10 min at power of 20% at 11000 rpm for 9 s by ultrasound, carefully discarding supernatant, adding 1mL boric acid buffer solution (pH of 8.2) into precipitate for resuspension, repeatedly performing ultrasonic treatment for 9 s after uniformly mixing by vortex, adding 20 mu L of carbodiimide aqueous solution which is now prepared into 15 mg/mL for activation, vigorously shaking at room temperature for 15 min, centrifuging for 10 min at 11000 rpm, carefully discarding supernatant, adding 1mL boric acid buffer solution into precipitate for resuspension, performing ultrasonic treatment for 9 s after uniformly mixing by vortex, adding 60 mu g of aflatoxin early warning molecular rabbit polyclonal antibody, performing shaking for 2 h at 10 ℃ by a shaking table of 11000 rpm, centrifuging for 10 min, re-dissolving the precipitate by 1mL of 0.1% bovine serum albumin-phosphate buffer solution, repeating ultrasonic step, shaking for 1 h at 4 ℃ to seal unbound sites on the surfaces of the microspheres; after the reaction is completed, the aflatoxin early-warning molecular rabbit polyclonal antibody marked by the signal material can be obtained and is preserved at 4 ℃ for standby. When in use, the aflatoxin early-warning molecular rabbit polyclonal antibody marked by the signal material obtained by the preparation is diluted 1000 times by adopting a conventional phosphate buffer solution containing 1% of sucrose, 0.5% of ovalbumin, 2.5% of tween-20 and 0.5% of PVPK30 for reuse.

(3) In concert with the scheme (2) above, the following are: and a goat anti-rabbit antibody is fixed on the nitrocellulose membrane quality control line.

The aflatoxin early warning molecule AFT-YJFZP008 nanometer antibody or monoclonal antibody as described above can be prepared by the method of example 2 and example 3.

The aflatoxin early-warning molecule AFT-YJFZP008 rabbit-source polyclonal antibody can be prepared by the method of example 4.

The quality guarantee period research result of the aflatoxin pollution early risk early warning intelligent perception card shows that the aflatoxin pollution early risk early warning intelligent perception card can be stored for at least more than 6 months at the temperature of 4 ℃.

Example 6 establishment of standard curve for fast determination of aflatoxin early-warning molecule AFT-YJFZP008 by aflatoxin pollution early-warning intelligent perception card

Taking the aflatoxin pollution early-stage risk early-warning intelligent sensing card finally prepared by the europium latex microspheres as an example, the establishment process of a standard curve for rapidly determining aflatoxin early-stage risk early-warning molecules AFT-YJFZP008 by the aflatoxin pollution early-stage risk early-warning intelligent sensing card is described.

The aflatoxin early-warning molecule AFT-YJFZP008 prepared in the embodiment 1 is prepared into solutions with serial concentrations of 0.001, 0.01, 0.1, 1, 10, 100 ng/mL and the like by using a conventional phosphate buffer solution, then the aflatoxin early-warning molecule rabbit polyclonal antibody marked by a signal material of an aflatoxin pollution early-warning intelligent perception card is sequentially dissolved by using the solution, the conventional immunochromatography reaction is carried out for 15 min, finally a fluorescent signal on the aflatoxin pollution early-warning intelligent perception card is read, the fluorescent signal is taken as an ordinate, and the AFT-YJFZP008 is taken as an abscissa, so that the result shows that the content of the aflatoxin early-warning molecule AFT-YJFZP008 can be rapidly and quantitatively measured by the aflatoxin pollution early-warning intelligent perception card, the detection limit can reach 0.1ng/mL, and the correlation coefficient can reach 0.99. And then the repeatability and the accuracy of aflatoxin early-stage risk early-warning intelligent perception card for rapidly determining aflatoxin early-warning molecules AFT-YJFZP008 are evaluated by adopting a conventional method, and the repeatability evaluation result shows that: the variation coefficients of the measurement results are all below 10%, which indicates that the repeatability of the measurement results of the method is good; the accuracy evaluation result shows that the aflatoxin early-warning molecule AFT-YJFZP008 prepared in the embodiment 1 is added into products such as peanut, corn and rice, the recovery rate range is 86% -103%, and the accuracy of the measurement result of the method is good. Therefore, the aflatoxin pollution early-stage risk early warning intelligent perception card method for rapidly and quantitatively determining AFT-YJFZP008 has good detection sensitivity and accuracy.

Method specificity evaluation: in order to evaluate the specificity of the immune detection method of aflatoxin early-warning molecule AFT-YJFZP008, several strains of fungi including fusarium oxysporum, aspergillus niger, aspergillus ochraceus, fusarium moniliforme and the like which have certain homology with the aflatoxin are researched and selected, and the cell disruption liquid of the fungi cultures is detected, so that the detection result shows that the method has no obvious cross reaction on the protein of fungi with homology with the aflatoxin, and the established immune rapid detection method of the aflatoxin early-warning molecule AFT-YJFZP008 has good specificity.

Example 7 early risk early warning Aflatoxin contamination Smart sensor card for fast sensing identification of Strain virulence comparing Aflatoxin

Firstly, preparing a to-be-identified strain to be tested liquid: according to the toxicity of the strain, 10 strains of the published patent documents Aspergillus flavus distribution, toxicity and infection research of China typical peanut production area, namely national institute of agriculture, namely, national institute of Chinese agricultural, namely, shuoshi, author Zhang Xing, page 33, namely, published toxicity producing strains HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, heNZY-2, huBha-24, jcnt-1, huNdx-7, GDZJ-108-19, HBHA-8-17 and the like are selected, the strain to be identified is cultured in a Chlamydia culture medium, the culture environment temperature is 15-35 ℃, the culture time is not less than 12 h ℃, the mixture of the culture medium and the culture is fully homogenized, and then the mixture is diluted by 1-10 times by sterile water, so as to obtain the liquid to be tested of the strain to be identified.

Second, the measurement of the strain to be identified is carried out: and measuring the liquid to be measured by using an aflatoxin pollution early risk early warning intelligent perception card measuring scheme, and reading and calculating the concentration of AFT-YJFZP008 in the sample to be measured.

According to the results of the above documents, the virulence of 10 strains, such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, heNZY-2, huBha-24, jcnt-1, huNdx-7, GDZJ-108-19, HBHA-8-17, etc., are as follows: 0. mu g/L, 0 mu g/L, 3.8 mu g/L, 4.9 mu g/L, 67.2 mu g/L, 81.7 mu g/L, 192.0 mu g/L, 204.4 mu g/L, 297.4 mu g/L and 1027.5 mu g/L, which indicate that HBZHX-21 and HBXY-36 are non-virulent strains, HBHA-1-4 and GDZJ-6 are weak virulent strains, and HBHA-8-17 and GDZJ-108-19 are strong virulent strains.

Thirdly, judging the identification result: the higher the content of aflatoxin early warning molecules AFT-YJFZP008 in the strain to be identified, the stronger the aflatoxin production capability, i.e. the virulence of the identified strain is. According to the early risk early warning intelligent perception card for aflatoxin pollution and the application technical scheme thereof, the concentration results of AFT-YJFZP008 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 and the like are sequentially determined: 0 ng/mL, 0 ng/mL, 7.2 ng/mL, 9.9 ng/mL, 42.5 ng/mL, 67.7 ng/mL, 112.0 ng/mL, 127.6 ng/mL, 176.0 ng/mL, 537.1 ng/mL, the results also indicate that: HBZHX-21 and HBXY-36 are non-virulent strains, HBHA-1-4 and GDZJ-6 are weak virulent strains, HBHA-8-17 and GDZJ-108-19 are strong virulent strains. The measurement result is consistent with the sequence of the strain virulence intensity published in the publication of the distribution, virulence and infection research of the Aspergillus flavus in the China typical peanut production area, and the identification result of the strain with strong virulence and the strain with weak virulence is consistent with the publication, so that the intelligent detection card and the application technical scheme thereof provided by the invention can be used for identifying the strain virulence of the aflatoxin, and the method is simple, easy to operate and strong in practicability.

Example 8 fast sensing identification of virulent Strain containing Aflatoxin in agricultural fields Using Aflatoxin pollution early risk early warning Smart sensor card

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

Step two, the determination of the sample to be identified is carried out: and (3) measuring the liquid to be measured by using an aflatoxin pollution early risk early warning intelligent perception card measuring scheme, and reading and calculating the concentration of AFT-YJFZP008 in the sample to be measured.

Thirdly, judging the identification result: if the concentration of aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified is high, the sample to be identified is proved to contain the strong virulence strain of aflatoxin. According to the technical scheme, the concentration results of AFT-YJFZP008 in the soil sample-1, the soil sample-2, the soil sample-3 and the soil sample-4 are sequentially: 0.1 ng/mL, 0.2 ng/mL, 12.7 ng/mL, 19.5 ng/mL. The result shows that 50 mu L of sample uniform dispersion liquid is taken as an example to be added into a 30 mL-containing culture medium, the amount of AFT-YJFZP008 contained in each milliliter of culture liquid in a soil sample-1 and a soil sample-2 is less than 1.0 ng, and the strong virulent strain without aflatoxin is the corresponding peanut post-partum pollution risk in the farmland; the amount of AFT-YJFZP008 contained in each milliliter of culture solution in soil sample-3 and soil sample-4 is above 10 ng/mL, and the strong virulence strain containing aflatoxin has higher risk of postpartum peanut pollution in corresponding farmlands.

Example 9 fast sensing identification of virulent Strain containing Aflatoxin in agricultural products Using Aflatoxin pollution early risk early warning Smart sensor card

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

Thirdly, judging the identification result: if the concentration of aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified is high, the sample to be identified is proved to contain the strong virulence strain of aflatoxin. According to the application technical scheme, the concentration results of AFT-YJFZP008 in the samples of peanut, corn, rice and wheat are sequentially: 16.2 ng/mL, 11.3 ng/mL, 0.4 ng/mL, 0 ng/mL. The result shows that the amount of AFT-YJFZP008 contained in each milliliter of culture solution in the peanut and corn samples is more than 10 ng, and the peanut and corn samples contain strong virulence strains of aflatoxin, so that the pollution risk is high; the rice sample contains AFT-YJFZP008 in an amount of below 1.0 in each milliliter of culture solution, does not contain strong virulent strain of aflatoxin, and has low pollution risk; the measured wheat sample contains AFT-YJFZP008 in an amount of 0 in each milliliter of culture solution, does not contain aflatoxin toxigenic strains, and basically has no risk of aflatoxin pollution.

Example 10 fast sensing identification of virulent Strain containing Aflatoxin in feed Using Aflatoxin pollution early risk early warning Smart sensor card

Firstly, preparing a sample to be identified and a liquid to be tested: 4 parts of feed samples to be tested are selected from the market, and are named feed-1, feed-2, feed-3 and feed-4 in sequence. Sequentially weighing farmland soil samples to be measured, crushing, transferring into sterile water, and vibrating at room temperature until the concentration is 0.5g/mL until the concentration is uniform, thereby preparing sample diluent to be measured. And 50 mu L of the sample diluent to be detected is taken, added into a Chlamydia medium containing 30 mL, placed at 28 ℃ for 200 rpm shake culture, and sampled after 24 h culture, so as to form the sample diluent to be detected of the sample to be identified.

Thirdly, judging the identification result: if the concentration of aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified is high, the sample to be identified is proved to contain the strong virulence strain of aflatoxin. According to the application technical scheme, the concentration results of AFT-YJFZP008 in the feed-1, the feed-2, the feed-3 and the feed-4 are as follows: 0.5 ng/mL, 19.6 ng/mL, 10.7 ng/mL, 72.2 ng/mL. The result shows that the amount of AFT-YJFZP008 contained in each milliliter of culture solution in a feed-1 sample is below 1.0 ng/mL, and the feed-1 sample does not contain strong virulence strain of aflatoxin and has low pollution risk; the amount of AFT-YJFZP008 contained in each milliliter of culture solution in the feed-2, the feed-3 and the feed-4 is above 10 ng/mL, and the peanut post-partum pollution risk in corresponding farmlands is high due to the strong virulence strain containing aflatoxin.

Example 11 correlation of Aflatoxin Pre-alarm molecule concentration in agricultural products with Aflatoxin contamination level of agricultural products

The agricultural product samples such as peanut, corn, rice, wheat, walnut, pistachio and the like are randomly selected to be 293 in parts, the concentration of aflatoxin early warning molecule AFT-YJFZP008 in the samples is measured by adopting the method of the embodiment 5, and the aflatoxin level is measured by adopting a high performance liquid chromatography-mass spectrometry method in the measurement of aflatoxin B and G in national food safety standards of GB 5009.22-2016. The measurement result shows that: when the concentration of AFT-YJFZP008 is less than 0.1 mug/g, the aflatoxin pollution level of a 90% sample is less than 1.0 mug/kg, which is low risk; when the concentration of AFT-YJFZP008 is higher than 1.0 mug/g, the aflatoxin pollution level of 75% sample is higher than 10 mug/kg, which is a high risk; the rest of the AFT-YJFZP008 concentration is measured to be 0.1-1.0 mug/g, and the risk is judged to be apoplexy.

Further adopting a conventional Chlamydia culture medium method to randomly culture 6 aflatoxin-producing strains from different producing areas, adopting the early warning molecule detection method and the aflatoxin detection method to track and monitor the growth regulation of AFT-YJFZP008 and aflatoxin in the culture process, finding that AFT-YJFZP008 can be detected after 16 hours of culture, and the aflatoxin is almost zero at the moment, and the result shows that the early warning molecule AFT-YJFZP008 is produced before the aflatoxin is produced.

Therefore, an agricultural product aflatoxin molecular early warning method is established based on an aflatoxin early warning molecule AFT-YJFZP008, when the concentration of the detected AFT-YJFZP008 is less than 0.1 mug/g, the risk is low, and the agricultural product can be safely eaten or stored in the aspect of guiding production; when the measured AFT-YJFZP008 concentration is more than 1.0 mug/g, the risk is high, and on the aspect of guiding production, the prevention and control and even reduction measures are required to be adopted to prevent and control the aflatoxin pollution hazard; when the concentration of the AFT-YJFZP008 is measured to be 0.1 mu g/g-1.0 mu g/g, the method is a medium risk, and on the aspect of guiding production, the method shows that the measured agricultural products need continuous tracking and monitoring. The accuracy of predicting the low risk of aflatoxin in agricultural products by the method is up to 90%, and the accuracy of predicting the high risk is more than 75%.

Embodiment 12 utilizing an Aflatoxin pollution early risk early warning intelligent perception card to rapidly perceive and early warn of Aflatoxin pollution risk level in agricultural products

By adopting the aflatoxin pollution early risk early warning intelligent perception card, agricultural products such as peanuts from 10 county-level peanut producing areas, corns from 3 county-level producing areas, rice from 3 county-level producing areas, wheat from 3 county-level producing areas, 1 walnut, 1 pistachio and the like are predicted. The prediction result shows that: 1) The AFT-YJFZP008 concentration of 1 county-level peanut in the production area is more than 1.6 mug/g, which belongs to high risk, and effective aflatoxin prevention and control or reduction measures need to be adopted for the product. 2) AFT-YJFZP008 concentration of 2 county-level producing area peanuts, 1 county-level producing area corn and 1 producing area walnut is in the range of 0.2 mug/g-0.9 mug/g, and is sequentially 0.53 mug/mL, 0.29 mug/mL, 0.85 mug/mL and 0.26 mug/mL, which belongs to medium risk, and continuous tracking and monitoring of pollution risk are needed.

As demonstrated by the above specific examples in combination,

the technical scheme of the invention can be used for early warning of the aflatoxin risk in agricultural products and guiding production in time, 1, the intelligent early warning perception card for early warning of the aflatoxin pollution, which can be used for fast and quantitatively detecting the aflatoxin early warning molecule AFT-YJFZP008,2, can be used for identifying and comparing the aflatoxin producing ability of aflatoxin producing strains of aspergillus, 3, can be used for identifying whether aflatoxin producing strains with strong toxicity exist in farmlands, agricultural products and feeds, 4, can be used for fast and intelligently sensing the aflatoxin pollution risk level in agricultural products, 5, is easy to operate, has strong practicability, is easy to popularize and apply, and 6, has important significance for promoting the high-quality development of agricultural industry and guaranteeing the food safety.

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

< 120 > an aflatoxin pollution early risk early warning intelligent perception card and application thereof

＜160＞ 1

＜210＞ 1

＜211＞ 18499

＜212＞ PRT

< 213 > Aspergillus flavus

＜400＞ 1

ALAALASERL EPRALASERP HESERGLYPH ELYSALAPHE ASPSERSERP HEPRLEPRLY 60

SALAPHEPRA SNALAPRASP LYSALAGLYV ALILEPRGLS ERLEHISGLN ASPTHRVALG 120

LYTHRPHEGL YLYSALAILE HISASPGLVA LSERPRVALG LYASPTHRAS PALALELEGL 180

ARGALAILEA SNASPTYRIL EASPSERGLN LEASPLYSAL ALELEPHEGL YALAALAGLY 240

SERALAGLAS PPRVALVALV ALLYSALALE THRASNGLYA LAGLYALAIL ELYSALALEV 300

ALSERHISAS PGLYTHRPHE VALALAASPA LALYSALAAS NASNTYRCYS SERASNGLNV 360

ALGLGLYPRT YRSERLETYR SERGLYARGA LAPRVALVAL GLNTYRALAL EASNARGALA 420

SERMETVALT RPGLGLALAG LNGLNVALSE RGLYLYSALA SERPRSERTY RLETHRALAT 480

HRPRARGALA VALGLGLNSE RLEASPALAI LEARGALAVA LGLYGLNALA THRGLARGAL 540

AVALILETHR ASPILEVALA SNGLNGLNAR GALAVALSER PRSERPHEGL ASPVALTRPS 600

ERGLNPRARG ALATYRGLNG LYTYRPHEHI SSERASNASP ASPLELEASN ARGCYSASPV 660

ALALATHRTH RASPVALTYR TYRSERGLYL YSCYSVALTH RALAPRSERG LYPRCYSGLY 720

GLNLYSCYST YRALALEVAL ASNHISGLPH ESERARGASP ALAASPALAC YSASNGLYGL 780

YGLYILEGLT YRASPSERPR ALAASPTHRP RLEGLPHELY SASPALAGLY SERPRLYSPR 840

VALVALGLNI LEGLYHISGL GLYASPVALG LYVALALAGL ILEGLNASNM ETARGASPAL 900

AGLYTYRGLT HRSERILETH RASPTYRTRP GLYARGASPA LAVALTYRAL ALEASPALAI 960

LETYRGLYIL EASPALAARG ASPGLARGAL ASERLEASPV ALGLPRSERL EPRTRPPRAS 1020

NASPGLYILE PHEARGTRPA RGASPPHEPH EASNHISVAL THRILELYSA SPPHEGLYTR 1080

PASPSERALA PHEASPGLNL YSGLYASNSE RLEGLYLECY SLEPRTHRAS PPHELYSASP 1140

PHEPRCYSAS PVALGLNARG PRPRASPLEA LAALAASNAS PALALYSASP PHETHRASPI 1200

LETHRALAGL YSERSERILE GLYCYSASPG LYVALASNPR GLNTHRGLYL YSASPGLYAL 1260

AGLYGLNMET PHEILEPRLE ASNPRASNAL ATYRSERPRA SNTHRLEASN LYSASPGLYA 1320

SPLEVALTHR GLNGLNASNG LLEGLNGLYL YSASPLEASP GLNASPILEG LLYSLYSASP 1380

LEASNASPGL YGLYSERSER VALGLYVALG LNASNARGLY SASPLEASNP RASNGLYSER 1440

GLNPHEILET HRPRGLYGLY LYSASPLEPR TRPTYRGLNC YSASNGLNGL ILEHISTHRL 1500

EVALSERGLY LELEARGARG ASPASNILEL EPRGLASNLE ASPASPGLYL EPRSERGLNP 1560

HEVALTYRGL LYSASPASNT HRCYSASNAL APRILEPRVA LSERPHEPRV ALALAPRTHR 1620

ASPTHRLYSA SPPRPHELYS ALAILEILET HRLESERALA ARGLEASPTH RPHEALATHR 1680

ILEASNTHRL EPHELYSASP PRPRILEASN METGLYPRIL EPRALATHRT HRASPLETHR 1740

ASNMETASPA RGARGASPPR TYRMETPHEH ISGLNALAAS NLEARGASPG LNCYSASNTY 1800

RSERLEGLNT YRTHRILEGL YASNLYSASP GLNGLLYSAR GGLNARGASP GLNILEILEG 1860

LCYSARGASP THRLEVALIL EPRPRGLYSE RARGASPTHR SERLECYSPR METALAPRPR 1920

ASNSERPHEM ETSERTHRLE PRMETTHRAL AASPPHEARG ASPTHRTHRG LYPHEILEGL 1980

THRASPPRLE LYSASPVALH ISGLYPHEAL ATHRARGASP VALVALGLAL APHEARGASP 2040

TYRALACYSP RTRPASNGLY GLYGLGLVAL SERLELYSGL ALAALASERA LAALALEALA 2100

ALAGLYTYRL YSGLALAGLY LEVALPRPHE GLNVALSERP RTHRTHRLYS GLGLTYRASP 2160

GLGLYLEARG METVALASNL YSGLYLEALA LYSGLGLYAS NGLSERVALG LNVALPRARG 2220

ASNHISALAL ESERSERASP ARGGLHISHI SGLLEALAIL EALASERLYS GLILEASNGL 2280

NILEGLNARG GLILESERPH EASNGLNALA TRPLEARGGL LESERALATH RVALMETASP 2340

HISLELESER GLNARGGLLE VALLEVALLE GLYARGGLPR GLYALAGLGL YVALCYSGLT 2400

HRTHRPRGLY VALLYSGLPR GLYILECYSG LTHRTHRPRG LYVALLYSGL ARGGLLEASP 2460

SERARGGLSE RGLPHEPHEI LEARGPHEAL ALESERTHRT RPALAARGPH EALAASNGLN 2520

METPRASNGL YCYSGLNASP LEILESERTH RCYSLYSPHE ALASERASPA SPALACYSGL 2580

GLLYSPHEGL GLILEALAPR TYRVALASNG LYLYSARGPH EGLGLYTYRL EPRASPALAA 2640

RGPHEGLASN SERASNVALL YSPHEGLASN SERASNVALL YSSERSERVA LVALARGPHE 2700

GLYLYSPRVA LGLYALAVAL GLYSERALAA LATHRALALE LYSPHEGLYT RPTRPSERAL 2760

AASPGLYALA TRPPRGLYAL ALEASPASPP HEVALVALTR PVALGLNLYS LYSPHEHISA 2820

SPSERSERAS NASPSERGLY ASNARGPHEH ISVALLETHR ALAGLNLESE RPHEPRARGP 2880

HELEASPGLA LALETHRTYR PRPRPRLYSP HEASNSERLE ALAASPARGP HEGLNTYRPR 2940

GLYASPLEPH EASPGLNGLY THRTHRILEA RGPHESERSE RCYSSERGLY THRARGPHES 3000

ERTHRVALAL AGLYSERARG PHESERVALA LAGLILELEP RGLYALALYS PHEVALGLYG 3060

LYALASERTH RASPALAPHE ALAASPPRLY SPHEVALTHR ASPASNGLYA SPSERLYSGL 3120

YALAASPARG GLYARGGLYA SPALAGLYSE RPRVALPHES ERPRASPSER LYSGLYPHEP 3180

HETHRALAPR GLYARGGLYG LYGLYGLYGL YTHRPHEGLY VALVALMETG LSERTHRHIS 3240

ARGGLYGLYS ERGLYALALE GLYLEALAPH ESERGLALAL YSGLYILEAS PVALALALYS 3300

PRTHRGLYAR GGLYILEGLN ILEASNASPP RSERILEASN ASPASPSERV ALMETILETY 3360

RALAPRALAV ALARGGLYLE ARGASNSERG LYVALHISGL YTHRPHESER SERARGPRGL 3420

GLNGLGLILE GLNLYSGLYL ETYRALAGLY HISARGGLYP RLEASNGLGL YGLYLETYRA 3480

LAGLARGGLY GLNTHRPRLE PRILELEVAL ALAASPGLYA RGGLYSERAS PCYSSERTHR 3540

THRALAGLYG LYCYSCYSGL YGLYTHRGLY CYSGLNPRAS NGLTHRLEVA LPHEGLYSER 3600

SERASPLEAL AARGGLYVAL ASPPHETHRG LASPPRLELE GLNGLYARGG LYVALGLYSE 3660

RASPALATRP THRVALSERG LSERGLYARG GLYVALLEAR GPRVALSERT HRGLYSERAR 3720

GGLYVALTYR ASPILEARGG LYTYRLYSPR SERALASERS ERGLYSERLY SGLYTYRPRT 3780

HRSERGLNGL NASNTRPVAL GLYTHRLELE LEPRARGHIS ALAGLYGLNC YSGLYGLTYR 3840

HISGLASNLY SHISPHEGLN LEILEASNTH RALAALATYR TRPLYSHISP HETHRSERLE 3900

GLGLLYSHIS PHEVALASPT HRPHEGLYLE HISGLYHISL YSHISGLYGL YPRASNPHEG 3960

LGLNLEPRIL EASNGLNPRA RGHISLEPHE GLYLEHISAR GHISTHRASP TYRSERSERG 4020

LNGLSERTHR SERTYRLYSH ISVALASPGL YPHEGLYILE HISTHRPHEA RGHISVALTY 4080

RASPALAVAL GLNASPLYSI LEALAPHEAL ASERTYRLEG LGLTYRALAA RGILEALAPR 4140

GLNPHEGLYA SPLELYSILE ALASERLEAS NASPSERTYR GLTHRLELYS LYSARGILEP 4200

HEARGLELEG LYTHRPRASP GLASPSERTR PPRGLYVALT HRSERPHEPR ASPTYRLYSI 4260

LELEPHEASP SERASNASNV ALALATHRGL YVALGLNVAL SERTHRGLYG LYTHRPHEGL 4320

YTHRARGILE ASNPRHISGL LESERILEAR GASPPRASPP HETYRASNGL ILETYRVALT 4380

HRGLSERLYS ARGILEGLNA LAPHEVALIL ETYRPRGLAS NPHEASPLYS ILESERALAT 4440

YRVALGLGLY SERSERARGI LESERHISHI SALAGLNTHR LELEGLGLYL EGLYTHRHIS 4500

ARGTYRLEGL SERCYSTHRG LARGILESER TYRLYSGLPR GLYILECYSG LTHRTHRPRG 4560

LYVALLYSIL EVALPRGLGL TYRVALPRIL ETHRLYSILE VALGLNVALG LYASPLEARG 4620

ILEVALTHRP RASPGLYLYS ILETYRASPS ERILETYRVA LARGLYSALA LYSGLSERSE 4680

RSERGLSERS ERASPSERSE RGLSERGLSE RGLSERGLSE RGLASPGLLY SLYSPHETHR 4740

ASPTHRPRVA LLETYRGLYP RLYSLYSMET GLASPASPLE ARGLYSPRLE GLYTHRGLYT 4800

HRASPLETRP PRLYSLYSTH RLELEPHETY RALASERSER HISGLALAIL ESERPHEASP 4860

SERCYSARGL YSTHRSERSE RSERTHRALA THRSERTHRS ERTHRSERTH RGLYALAALA 4920

ALALEPRTHR ALAALAPHEG LYALAVALGL GLYGLYLEME TLEGLYVALV ALLEGLYVAL 4980

LEGLYLELEA SPARGPRPRV ALILEPRLEP RPRSERASPS ERASPVALTH RALAPHEARG 5040

LEGLGLLEAS NGLNARGLEG LGLNTYRARG METGLNLESE RGLYLELESE RGLNASNGLY 5100

GLNGLYSERL YSLEPHESER ILEPRALAAS PALAGLYASP ASPTYRLYSP RLYSLEPHES 5160

ERTYRLEASP THRGLNLEAS NARGLEPHET YRASNSERLE THRPRALAGL GLNGLNPHEV 5220

ALVALASPAL AILEARGLEL YSASPLEVAL LESERLELEA SNLESERSER PHEASPALAS 5280

ERGLYTYRIL EASPARGLEA SNTHRGLYAL AVALILEPRV ALLEVALARG LEPRASPILE 5340

CYSASNTHRC YSPHELYSLE SERGLNLEGL SERGLYLYSL ESERSERILE ALALEPRARG 5400

LETHRASPLE GLILEASNAR GLETHRGLYA SNLEGLYGLY GLASPTYRGL NASPLYSMET 5460

PRMETPRILE LEVALALAAS PGLYARGASN ALAGLYILEG LNTHRSERAR GASNALAHIS 5520

GLYGLNGLIL ELELEARGAS NALALEGLNT HRMETTYRAS PTHRGLNASP LYSASNASPP 5580

RVALALAVAL PHEASPGLYS ERVALILEPR LYSASNPHEA SPASNASPGL NHISARGASN 5640

PHEGLNGLLE PHEGLYILEL YSASNGLYAS PGLNSERPRP RSERALALEG LYPRLEPRSE 5700

RVALILEGLA RGASNHISAS NVALLESERA LAILEPRGLN GLPRTYRARG ASNHISTHRA 5760

LAGLYILEGL ALAARGASNL EGLTHRGLAR GGLNLEARGA SNLEGLVALL ESERLETHRL 5820

YSASNLEVAL GLYTHRSERG LYPHETHRSE RALAARGASN ASNVALILEI LEGLNLEASN 5880

ARGASNPRAS PLESERSERT HRSERASPTH RTHRASPVAL ILEARGASNS ERILELEGLG 5940

LYPRASPVAL LYSASNSERV ALVALGLYIL ELYSPRTHRV ALGLYLETHR SERARGASNV 6000

ALGLYLEVAL SERVALSERL EASPGLYLYS ASNVALLEAS PTYRGLYALA ARGASNVALV 6060

ALLEASPTHR THRALALESE RALAASNTHR LYSASNTRPH ISARGLESER PHETHRTYRA 6120

SNCYSTHRPR SERALAASNT YRGLNTYRIL EPRALATYRL YSASNTYRPH EALAGLTHRG 6180

LGLNVALMET PHEGLNPRGL YHISILEVAL ARGASNTYRI LEVALVALAS PALAASPSER 6240

SERPRLEGLN ILEVALILEA SPGLYPHEAR GPRPRMETGL ASPILEVALS ERPRTHRLEG 6300

LASPLEILEH ISLEALALEA RGARGPRPRG LNPRASPGLY ALATHRCYSI LELYSGLYAS 6360

NGLCYSGLAL APHECYSVAL ASPGLYVALC YSLYSPRGLN GLYASPPRLY SPRGLNSERI 6420

LEASPTHRIL EVALGLYTHR ASNLEHISME TASPILELES ERASPLEALA ALAALALEAL 6480

AGLYSERILE GLYVALALAP RSERSERASN LEASPPRTHR ARGLYSPRVA LALAASPALA 6540

ALAVALVALA SNALACYSGL SERPHEPRLE SERPHEASPT HRASPVALSE RARGARGPRT 6600

RPVALGLYGL YGLNILEVAL ASNSERILEP RALASERVAL GLLYSILEAL AVALLEGLGL 6660

NVALARGGLN ASPGLYHISP HESERVALPR SERTYRALAG LYHISVALAL ATHRMETTHR 6720

SERVALSERL EARGARGGLN ASPLEPHEGL ALAILEGLAL AGLYARGGLN ILEGLNTHRS 6780

ERARGGLNLE PRPRSERLEP RTYRTHRPHE TYRTHRSERT YRTHRSERGL ASPSERTYRL 6840

YSLYSGLNLE SERGLASPGL YVALASPVAL VALVALVALA LAGLARGARG ASPGLGLHIS 6900

GLNLEASPAS PASPPHEGLG LYILEASPAS PGLMETASPG LSERGLNSER ARGARGPRAS 6960

PLESERTHRP HEPHEALATH RLESERGLIL ESERPRASPG LALAARGARG PRPHEILEIL 7020

EALAARGARG PRGLYASPAR GSERHISSER ARGSERALAA LAALALESER THRSERGLLY 7080

SSERALAALA ALALESERTH RSERGLLYSA SPTRPLEGLN VALARGSERA LAALAASPGL 7140

YLEALASERA LAILETHRSE RLYSSERALA GLYTYRTHRP RLELYSSERA LAMETTHRLE 7200

PRARGSERAL ATHRVALMET THRTYRTRPL YSTHRGLGLG LYALAPRLEP RGLYCYSPRA 7260

RGTHRTHRLE GLNARGSERA SPASPALAIL ETHRARGTHR ASNPRASPAL ALELYSALAS 7320

ERALAVALPR GLYALASERA LALYSSERGL YASNTYRSER TYRGLYVALA RGSERGLYSE 7380

RSERPHESER SERILEGLYS ERALAILESE RMETSERLYS SERGLYSERV ALALALEGLY 7440

LYSSERHISL ESERVALVAL ASPGLYGLYG LASPGLYGLN ASNILEPRLE HISPRLEILE 7500

GLNPRGLARG SERILEALAV ALLYSALAPR SERPRTHRGL PRGLYSERPR ALASERPRGL 7560

YGLYSERGLN PRARGSERIL EILESERARG METLEVALTH RASPPRLYSS ERLEGLYASP 7620

VALCYSMETA SPLEGLNTHR ILETHRTHRS ERSERASPPR ASPPRLYSAR GSERASNILE 7680

THRGLILELE PRALAGLYTH RPRLEPRGLY THRALAALAT HRALAARGGL NASNPRASNP 7740

RALAALAALA ALASERTHRG LYGLYGLNAS PGLYGLYPRA SNASPALAVA LPRARGSERP 7800

RALAALAVAL GLNGLYILET YRGLYASNAR GSERSERGLL YSPRSERILE THRILEASPG 7860

LYASNASNIL EASNLYSSER SERGLYALAV ALTHRGLYGL NSERTHRARG SERSERGLYT 7920

HRGLYTHRSE RTHRGLYALA ALAALATHRG LYTHRGLTHR ASNALAALAS ERVALALALY 7980

SLEGLNMETG LYVALSERAL AALAGLYILE ALAGLYLEAL ALEGLYILET RPALALESER 8040

SERHISPRIL EGLVALPRVA LLYSSERTHR ASPTHRSERI LEASNVALAR GSERVALTHR 8100

SERGLYPHEV ALASPGLYIL ELYSSERVAL THRSERGLYP HEVALASPGL YILELYSASP 8160

GLYLEARGTH RALALEALAA SPTYRALALE CYSALAGLAL ATHRASNMET CYSARGTHRA 8220

LASERASNPH EASPGLNPRH ISSERASPGL SERALALEGL NHISLEARGT HRALAVALPR 8280

ILEASNGLYP RASPSERPRG LYTHRPRGLG LYVALLYSTH RASPTYRSER VALCYSGLYG 8340

LTHRTHRILE PHELYSTHRG LYTYRVALAS NTYRASNVAL ASPTHRTHRA SNLEARGTHR 8400

ILEPHEGLYT RPASPILEAL AGLGLYGLNL YSTHRILESE RASNVALVAL ASPASNGLLE 8460

ALAARGTHRI LEVALSERPR ASPGLYPHEA SNTRPASPTY RGLYSERTHR ARGTHRLEGL 8520

YILEASPILE ALAARGTHRL ESERTHRASN GLGLGLYTYR GLTHRSERAL AVALARGTHR 8580

METLEVALGL YMETASPVAL THRHISPRSE RPRGLYSERS ERALAASNAL APRSERVALA 8640

LAGLYMETVA LALASERVAL ASPSERTHRL ESERGLNTRP PRALAGLILE ARGVALGLNA 8700

RGTHRASNTH RGLNVALPRA SPALACYSTH RGLNCYSPHE GLNLYSTHRG LNGLYPRHIS 8760

SERTHRPHEA SPARGTHRSE RGLYSERGLY SERSERSERP RGLPRARGTH RTHRASPVAL 8820

GLYTHRPHEG LYGLNLYSTH RTHRGLYALA PHEASPGLSE RGLYPRPRLE SERGLNLYST 8880

HRTHRASNGL YILEVALSER THRASNGLSE RGLYARGTHR THRSERGLNT RPASNVALLE 8940

ASPLELYSTH RTHRTHRLEA SPGLNGLYHI STYRGLNSER ARGTHRTHRT YRASNVALVA 9000

LALAGLNTHR LYSTHRVALA SNVALASNAS NLELYSVALA LALEVALTYR GLYASPARGV 9060

ALALATHRIL EGLYSERALA THRPHEALAA RGVALCYSAS NLEILEGLYL EMETGLYLEA 9120

RGVALASPPH ETYRASNASN LELYSVALGL LEGLNSERLY SVALGLARGT HRGLYTYRAL 9180

AALAPHEARG VALGLSERAL ASERALAASP LEILESERTH RILETHRLYS VALPHEASPA 9240

LAGLYHISTH RVALPRALAP HEGLNPRGLT HRMETPHEAR GVALPHEGLA LAGLYHISGL 9300

VALPRALATY RGLNPRGLTH RALATYRGLI LEPHEHISAR GVALGLYGLG LGLTHRPRAL 9360

ALEVALHISA SPLEASNTHR ALAMETARGV ALGLYPHELE ALASERVALG LTHRPRALAS 9420

ERILEGLALA ALASERGLLE SERLYSVALG LYGLYTHRLE ALATYRVALS ERVALGLILE 9480

GLLYSVALLY SVALGLYTHR ILEILETHRG LYASPPRLEA SPPRPRVALL ELYSVALILE 9540

PRLEGLNGLY CYSASPALAA SPGLTYRGLY ARGVALLELE HISPRLELET HRALAALAAL 9600

ALELEGLYAL ASERALAARG ALAGLNSERV ALVALGLYTH RPRPHEGLYP HEALASERGL 9660

YTHRTHRGLY GLYGLYASNA LAALAPRALA ALAPRLYSVA LASNGLYVAL GLTYRGLYGL 9720

THRARGVALG LNLEASPGLG LYLELYSARG VALSERILET RPTHRGLSER TYRGLYGLYA 9780

RGVALSERAS NASPLEALAA RGVALSERGL NILESERGLY ASNARGPRLE ASPALALEAS 9840

PGLNGLYTHR ARGVALSERT YRTHRGLTYR ASPSERTYRT YRASPHISTY RASNLYSVAL 9900

THRASNSERP RSERASNLEV ALTRPTYRSE RILESERTHR ARGVALVALA LAVALASPTH 9960

RALASERASN LYSVALVALA SNTYRTYRSE RASPASPPRT HRGLYMETSE RASPSERGLY 10020

GLASPALAPH EASPMETARG LYSVALTYRA LATHRPRASP GLNASPILEG LHISGLYARG 10080

TRPASNGLTH RILETYRVAL ILEILETHRS ERPHESERAS PTHRLETHRI LEGLNPRTYR 10140

ASPTRPASNG LPHEARGLYS TRPASNPHEI LEMETASNSE RARGTRPARG HISTYRTYRL 10200

EARGTYRALA GLYGLTYRGL PHEGLNALAA SPLEPHELYS TYRCYSALAS ERALAGLNGL 10260

ASPASNALAT HRLEGLNALA LELEARGTYR ASPLEASNLE GLASNLYSTY RGLYPRSERP 10320

HETHRALAPH EPHEGLNGLG LNASNGLLYS TYRLYSGLPR GLYALAGLGL YVALCYSGLT 10380

HRTHRPRGLY VALLYSTYRL EALASERTHR GLNMETGLPR THRASPALAA RGTYRLEASP 10440

GLNGLNILET HRALAGLTHR LYSTYRLEAS PTHRLEPRGL ILELYSTYRL ETHRASNSER 10500

GLNALALEAL AASPLEPRTY RPHEALAGLL YSTYRLEVAL ASPGLNLEAS NPRGLGLYLY 10560

STYRGLNGLY ALASERGLNC YSPRPHEARG TYRGLNPRHI STHRVALTHR THRVALSERA 10620

LAGLYALASE RASPPRARGG LYSERPRGLG LYGLYGLYAR GTYRVALASP ALAGLYGLYP 10680

HEGLPRSERI LELYSTYRVA LTHRSERASN ALAVALSERV ALGLYVALTH RHISPHEALA 10740

GLYSERARGA LAALAALALE ALAGLLEVAL TRPSERGLYA SNARGALAAL AALAPRLYSS 10800

ERALAALALE ASPALALEGL NGLNSERILE TYRLEGLNPR LYSALAALAT HRTYRCYSPR 10860

GLASNILEGL LYSALAGLAS PTYRLELEAS NPRSERPRLY SALAGLHISC YSPHEASPTY 10920

RASPLESERT YRLYSPRALA ASPLYSALAG LASNGLNALA VALALAVALG LYARGALAGL 10980

YALAVALALA ALAVALVALT YRASNASNGL LYSALAGLYL YSPRTHRLEG LYPHELEASN 11040

PRLELETYRS ERGLYALALE LYSALAGLYS ERSERPRTHR ASPILEILES ERGLYILESE 11100

RASPLYSALA ILEHISASPG LVALSERPRV ALGLYASPTH RASPALALEL EGLARGALAI 11160

LEMETGLYAL AGLGLALAAL ALYSALALES ERGLMETILE LEGLNSERGL LYSALALEVA 11220

LGLGLYSERT HRPHEALALY SALALETYRS ERSERALAAL ATHRGLYTHR TYRALASERS 11280

ERTHRTHRVA LTYRLYSALA ASNGLGLNPR THRTRPVALT YRARGALAAS NPHEGLVALG 11340

LTHRPRARGA LAASNASNTY RCYSSERASN GLNVALGLGL YPRTYRSERL ETYRSERGLY 11400

ARGALAPRVA LVALGLNTYR ALALEASNAR GALAGLNASN ASPPRASNAL APHEGLYVAL 11460

VALALAALAA RGALASERAL AILEGLNLEA SPGLYILEIL ETYRARGALA SERMETVALT 11520

RPGLGLALAG LNGLNVALSE RGLYLYSALA SERASNSERL EGLNTYRVAL ASNVALGLNV 11580

ALLYSALATH RGLYASPVAL LEPHEASNTH RLYSALAVAL GLYGLNALAT HRGLARGALA 11640

VALHISGLAS PLEASPVALA LAALAILEAS PALAALAGLV ALARGALAVA LLELELEASP 11700

GLALAASPVA LPHEMETGLG LARGALAVAL SERPRSERPH EGLASPVALT RPSERGLNPR 11760

ARGCYSGLNS ERVALPHEAS NPRASNILEP RLYSASPALA TYRSERPRHI SGLILETYRS 11820

ERARGASPPH ETHRASPILE THRALAGLYS ERSERILEGL YCYSASPGLY VALASNPRGL 11880

NTHRGLYLYS ASPGLYLEGL GLYSERPHEL YSASPLYSAS PPRGLLYSAS PPRLYSALAI 11940

LEGLLEPRAR GASPGLNILE ILEGLCYSAR GASPSERGLY LEVALMETLY SASPSERPRL 12000

ETYRPRTYRA RGASPVALHI SGLYPHEALA THRARGASPV ALLYSSERME TLYSASPVAL 12060

VALVALVALG LYGLYGLYAL ASERGLYALA TYRALAALAV ALARGASPTY RGLNVALGLM 12120

ETVALASNLY SGLALAGLYL EVALPRPHEG LNVALSERPR THRTHRLYSG LGLPRSERPH 12180

EGLNPRASPA SPVALTHRLE LELESERGLN ASPPRGLYHI STRPGLLYSG LGLYILESER 12240

ILEHISTHRC YSASPGLNAR GGLHISHISG LLEALAILEA LASERLYSGL ILEPRVALGL 12300

YTYRSERALA ALAASPILEA SPTHRASNAR GGLLEASPTH RGLNHISILE HISPRPRASP 12360

SERTYRPHEV ALSERPRLET HRARGGLPRG LYILECYSGL THRTHRPRGL YVALLYSGLP 12420

RSERASNASP PRASNPRPRG LTHRTYRSER LYSGLSERLE GLASPILEAR GLYSGLTYRL 12480

EVALALAASN GLYVALGLNA LAGLNALALE VALPRLYSPH EGLPRPRALA VALTYRASNA 12540

SPGLLELYSP HEGLYALATH RGLYASPGLT YRARGPHEGL YLYSPRVALG LYALAVALGL 12600

YSERALAALA THRALALELY SPHELEASPG LALALETHRT YRPRPRPRLY SPHEASNVAL 12660

ASPGLTHRAL APHETHRGLY ALATRPGLYA RGPHEARGGL NASPLEILES ERGLILELYS 12720

PRCYSCYSGL GLLYSPHETH RALAVALPHE THRPRSERIL EVALGLARGP HETHRASPTH 12780

RPRVALLETY RGLYPRLYSP HEVALTHRAS PASNGLYASP SERLYSPHEV ALTHRASNME 12840

TGLNALAALA LELELYSGLY PHEPRASPVA LALAALAHIS SERLETHRPR ARGGLYGLYS 12900

ERILELEPRM ETGLNGLVAL ALALETHRTH RARGGLYILE ASPVALALAL YSPRTHRGLY 12960

ARGGLYILEM ETLEASPTHR GLYARGGLYL YSGLSERCYS LYSGLYLETY RALAGLYHIS 13020

ARGGLYMETV ALPHESERIL EASPALAGLN GLYGLLYSGL YPRALAARGA RGARGGLYGL 13080

NLEGLYPHET RPGLYASNLY SGLYSERILE VALGLYPRAR GTRPLYSLEP RPHEMETGLY 13140

PRPHELEGLN SERVALASNP RLYSGLYTHR VALPHEPRSE RGLTHRGLGL YGLSERMETA 13200

LASERARGGL YVALASPPHE THRGLASPPR LELEGLNGLY ARGGLYVALL YSILESERGL 13260

YTRPASPVAL GLTHRLEGLY ASPGLILETH RHISVALGLY GLLYSPHETH RLYSGLYTYR 13320

LYSPRSERAL ASERSERGLY SERLYSHISA LAGLYGLNCY SGLYGLTYRH ISGLASNLYS 13380

HISPHETHRS ERLEGLGLLY SHISPHEVAL ASPTHRPHEG LYLEHISGLY HISLYSHISG 13440

LYGLYPRASN PHEGLGLNLE PRILEASNGL NPRARGHISG LYILEPRGLY GLYGLYILEA 13500

LATHRGLYAL AGLGLYILEL YSHISMETPH EGLYLEVALA LASERGLASP ALAGLYARGH 13560

ISPRVALGLV ALALAGLGLG LALASERLYS PRLYSHISVA LASPGLYPHE GLYILEHIST 13620

HRPHEARGHI SVALGLNLEL EGLNLEASNM ETGLTYRASP ASPASPILEL ECYSARGLYS 13680

SERLYSHISV ALTYRASPAL AVALGLNASP LYSILEALAP HEALASERTY RLEGLGLTYR 13740

ALAARGILEA SPALATHRTH RASNPRGLYM ETARGILEAS PTYRILEGLY GLYGLYASPL 13800

EPHEARGILE GLILEGLASN SERILEARGI LEGLASNGLN SERASPALAA SPGLYTYRSE 13860

RSERCYSSER THRLELYSIL EPHEGLGLNL EGLGLYMETS ERLESERLYS ILEPHESERT 13920

YRLYSMETAS NSERTHRLEA RGTYRLEPRP HEARGILEGL YLEHISPHEA RGTHRARGIL 13980

EHISLETHRV ALPRGLASPL EARGILEGLN ASPGLYSERG LNVALLYSIL EGLNGLYILE 14040

SERASNPRSE RGLYALALES ERSERGLYGL YLEGLYGLPR LYSILEGLNS ERLYSLEARG 14100

GLYLEVALGL NARGILEARG ASPALAMETA RGGLNARGIL EARGLEHISL EGLARGTHRG 14160

LYGLNLEGLY VALGLYSERA SPGLYASNPR VALVALALAG LYARGILESE RALATYRVAL 14220

GLGLYSERSE RARGILEVAL PRGLGLTYRV ALPRILETHR LYSILETYRS ERPHEPHEVA 14280

LGLYGLYALA VALPRGLASN LEARGILETY RVALTHRGLY GLSERTYRAL AGLYARGLYS 14340

ASPILEARGH ISGLYHISLY SLYSGLHISA SPLYSSERLY SPHETHRASP THRPRVALLE 14400

TYRGLYPRLY SLYSGLYASP ALAPRTHRIL EASPTHRSER ASNTYRPHEL EPHEGLYLYS 14460

LYSMETGLAS PASPLEARGL YSTYRTHRVA LPRSERTHRC YSGLYVALLY SLEGLMETTY 14520

RGLNGLYGLY ILEGLLESER ALALELEGLN METILEGLNA SPALAILEAR GLEPHESERT 14580

YRLEASPTHR GLNLEASNAR GLEGLYILET HRTYRTHRTH RTYRSERLYS LELYSASPLE 14640

VALLESERLE LEASNALALE GLNGLYGLYA RGLEASNTHR GLYALAVALI LEPRVALLEV 14700

ALARGLEASN VALILEASPP HEPRLYSLEG LNVALARGAL AALAALAARG ARGLESERAL 14760

AGLYSERARG LESERGLLEG LTRPILEARG LESERGLNLE GLSERGLYLY SLESERSERI 14820

LEALALEPRA RGLETHRASP LEGLILEASN ARGLEVALAL AHISSERVAL ALATHRTYRA 14880

LAARGLEVAL CYSPHEPHEP RTHRLYSLEV ALGLNASNAS PPHEASNTHR LELEARGMET 14940

ALAPRMETSE RGLGLASPLE ALATRPPHEA RGSERTHRPH EHISPRILEP RLYSMETGLY 15000

SERLESERAS PVALARGMET LYSSERILEG LGLLYSGLYG LGLYMETTHR ASNASPTYRI 15060

LESERALALE THRLYSASNA LAPHEILETH RASNTYRPRS ERGLGLNARG ASNALAGLYI 15120

LEGLNTHRSE RARGASNPHE SERARGPRLY SASNHISGLY THRSERTHRV ALALAPRGLN 15180

VALGLNALAS ERVALTYRAR GASNHISASN VALLESERAL AILEPRGLNG LPRTYRARGA 15240

SNILEASNME TLELETYRGL YTHRASPASP CYSSERGLYL YSASNLEASP GLLETRPILE 15300

VALGLYHISG LYALAVALAL AARGASNLEV ALGLYTHRSE RGLYPHETHR SERALAARGA 15360

SNMETHISAS PVALILEGLY ASNASPGLYT HRVALPRSER GLPHEARGAS NASNVALILE 15420

ILEGLNLEAS NARGASNSER METTHRASPC YSCYSILEGL THRTYRLEME TLYSSERGLA 15480

RGASNTHRLE ALAPHEPHES ERGLYASNGL VALILEASNA SPGLYPRSER SERLYSASNT 15540

HRPRVALPRS ERCYSPHEHI SPHEPHEILE TYRLYSGLYC YSTRPMETPH ELETYRARGA 15600

SNTYRPHEAL AGLTHRGLGL NVALMETPHE GLNPRGLYHI SILEVALARG PRASPGLYTH 15660

RGLYPHEARG LYSPRLETRP ARGHISTYRP HEGLNASNTH RGLNGLYILE ILEPHEVALV 15720

ALASPSERAS NASPARGPRV ALVALGLNVA LLEMETPRGL GLYMETASPS ERASPGLSER 15780

GLNALAILEL EASNASNILE GLYALAASPG LYGLNSERAL AGLNGLYALA SERPRGLYVA 15840

LVALILEALA SERPRSERLY SGLNASPLEP HEGLALAILE GLALAGLYAR GGLNTHRTYR 15900

ALASERCYST RPGLYGLYVA LGLYGLNGLY GLCYSARGGL YSERSERASN CYSLYSARGC 15960

YSTRPSERGL YVALPHETYR SERASNTRPI LEGLNGLLEL EARGARGSER METGLYLEGL 16020

SERARGSERA LAALAASPGL YLEALASERA LAILETHRSE RLYSSERALA ILESERGLNT 16080

YRGLYASPSE RPHEALALYS SERALAMETT HRLEPRARGS ERALAVALGL NSERASPVAL 16140

TRPARGSERA SPGLYGLNCY SSERASPLEL ELYSSERASP LYSLEASNVA LILEASPPHE 16200

PRLYSSERAS PTYRASPALA PHEILEARGS ERASPTYRGL NGLCYSALAA SPALAPRGLY 16260

GLNLYSSERA SPTYRSERAL ALEGLNSERG LNGLYLEILE LESERLEARG SERGLMETLE 16320

ALAGLGLNAS PLYSSERGLS ERASNPRGLY VALMETSERT HRARGSERGL YALAASPTHR 16380

HISLYSSERI LEVALILEAR GSERILETYR ALAILEASNS ERGLYARGSE RLEPRLEILE 16440

VALGLYASNS ERASPGLNGL GLYLYSSERG LNSERASPPH EGLSERGLPH ESERTHRALA 16500

LYSSERSERG LYALAVALTH RGLYGLNSER THRARGSERS ERSERALATY RGLSERLETH 16560

RSERALAVAL LYSSERTHRA SPTHRSERIL EASNVALARG SERVALVALG LASNASNASN 16620

ASPGLYLETH RALAALATYR ARGTHRALAL EPHEASPSER HISGLTYRAR GTHRALASER 16680

ASNPHEASPG LNPRHISSER ASPGLSERAL ALEGLNHISL EARGTHRCYS HISARGCYSC 16740

YSTHRTHRPH EALAPRASPA LATHRGLCYS GLASNCYSLY SHISTHRARG THRASPTYRS 16800

ERVALCYSGL YGLTHRTHRI LEPHELYSTH RGLYGLTHRT HRGLNILEHI SALAARGTHR 16860

GLYPRSERIL EGLNASPARG THRILESERA SNVALVALAS PASNGLLEAL AARGTHRLYS 16920

SERLEPRARG THRLEPRPRL EGLNTYRARG ASPLEASPLE LEPRLEHISG LNASNLEILE 16980

LYSTHRLEVA LSERTHRGLY ARGTHRPRAL AALAHISARG ALAARGTHRS ERGLYSERGL 17040

YSERSERSER PRGLPRARGT HRTHRASPVA LGLYTHRPHE GLYGLNLYST HRTHRGLMET 17100

THRGLNARGT HRTHRSERAS NPRGLTHRAR GTHRVALGLY SERSERCYSP RTYRCYSASP 17160

SERGLNALAP RGLNVALARG THRVALASNG LYGLYPHEGL NILEALAARG THRVALASNV 17220

ALASNASNLE LYSTHRVALT YRALAPHEAS PVALSERGLA SPGLYSERTY RLELYSTHRT 17280

YRGLVALVAL GLYASNVALT YRLYSVALAL ALEVALTYRG LYASPARGVA LALAPRASNS 17340

ERGLYALATY RLEASNGLAL AASPPHEARG VALALASERL ELEGLNARGV ALALATHRIL 17400

EGLYSERALA THRPHEALAA RGVALCYSAS NLEILEGLYL EMETGLYLEA RGVALASPAS 17460

NVALVALALA SERPHELYSV ALGLTYRSER ASPALAALAL YSVALPHEGL ALAGLYHISG 17520

LVALPRALAT YRGLNPRGLT HRALATYRGL ILEPHEHISA RGVALGLYSE RILEGLPHET 17580

HRALALEPRG LNLEGLNSER LEASPPHETH RLYSVALILE PRGLILEASP METPRSERHI 17640

SSERSERSER GLYTRPLYSV ALLEASPARG ASPPRASNHI SALALYSVAL LEPHELEGLY 17700

ARGVALLEIL EALAASPMET CYSARGARGV ALLEPRGLNV ALILEGLALA THRASNARGV 17760

ALGLNASNGL YALAVALTHR TRPGLSERAS PPRASNARGV ALGLNASNGL YALAVALTHR 17820

TRPGLSERAS PPRASNARGL YSVALSERAS NASPLEALAA RGVALTHRAL AMETARGTYR 17880

TRPTRPLECY SGLILEALAT YRCYSPHEAL ASERVALGLY GLYLYSVALV ALTHRASPSE 17940

RPHEARGVAL TYRSERVALA SPASNSERLY STRPASPASN LEASPSERAL AALALEASNT 18000

HRLYSTRPPH EALAGLASPP RSERARGTYR CYSALASERA LAGLNGLASP ASNALATHRL 18060

EGLNALALEL EARGTYRCYS GLYVALGLYV ALASNILELE TYRGLARGTY RGLALAALAI 18120

LEGLNGLYVA LALAALATHR ASPLYSTYRP HETYRGLYAS PASNTYRALA THRLEARGTY 18180

RGLYALATYR SERVALCYSS ERPRLYSTYR GLYGLTHRGL LYSSERGLYL EGLSERILEA 18240

LAALAALAAR GTYRILEALA ARGPRASPIL EMETLYSTYR LEASPGLNGL NILETHRALA 18300

GLTHRLYSTY RLEVALASPG LNLEASNPRG LGLYLYSTYR GLNPHEPRGL NTHRPRSERA 18360

RGTYRARGHI SLEPRPRGLT HRVALTHRGL YILELEGLYA RGALATHRPH ETRPTRPILE 18420

ASNSERILEL ELYSTYRTHR ALAGLGLYTY RGLALAALAT HRLYSTYRVA LASPALAGLY 18480

GLYPHEGLPR SERILELYS 18499

Claims

1. Aflatoxin pollutes early risk early warning intelligent perception card, its characterized in that: the aflatoxin early warning molecular kit comprises an aflatoxin early warning molecular nano antibody or an aflatoxin early warning molecular monoclonal antibody marked by a signal material, a sample pad, a nitrocellulose membrane, a water absorbing pad, a bottom plate and a goat anti-rabbit antibody, wherein one surface of the bottom plate is sequentially stuck with the water absorbing pad, a detection pad and the sample pad from top to bottom, adjacent pads are overlapped and connected at a joint, the detection pad takes the nitrocellulose membrane as a base pad, a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with the goat anti-rabbit antibody, the detection line is coated with the aflatoxin early warning molecular nano antibody or the aflatoxin early warning molecular monoclonal antibody, the aflatoxin early warning molecular rabbit antibody marked by the signal material is loaded on the sample pad or is independently loaded, and the aflatoxin early warning molecule refers to AFT-YJFZP008 peptide, and the amino acid sequence of the aflatoxin early warning molecule is shown as SEQ ID NO. 1.

2. The smart sensor card of claim 1, wherein: the signal material refers to conventional europium latex microspheres or gold nanoparticles.

3. The smart sensor card of claim 1, wherein: the signal material marked aflatoxin early-warning molecular rabbit polyclonal antibody is a product obtained after the signal material is coupled with the aflatoxin early-warning molecular rabbit polyclonal antibody by a conventional marking method.

4. The application of the aflatoxin pollution early risk early warning intelligent perception card as claimed in claim 1, which is characterized in that: the method is used for rapidly sensing the aflatoxin production capacity of the aspergillus strain, or identifying whether a strong virulent aflatoxin toxigenic strain exists in a sample, or intelligently sensing the aflatoxin pollution risk level in agricultural products.

5. The use according to claim 4, characterized in that: the application method comprises the following steps: culturing and preparing a strain to be identified or a sample to be identified to be tested liquid by using a conventional Chlamydia cell culture medium, and then determining the content of aflatoxin early warning molecules AFT-YJFZP008 by using an aflatoxin pollution early risk early warning intelligent perception card, wherein the specific method comprises the following steps: when the sample pad is loaded with the aflatoxin early-warning molecular rabbit polyclonal antibody marked by the signal material, the strain to be authenticated or the liquid to be tested of the sample to be authenticated or the liquid to be tested of the agricultural product sample is added to the sample pad of the aflatoxin pollution early-warning intelligent perception card, and the reaction is carried out for a period of time, and the analysis result is read;

When the aflatoxin early warning molecular rabbit polyclonal antibody marked by the signal material is not loaded on the sample pad, the strain to be authenticated or the liquid to be tested of the sample to be authenticated or the liquid to be tested of the agricultural product sample is added into the aflatoxin early warning molecular rabbit polyclonal antibody marked by the signal material, then the aflatoxin pollution early risk early warning intelligent perception card is inserted into the strain to be authenticated or the liquid to be tested of the sample to be authenticated, the reaction is carried out for a period of time, and the analysis result is read.

6. The use according to claim 4, characterized in that: the application method further comprises the following steps: judging the detection result: according to the measurement result of the aflatoxin pollution early risk early warning intelligent perception card, the content of aflatoxin early warning molecules AFT-YJFZP008 in a unit volume of strain to be measured is obtained, the aflatoxin production power of the strain to be identified is analyzed, and the higher the concentration of the aflatoxin early warning molecules AFT-YJFZP008 in the strain to be identified is, the stronger the aflatoxin production capacity, namely the production power of the strain to be identified is;

obtaining the content of aflatoxin early warning molecule AFT-YJFZP008 in a unit volume of to-be-identified sample to be detected liquid according to the measurement result of an aflatoxin pollution early risk early warning intelligent perception card, and judging whether the to-be-identified sample contains a strong virulence strain of aflatoxin or not;

According to the determination result of the early-risk early-warning intelligent perception card for aflatoxin pollution, the content of aflatoxin early-warning molecules AFT-YJFZP008 in the to-be-detected liquid of the agricultural product sample in unit volume is obtained, and the risk of aflatoxin pollution in the agricultural product is predicted.

7. The use according to claim 4, characterized in that: preparing a to-be-detected liquid of a strain to be identified: culturing and diluting the strain to be identified to obtain a solution to be detected of the strain to be identified; the method comprises the following steps: culturing the strain to be identified in a conventional Chlamydia medium or other culture mediums suitable for strain growth, wherein the culture environment temperature is 15-35 ℃, the culture time is not less than 12 h, fully homogenizing a mixture of the culture medium and the culture, and diluting 1-10 times by using sterile water to obtain a solution to be detected of the strain to be identified;

preparing sample liquid to be identified: culturing a sample to be identified to obtain a sample to be identified to be tested; the method comprises the following steps: weighing a sample to be identified, transferring the sample to sterile water, vibrating the sample to be identified to be uniform at room temperature, preparing a sample diluent to be identified, adding 10-1000 mu L of the sample diluent to a conventional Shake liquid culture medium containing 6-600 mL, placing the culture medium to be subjected to shake culture at 200+/-50 rpm at 15-35 ℃, and sampling after culturing 6-24 h to form the sample diluent to be identified.

8. The use according to claim 4, characterized in that: the sample is farmland, agricultural products, chinese medicinal materials or feed products.