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Aspergillus flavus toxin-producing bacterium reference substance containing aflatoxin early-warning molecules and preparation method and application thereof

A kind of aflatoxin and reference substance technology, applied in the preparation method of peptides, chemical instruments and methods, fungi, etc., can solve the problems of deviation of test results and poor stability, and achieve long shelf life, good stability and practicality strong effect

Active Publication Date: 2021-10-08
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical applications, we found that the pure product of aflatoxin early warning molecule has poor stability when used as a standard substance, which may easily lead to the deviation of test results.

Method used

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  • Aspergillus flavus toxin-producing bacterium reference substance containing aflatoxin early-warning molecules and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of Aflatoxin Early Warning Molecule AFT-YJFZ01

[0048] 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. Randomly selected public literature "Research on the Distribution, Toxicity and Infection of Aspergillus flavus in Typical Peanut Production Areas in China" - Master's Degree Thesis of Chinese Academy of Agricultural Sciences, author Zhang Xing, page 33 - Published toxin-producing strains HLJ-1, HeNZY-2, HuBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-122-2, JSnt-1, HuNdx-7, HBHA-8-17 and other 10 strains were inoculated into the above observation After culturing at 28°C at 200rpm / min for 5 days, the cells were fully homogenized and broken by conventional methods, and then purified by conventional protein purification systems, protein electrophoresis, immunoaffinity and other ...

Embodiment 2

[0069] Example 2 Preparation of Aflatoxin Early Warning Molecule AFT-YJFZ01 Nanobody

[0070] 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.

[0071]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 venous blood of the immunized alpaca, extract total RNA, synt...

Embodiment 3

[0073] Example 3 Preparation of Aflatoxin Early Warning Molecule AFT-YJFZ01 Monoclonal Antibody

[0074] 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.

[0075] 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 cell...

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Abstract

The invention discloses an aspergillus flavus toxin-producing bacterium reference substance containing aflatoxin early-warning molecules and application of the aspergillus flavus toxin-producing bacterium reference substance. The aflatoxin early-warning molecule AFT-YJFZ01 is prepared from aspergillus flavus toxin-producing bacteria, the aflatoxin early-warning molecule AFT-YJFZ01 produces the aflatoxin early-warning molecule AFT-YJFZ01, and the amino acid sequence of the aflatoxin early-warning molecule AFT-YJFZ01 is shown as SEQ ID NO.1. The content of the AFT-YJFZ01 in the aspergillus flavus toxin-producing bacterium reference substance containing the aflatoxin early-warning molecules is calibrated through detection, and the aflatoxin toxin-producing bacterium reference substance containing the aflatoxin early-warning molecules is used as a reference substance. The aspergillus flavus toxin-producing bacterium reference substance can be used for identifying and comparing aflatoxin pollution risk levels of products such as farmland soil, agricultural products and feeds; and the stability is good, shelf life is long, operation is easy, practicality is high, and popularization and application are easy.

Description

technical field [0001] The invention relates to an aspergillus flavus toxin-producing bacteria reference material containing an aflatoxin early warning molecule, a preparation method and an application thereof. Background technique [0002] 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. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/38C07K1/14C12N1/14G01N33/577G01N33/569G01N33/58G01N33/558G01N33/543G01N33/68
CPCC07K14/38C12N1/14G01N33/577G01N33/56961G01N33/581G01N33/54393G01N33/558G01N33/582G01N33/6827G01N2333/38G01N2469/10
Inventor 李培武张奇毛劲白艺珍姜俊
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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