Genetic engineering recombinant high-purity anti-mycotoxin monoclonal antibody for inhibiting and killing mycotoxin as well as composite monoclonal antibody preparation method and combined preparation thereof

A mycotoxin and genetic engineering technology, applied in antifungal agents, antibodies, antifungal/algae/lichen immunoglobulins, etc., can solve the problems of feed additives without mycotoxins, unstable treatment effects, and mycotoxin hazards. Achieve natural safety toxic side effects and residues, improve biological activity and consistency, and ensure food safety

Pending Publication Date: 2015-06-10
SHENZHEN JASON INTELLIGENT BIOTECH CO LIMLTED PRC
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  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 4. Oxidation treatment method: the treatment effect is unstable, and the nutrients such as vitamins in the feed are seriously lost
[0010] 5. Antioxidants: Aflatoxin poisoning cannot be completely eliminated, and the concentration of antioxidants such as BHT exceeds 30 times the conventional use to show a better detoxification effect; in this way, it will inevitably have a toxic effect on animals
[0011] 6. High-temperature treatment method: high energy consumption and great damage to the nutrients in the feed, so there are few practical applications
The ideal reaction conditions for detoxification enzymes are in vitro reaction systems with neutral pH value and high moisture content, which are suitable for vegetable oil, soy sauce, beer, milk, etc. It is difficult to directly apply them in low moisture feed and raw materials
[0014] In view of the fact that countries in the world have so far no ideal method to deal with mycotoxins in feed and food, there is no safe and effective feed additive for degrading mycotoxins, and there is no specific drug for the treatment of mycotoxin poisoning. Even the most prestigious [Mycotoxin antidote] produced in Germany only adopts the traditional dextran adsorption method; market feedback information shows that the existing methods have failed to solve the increasingly serious problem of mycotoxin pollution
Therefore, the harm of mycotoxins is becoming more and more serious; it has brought huge economic losses to the feed industry and animal husbandry production in my country and the world and damaged national health

Method used

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  • Genetic engineering recombinant high-purity anti-mycotoxin monoclonal antibody for inhibiting and killing mycotoxin as well as composite monoclonal antibody preparation method and combined preparation thereof
  • Genetic engineering recombinant high-purity anti-mycotoxin monoclonal antibody for inhibiting and killing mycotoxin as well as composite monoclonal antibody preparation method and combined preparation thereof
  • Genetic engineering recombinant high-purity anti-mycotoxin monoclonal antibody for inhibiting and killing mycotoxin as well as composite monoclonal antibody preparation method and combined preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0051] The preparation method of the genetically engineered recombinant high-purity anti-mycotoxin compound monoclonal antibody for inhibiting mycotoxins used in the present invention comprises the following steps:

[0052] 1. Screen representative mycotoxins

[0053] Mycotoxins mainly refer to the toxic metabolites produced by molds in the process of contaminating substrates. Due to the diversity of mold species, their toxic metabolites are also diverse. There are more than 200 known mycotoxins. Among them, the main mycotoxins that are most harmful to animals and humans are: aflatoxin (mainly AFB1), zearalenone, deoxynivalenol (deoxynivalenol), ochratoxin A, citrinin, and fumonisins B1, penicillin acid. Their characteristics and hazards are as follows:

[0054] (1) Mycotoxins: Aflatoxins are the most harmful and common mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus through the polyketide pathway, and their toxicity is much higher than that of cyanide...

experiment example 1

[0156] Antibody binding titer detection of multiple mycotoxin compound monoclonal antibody IgC to common mycotoxins

[0157] Detection of anti-multiple mycotoxin compound monoclonal antibody IgC against aflatoxin AFB1, zearalenone, deoxynivalenol, ochratoxin A, citrinin, fumonisin B1, penicillin, T Antibody binding titers of 8 antigens including -2 toxins:

[0158] Eight kinds of mycotoxins including aflatoxin AFB1, zearalenone, deoxynivalenol, ochratoxin A, citrinin, fumonisin B1, penicillic acid and T-2 toxin were used as the Detect the antigen, and use the "ELISA" method (enzyme-linked immunoassay) to detect the antibody titer of the prepared anti-multiple mycotoxin complex monoclonal antibody IgC soluble antibody protein solution, and the results are shown in the table below.

[0159]

[0160] Note: The concentration of the anti-multiple mycotoxin complex monoclonal antibody IgC soluble antibody protein solution in the test sample is 1mg / mL.

[0161] It can be seen fr...

experiment example 2

[0163] Anti-Multiple Mycotoxin Compound Monoclonal Antibody IgC Degradation Effect of Common Mycotoxins

[0164] 1. Materials

[0165] (1) Anti-multiple mycotoxin compound monoclonal antibody IgC soluble protein dry powder

[0166] (2) Standard mycotoxin working solution (pH 5.0)

[0167] Aflatoxin AFB1, zearalenone, deoxynivalenol, ochratoxin A, citrinin, fumonisin B1, penicillic acid, T-2 toxin and other 8 mycotoxin standard products were purchased from Singapore Pribo Laboratory . Prepare mycotoxin working solution with pH 5.0 phosphate buffer. The content of mycotoxins in the working solution is set according to the national limit standard of mycotoxins in feed and the actual production situation.

[0168] (3) Instruments and kits:

[0169] THZ-82 constant temperature water bath shaker; Bio-Rad iMarkTM microplate reader; SK-1 fast mixer; Kubota 2420 high-speed centrifuge; aflatoxin B1, zearalenone, deoxynivalenol, ochratoxin A , citrinin, fumonisin B1, penicillin, T-...

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Abstract

The invention relates to a genetic engineering recombinant high-purity anti-mycotoxin monoclonal antibody for inhibiting and killing mycotoxin as well as a composite monoclonal antibody preparation method and a combined preparation thereof. The preparation method comprises the following steps: screening mycotoxin; connecting the mycotoxin with vector carrier to prepare immune antigen; performing immune and cell fusion to prepare a hybridoma cell; performing gene cloning, constructing anti-mycotoxin monoclonal antibody Ig and expressing in engineering bacteria; fermenting and amplifying recombinant engineering bacteria, culturing to prepare bacteria of the anti-mycotoxin monoclonal antibody Ig; performing cell crushing, filtering and removing bacteria and viruses, centrifuging, purifying and performing chromatography to prepare anti-mycotoxin monoclonal antibody Ig and soluble antibody protein of composite monoclonal antibody IgC. The anti-mycotoxin monoclonal antibody and the composite monoclonal antibody IgC can be used as a grain mycotoxin remover, feed components and can be prepared into an animal drug; the anti-mycotoxin monoclonal antibody Ig, the composite monoclonal antibody IgC and the combined preparation thereof can be used for effectively degrading the mycotoxin in feed and food, preventing and treating mycotoxin toxic disorders.

Description

technical field [0001] The present invention relates to a new method for suppressing mycotoxins in food, feed and food, more specifically, to a method for preparing mycotoxin-inhibiting genetically engineered recombinant high-purity anti-mycotoxin monoclonal antibody and compound monoclonal antibody and its combination preparation. Background technique [0002] In recent years, mycotoxin poisoning has brought more and more harm to animal husbandry. According to a survey by the Food and Agriculture Organization of the United Nations, 25% of the world's traded grains are contaminated with mycotoxins. In my country, the proportion of feed and raw materials contaminated with mycotoxins exceeds the standard as high as 60% to 70%. According to statistics, more than 66% of feed materials are contaminated with at least one mycotoxin, 35% of feed materials are contaminated with more than two types of mycotoxins, almost all peanut meal is contaminated with mycotoxin, and corn meal, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14A61K39/395A61P31/10A23K1/18A23K1/16
Inventor 包晟张善钧曹天福汪玲包海威
Owner SHENZHEN JASON INTELLIGENT BIOTECH CO LIMLTED PRC
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