Mycotoxin magnetochemiluminescence immunoassay kit based on bifunctional fusion protein and application of mycotoxin magnetochemiluminescence immunoassay kit
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A mycotoxin and fusion protein technology, applied in enzyme fusion, analytical materials, immunoglobulin, etc., can solve problems such as low coupling efficiency, reduced sensitivity, and heterogeneous conjugates, and achieve simple expression, stable process, and process flow long effect
Active Publication Date: 2022-06-07
ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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At present, the preparation method of immunoenzyme labeling reagents is to use chemical labeling methods, such as using bifunctional reagents, glutaraldehyde, periodate, SMCC reagent {4 (N maleimidomethyl) cyclohexane 1 carboxylate succinate imide ester, etc.)}, 2IT reagent (iminothiophene hydrochloride), etc., to couple the antibody to the enzyme, but the chemical coupling method is complicated to operate and the coupling efficiency is low. Because the coupling site is not fixed and the conditions are severe , easily lead to a decrease in antibody or enzyme activity, the binding of antibody and enzyme is not uniform, it is necessary to separate and remove unbound enzyme and antibody, and the removal of unbound antibody is very important, because the free antibody will compete with the enzyme-labeled antibody for the corresponding Antigen, reducing the amount of enzyme-labeled antibody bound to the solid phase, thus reducing the sensitivity of detection
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Embodiment 1
[0044] Example 1 Preparation of aflatoxin B1 nanobody-alkaline phosphatase bifunctional fusion protein
[0045] AFB1-scFV-AP bifunctional fusion protein is obtained by using alkaline phosphatase as a bioluminescent catalyst, and the aflatoxin B1 nanobody (AFB1 scFv) is fused and expressed with alkaline phosphatase (AP).
[0046] AFB1-scFV-AP bifunctional fusion protein was prepared by the following method:
[0047] (1) Design and synthesis of AFB1 Nanobody gene: According to the amino acid sequence SEQ ID NO:7 of AFB1 Nanobody, the efficient expression of the gene in E. coli was achieved by optimizing the structure of the gene, including the use of E. coli preferred codons to eliminate possible The secondary structure of the GC / AT was balanced, etc., and the nucleotide fragment of the AFB1 Nanobody gene was designed and synthesized (the nucleotide sequence is shown in SEQ ID NO: 8).
[0048] (2) Construction of AFB1-scFV-AP expression vector: The aflatoxin B1 nanobody gene sy...
Embodiment 2
[0055] Example 2 Preparation of deoxynivalenol enol nanobody-alkaline phosphatase bifunctional fusion protein
[0056] Using the method of Example 1, a DON-scFV-AP bifunctional fusion protein was prepared, the amino acid sequence of which is shown in SED ID NO:2.
Embodiment 3
[0057] Example 3 Preparation of Zearalenone Nanobody-Alkaline Phosphatase Bifunctional Fusion Protein
[0058] Using the method of Example 1, a ZEN-scFV-AP bifunctional fusion protein was prepared, the amino acid sequence of which is shown in SED ID NO:3.
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Abstract
The invention discloses a fungaltoxin magnetochemiluminescence immunoassay kit based on bifunctional fusion protein and application of the fungaltoxin magnetochemiluminescence immunoassay kit. The kit comprises streptavidin magnetic particles, a biotin-labeled mycotoxin antigen, a mycotoxin standard substance solution, a mycotoxin nano antibody-alkaline phosphatase bifunctional fusion protein, a sample diluent, a scrubbing solution and a substrate solution. According to the kit, chemiluminescence, magnetic particle separation and genetic engineering technologies are gathered together, rapid, accurate and full-automatic detection of mycotoxin can be achieved, edible oil can be directly subjected to sample adding detection, extraction, centrifugation and other operations are not needed, the operation error of detection personnel is reduced, the requirement for the ability of the detection personnel is lowered, and the detection efficiency is improved. The detection efficiency of a large batch of samples at a basic level is remarkably improved, and a powerful means is provided for mycotoxin detection and monitoring.
Description
technical field [0001] The invention belongs to the technical field of food safety detection, and relates to a mycotoxin magnetic chemiluminescence immunoassay kit based on a bifunctional fusion protein and an application thereof. Background technique [0002] Mycotoxins are secondary metabolites produced by toxin-producing fungi under certain environmental conditions, which widely contaminate plant-derived products such as agricultural products, food, Chinese medicinal materials and feed. The more common mycotoxins in agricultural products, food and feed are: deoxynivalenol (deoxynivalenol, also known as vomitoxin, DON), aflatoxin B1 (aflatoxin B1, AFB1), aflatoxin B2 (AFB2) , Aflatoxin G1 (AFG1), Aflatoxin G2 (AFG2), Ochratoxin A (ochratoxin A, OTA), Fumonisin B1 (fumonisin B1, FB1), Fumonisin B2 (FB1), Gibberella zeae Enone (zearalenone, ZEN), T-2 toxin (T-2toxin), HT-2 toxin (HT-2toxin) or aflatoxin M1 (aflatoxin M1, AFM1), patulin (patμLin) and the like. Mycotoxins ca...
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