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Aflatoxin B1 nano antibody immune adsorption material as well as preparation method and application thereof

An immunoadsorbent material and aflatoxin technology, applied in the field of preparation of immunoaffinity materials, can solve the problems of product instability, poor specificity and high price, and achieve the effects of high specificity, easy expression and low price

Inactive Publication Date: 2020-05-15
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, most of the immunoaffinity columns on the market are prepared from monoclonal antibodies and polyclonal antibodies, which have problems such as high price, poor specificity, and unstable products.

Method used

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  • Aflatoxin B1 nano antibody immune adsorption material as well as preparation method and application thereof
  • Aflatoxin B1 nano antibody immune adsorption material as well as preparation method and application thereof
  • Aflatoxin B1 nano antibody immune adsorption material as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) Construction of pET-30a-G8-Halo vector

[0020] anti-AFB 1 Nanobody-encoding gene vector pET-25b-G8-DIG3 (the sequence disclosed in "One-step ELISA detection of aflatoxin B1 based on nanobody-alkaline phosphatase fusion protein", self-made in the laboratory) as a template , using G8-F0 and G8-R0 in Table 1 as primers for PCR amplification to obtain PCR products. PCR reaction 50ul system: ddH 2 O37.5ul, template (pET-25-G8-DIG lng / ul) 1ul, dNTP (2.5mM) 4ul, 5×PS Buffer 5ul, G8-F0 1ul, b72℃30s, a total of 25 cycles, the last 72℃10min fully extended. The G8(F0 / R0) PCR product was separated by 1% agarose gel electrophoresis to 409bp, and was produced with TaKaRa Fragment Purification Kit.

[0021] Table 1 PCR primers

[0022]

[0023]

[0024] Note: The underline is the enzyme recognition site

[0025] The PCR product encoding G8 and the plasmid vector pET-30a-T9-Halo ("One-step oriented immobilization of nanoparticles and its application for immunoglobulin ...

Embodiment 2

[0028] Example 2: Fusion Expression of G8-Halo Nanobodies

[0029] Transform the correctly sequenced pET-30a-G8-Halo vector into BL21(DE3) competent cells, pick a single colony from the plate and inoculate it in LB-K test tube medium, and culture it overnight at 220rpm at 37°C; Inoculate in 50mL LB-K medium according to 1% inoculation amount, shake culture at 37°C and 220rpm, when OD 600 When = 0.5, IPTG inducer was added to the culture to a final concentration of 0.5 mM, and then placed at 18° C. for induction culture for 12 h.

[0030] Centrifuge the culture after induction of expression in the previous step at 8000rpm for 20min, collect the cell pellet and resuspend the cell pellet with 1 / 10 volume of PBS buffer, and ultrasonically break the cell suspension for 10min until the broken cell suspension is clear, 4°C, 8000°C After centrifuging at rpm for 20min, the supernatant and the precipitate were collected respectively; SDS-PAGE was used to analyze the expression of the t...

Embodiment 3

[0031] Example 3: Immobilization and coupling of Halo-Tag protein

[0032]Take 50 μL of Halolink Resin (purchased from Promega, gel volume, the same below) into a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 1 min and discard the supernatant. Add 400 μL of binding buffer (100 mM Tris, 150 mM NaCl, 5% glycerol, pH 7.6) to the centrifuge tube in the previous step and mix thoroughly, centrifuge at 3000 rpm for 2 min, discard the supernatant, and repeat the operation 2 times. Resuspend Halolink Resin in 100 μL binding buffer, add 50 μL Halo-Tag-labeled protein and mix well, shake at room temperature for 2 hours to keep Halolink Resin suspended in the binding buffer. After centrifugation at 3000rpm for 2min, the supernatant was taken, and the protein concentration in the supernatant was measured by the Bradford method to calculate the coupling amount. Add 1mL wash buffer (100mM Tris, 150mM NaCl, 5% glycerol, 1mg / mL BSA pH 7.6) and mix thoroughly, shake at room temperature fo...

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Abstract

The invention relates to a gene engineering antibody technique and a molecular cloning technique and in particular relates to an aflatoxin B1 nano antibody immune adsorption material which is an agarose gel coupled with an aflatoxin B1 nano antibody fusion protein. The amino acid sequence of the aflatoxin B1 nano antibody fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence of the protein is shown in SEQ ID NO.2. A gene engineering technique is adopted, a single-domain heavy chain antibody specifically aiming at aflatoxin B1 is fused with a self-coupled tag protein, then purification-free specific one-step orientated coupling of a recombinant expression ligand can be achieved, preparation procedures of an affine adsorption material can be greatly simplified, the cost can belowered, and meanwhile, occupation of a ligand combination site of a conventional coupling method is avoided, so that the adsorption capacity of an immune affine adsorption material can be increased.

Description

technical field [0001] The invention relates to genetic engineering antibody technology and molecular cloning technology, in particular to the preparation of immunoaffinity materials for aflatoxin B1 single domain heavy chain antibody. Background technique [0002] Aflatoxin (Aflatoxin, AF) is a secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus, which has teratogenic, carcinogenic and mutagenic hazards, especially aflatoxin B 1 most serious hazards. Aflatoxin B in food is regulated in many countries 1 Limit standards have been established, but most of the mycotoxins are detected in trace amounts, and the contaminated object matrix is ​​often more complicated, which increases the difficulty of detection. Therefore, the quality of its pre-processing technology largely determines the accuracy of the test results. In recent years, immunoaffinity chromatography columns have been widely used in the purification of mycotoxins. Immunoaffinity columns...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K1/22G01N33/531
CPCC07K1/22C07K16/44C07K2317/569C07K2319/00C12N15/70G01N33/531
Inventor 钟引凤涂追黄云祥刘贝付金衡李燕萍何庆华
Owner NANCHANG UNIV
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