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Nanobodies capable of specifically binding anti-zearalenone antibodies and applications thereof

A zearalenone and nanobody technology, applied in the fields of application, specific peptides, biochemical equipment and methods, etc., can solve problems such as difficulty in extracting toxin small molecules, physical harm to operators, environmental pollution, etc., and achieve good results. , the effect of improving sensitivity and reducing harm

Active Publication Date: 2018-08-17
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of using standard products is first of all that it is difficult to extract small molecules of toxins. Now most of our country uses imported products, and the import of some products is restricted, which relatively increases the cost. The most important thing is that the use of standard products of toxin molecules can easily cause environmental damage Pollution and hazards to the operator's body

Method used

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  • Nanobodies capable of specifically binding anti-zearalenone antibodies and applications thereof
  • Nanobodies capable of specifically binding anti-zearalenone antibodies and applications thereof
  • Nanobodies capable of specifically binding anti-zearalenone antibodies and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Construction of camel-derived natural single domain heavy chain antibody library

[0025] 1) Separation of camel-derived leukocytes: add lymphocyte separation solution to the centrifuge tube, then slowly add an equal volume of blood sample, centrifuge at 1000g for 50min; carefully draw the leukocytes suspended in the middle layer into a new centrifuge tube, add 1 / 2 volume centrifuge at 1000g for 15min; discard the supernatant, wash the leukocytes on the wall of the centrifuge tube with PBS, and centrifuge at 1000g for 10min; discard the supernatant, add 500μL PBS to resuspend the leukocytes and count them; add the lysate (RNAiso ) are saved for future use.

[0026] 2) Extraction of total RNA: Add 1 / 4 volume of chloroform to the above lysate, shake vigorously for 20s to fully emulsify, let stand at room temperature for 5min; centrifuge at 12000g at 4°C for 15min, transfer the supernatant to another fresh centrifuge tube; Add an equal volume of isopropanol, mi...

Embodiment 2

[0049] Example 2. Affinity panning and identification of Nanobodies

[0050] 1) Affinity panning of Nanobodies: First, dilute the anti-ZEN monoclonal antibody with PBS (pH 7.4) to a final concentration of 10 μg / mL, and coat at 4°C overnight. The next day, after washing 5 times with PBST (10 mM PBS, 0.1% Tween-20 (v / v)), 5% BSA-PBS (or 5% OVA-PBS) was added to block for 1 hour at 37°C. Then wash 6 times with PBST, add 100 μL camel-derived natural single domain heavy chain antibody library (titer about 2.0×10 11 cfu), incubated at 37°C for 2 hours. Unbound phages were discarded, washed 10 times with PBST, added 100 μL of Glycine-HCl (0.2M, pH 2.2) to elute for 7 min, and immediately neutralized with 15 μL of Tris-HCl (1M, pH 9.1). Take 10 μL of the eluted phage to determine the titer, and the rest is used to infect 25 mL of the E.coli TG1 strain grown to the logarithmic phase for amplification. On the third day, the amplified phage was precipitated with PEG / NaCl, and the tite...

Embodiment 3

[0054] Example 3. Sequencing of Nanobody Encoding Gene and Determination of its Amino Acid Sequence

[0055] DNA sequencing was performed on the phage clones displaying positive nanobodies identified by indirect competitive ELISA, and the amino acid sequence of the nanobodies could be obtained according to the DNA sequencing results and the codon table, as shown in SEQ ID NO.1.

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Abstract

The invention belongs to the biological technical field, and in particular to preparation and application of nano antibody capable of specifically combining with anti-zearalenone antibody and with an amino acid sequence of SEQ ID No.1; and the invention also relates to the nucleotides encoding the amino acid. The nano antibody of the invention can replace the expensive toxic Zen standard, and can be used in immunological detection of Zen as a competitive antigen or solid coating antigen. The nano antibody has similar immunological properties to natural Zen molecule, and good effect. Compared to a traditional antigen mimicking epitope based on polypeptide and a traditional anti-idiotype antibody based on IgG, the nano antibody has the advantages of more stable structure, acid resistance, alkali resistance, high temperature resistance and high detection sensitivity, so that the immune detection stability has been greatly improved, and the tolerance on the environment has also been improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the preparation and application of a nanobody (Variable domain of heavy chain of heavy chain antibody, VHH) that can specifically bind to an anti-zearalenone Monoclonal antibody (anti-ZEN McAb) . Background technique [0002] Zearalenone (Zearalenone, ZEN) is an estrogen-like mycotoxin produced by Fusarium fungus. Lactone 161~163℃, insoluble in water, soluble in alkaline solution, ether, benzene, methanol and ethanol, etc. Zearalenone is widely found in moldy corn, sorghum, wheat, oats, barley and other cereal crops and milk. The most common genus of zearalenone-producing bacteria is Fusarium graminearum, and Fusarium tritina, Fusarium equiseti, Fusarium nivalorum, Fusarium pink, etc. ZEN has estrogen-like effects, reproductive and developmental toxicity, immunotoxicity, cytotoxicity, and hepatotoxicity, and has a certain impact on tumors. Therefore, once grains are contaminated b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N15/13G01N33/64
Inventor 许杨何庆华王显显
Owner NANCHANG UNIV
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