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Nanobody against deoxynivalenol antibody

A deoxynivalenol, nanobody technology, applied in biochemical equipment and methods, applications, instruments, etc., can solve the problems that restrict the application and promotion of immunological detection methods, the health and environmental threats of detection personnel, and the high price. problem, to achieve good effect, cost saving, harm reduction effect

Inactive Publication Date: 2017-11-03
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of establishing an immunological detection method, it is unavoidable to prepare competing antigens or solid-phase coated antigens with DON standard products as raw materials. DON standard products are not only expensive but also have strong toxicity. It poses a great threat to the health and environment of the country, which restricts the application and promotion of immunological detection methods to a certain extent.

Method used

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  • Nanobody against deoxynivalenol antibody
  • Nanobody against deoxynivalenol antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Construction of camel-derived natural single domain heavy chain antibody library

[0027] 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 1000 g for 50 min; carefully draw the leukocytes suspended in the middle layer into a new centrifuge tube, add 1 / 2 volumes of PBS, centrifuge at 1000g for 15min; discard the supernatant, wash the leukocytes on the wall of the centrifuge tube with PBS, centrifuge at 1000g for 10min; discard the supernatant, add 500 μL PBS to resuspend the leukocytes and count; add at a volume ratio of 1:15 The lysate (RNAiso) was saved for future use.

[0028]2) Extraction of total RNA: add 1 / 4 volume of chloroform to the above lysate, shake vigorously for 20 s to fully emulsify, let stand at room temperature for 5 min; centrifuge at 12000g at 4°C for 15 min, transfer the supernatant to another fresh centrifuge Add an equal ...

Embodiment 2

[0050] Example 2. Affinity panning and identification of Nanobodies

[0051] 1) Affinity panning of Nanobodies: First, dilute the anti-DON monoclonal antibody with PBS (pH 7.4) to a final concentration of 100 μg / mL, and coat at 4°C overnight. The next day, after washing 5 times with PBST (10mM PBS, 0.1% Tween-20 (v / v) ), add 5% BSA-PBS (or 5% OVA-PBS) 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), incubate 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 8 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 E. coli The TG1 strain was amplified. On the third day, the amplified phage was precipitated with PEG / NaCl, and the titer of the phage was determined.

[0...

Embodiment 3

[0055] Example 3. Sequencing of Nanobody Encoding Gene and Determination of its Amino Acid Sequence

[0056] The phage clones displaying positive Nanobodies identified by indirect competition ELISA were subjected to DNA sequencing, 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 field of biotechnology, and specifically relates to the preparation and application of a nanobody that can specifically bind to an anti-deoxynivalenol antibody. Its amino acid sequence is SEQ ID NO.: 1, and it also relates to a nucleotide encoding the amino acid. The nanobody of the present invention can replace the expensive and highly toxic DON standard product, and can be used as a competitive antigen or solid-phase coated antigen in the immunological detection of DON. The nanobody has similar immune response characteristics to natural DON molecules, and the effect is good. . Compared with traditional polypeptide-based antigen mimic epitopes and IgG-based traditional anti-idiotypic antibodies, nanobodies have more stable structures, acid, alkali and high temperature resistance, and high detection sensitivity, so their immunological detection stability has been greatly improved. At the same time, the tolerance to the environment has also been improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the preparation and preparation of a nanobody (Variable domain of heavy chain of heavy chain antibody, VHH) that can specifically bind to an anti-deoxynivalneol Monoclonal antibody (anti-DON McAb). its application. Background technique [0002] Deoxynivalneol (deoxynivalneol, DON) is a trichothecene toxin, mainly produced by Fusarium, widely present in barley, wheat, corn, oats, rice and other crops and their products. DON has cytotoxicity, embryotoxicity and immunotoxicity, and can cause symptoms such as anorexia, vomiting, diarrhea, fever and slow growth, which seriously threaten the health of humans and animals. Due to the high contamination rate and high toxicity of DON, the detection of DON in food is of great significance. [0003] At present, the methods for detecting DON in food mainly include thin layer chromatography, high performance liquid chromatography, gas chromatogr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/42C12N15/13G01N33/577G01N33/543
Inventor 许杨何庆华邱雨楼
Owner NANCHANG UNIV
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