Polypeptide specifically combined with benzothiostrobin antibodies and application thereof

A technology of benthiazoxan antibody and benthiazolin is applied to the application field of detection of benthiazolin, can solve problems such as peptides that have not yet been seen, and achieve the effects of high accuracy, high sensitivity and strong specificity

Active Publication Date: 2015-10-21
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there have been no studies and reports on the peptides a...

Method used

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  • Polypeptide specifically combined with benzothiostrobin antibodies and application thereof
  • Polypeptide specifically combined with benzothiostrobin antibodies and application thereof
  • Polypeptide specifically combined with benzothiostrobin antibodies and application thereof

Examples

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Embodiment 1

[0028] Example 1 Panning and preparation of benzathystrobin antibody specific binding polypeptide

[0029] 1. Panning of phage clones that specifically bind to benzothiastrobin antibody

[0030] According to the cycle of "adsorption-washing-elution-amplification", after three rounds of panning, the specific operation is as follows:

[0031] (1) Take 100 μL of 10 μg / mL protein A column-purified penthiostrobin antibody and add it to the microtiter plate, and coat at 4°C overnight, with a total of three wells;

[0032] (2) Take a small amount of Escherichia coli ER2738 and spread it on the LB+Tet plate, culture overnight at 37°C;

[0033] (3) Wash the ELISA plate in step (1) 5 times with PBST, add 300 μL 3% BSA, incubate at 37°C for 1 hour, wash 5 times with PBST, store at 4°C for later use;

[0034] (4) Add 2×10 11 phage (100 μL), shake slightly at room temperature for 1 hour;

[0035] (5) Take 20mL of LB medium and add it to a 250mL Erlenmeyer flask, add a single colony of ER...

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Abstract

The invention belongs to the technical field of biology and relates to polypeptide specifically combined with benzothiostrobin antibodies, including an application of the polypeptide to the detection of benzothiostrobin. According to the polypeptide, a phage display technique is adopted, and the benzothiostrobin antibodies which are purified by a protein A column are used for screening a phage display random octapeptide library for three rounds, so as to screen phage clones combined with the benzothiostrobin antibodies; a plurality of phage clones are selected at random for amplification and plasmid extraction, positive phage clones are selected by adopting a phage ELISA (enzyme-linked immuno sorbent assay) method, and the positive clones are sequenced, so as to obtain a polypeptide sequence. The invention further relates to the application of the phage display polypeptide to the detection of the benzothiostrobin. A phage enzyme-linked immunoassay method established by using the phage display polypeptide can be used for quickly, sensitively and conveniently detecting benzothiostrobin residues in the environment and agricultural products with low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a polypeptide with specific binding to penthiostrobin antibody, including the application of the polypeptide in detecting penthiostrobin. Background technique [0002] Benzothiostrobin is a new type of Strobilurins fungicide developed by Central China Normal University. Penthiostrobin has the advantages of wide bactericidal spectrum, high bactericidal activity, low application rate, long-lasting effect, etc. It has protective and therapeutic effects, and has excellent control effects on crop powdery mildew and downy mildew, and has a good market Development prospects. In order to prevent the potential risks in the application of penthiostrobin, a sensitive, rapid and selective residue detection method is needed. [0003] At present, the residue detection of fenthiastrobin includes instrumental analysis method and immunoassay method. The immunoassay method has the advantages of fast,...

Claims

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Application Information

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IPC IPC(8): C07K7/06G01N33/68
Inventor 王鸣华华修德冯璐施海燕
Owner NANJING AGRICULTURAL UNIVERSITY
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