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Polyclonal antibody performing specific antigen-antibody reaction with CP4-EPSPS protein and application thereof

A CP4-EPSPS and polyclonal antibody technology, which is applied in the application field of detecting CP4-EPSPS protein residues and degradation in processed foods, can solve the problems of high detection cost, difficulty in large-scale popularization and application, complicated operation steps, etc., and achieve rapid detection. Effect

Inactive Publication Date: 2010-12-15
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to: for the unprocessed product existing in the double-antibody sandwich ELISA method commonly used in the current CP4-EPSPS protein detection technology, the operation steps are cumbersome, the detection cost is high, and it is limited by factors such as instruments and sites. Practical problems that make it difficult to promote and apply on a large scale, providing a polyclonal antibody that can detect CP4-EPSPS protein residue and degradation in processed foods using immunoblotting and colloidal gold test strip technology

Method used

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  • Polyclonal antibody performing specific antigen-antibody reaction with CP4-EPSPS protein and application thereof
  • Polyclonal antibody performing specific antigen-antibody reaction with CP4-EPSPS protein and application thereof
  • Polyclonal antibody performing specific antigen-antibody reaction with CP4-EPSPS protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Preparation of CP4-EPSPS protein polyclonal antibody:

[0042] a) synthesizing the CP4-EPSPS protein polypeptide fragment of SEQ ID No: 1;

[0043] b) activating KLH with the polypeptide fragment to form Pep-KLH with a sulfo-SMC cross-linking agent; KLH was purchased from Pierce.

[0044] c) Immunizing purebred New Zealand male white rabbits with the obtained Pep-KLH and separating antiserum from their blood, and obtaining polyclonal antibodies to CP4-EPSPS protein that meet the requirements through separation and purification techniques.

[0045] The titer was determined by ELISA method, and the titer was >16000.

Embodiment 2

[0047] CP4-EPSPS protein in transgenic soybean meal and wild-type soybean meal is detected with the polyclonal antibody that embodiment 1 obtains

[0048] (1) Extract the total protein of the sample, grind 0.075g sample with 1.5ml TPBS extract containing 2% SDS until homogenized, centrifuge at 12000g for 15min, transfer the supernatant to a new centrifuge tube, repeat centrifugation 1-2 times.

[0049] (2) Protein quantification: Follow the operation instructions of the BCA protein quantification kit to determine the concentration of the sample.

[0050] (3) SDS-PAGE gel electrophoresis: After mixing the extracted total protein with SDS-PAGE loading buffer, bathe in boiling water for 5 minutes, centrifuge at 7500g for 5 minutes, 1-2 times. Then, the samples were subjected to denaturing polyacrylamide discontinuous gel electrophoresis (the loading amount was not less than 20 μg). SDS-PAGE was prepared according to the following recipe. 12% separating gel 10ml: ddH 2 O 3.3ml,...

Embodiment 3

[0058] The polyclonal antibody detection limit test that obtains with embodiment 1

[0059] The samples to be tested were respectively selected from transgenic soybean flour with transgenic content (w / w) of 20%, 10%, 5%, 1% and 0.5%.

[0060] (1) Extract the total protein of the sample, grind 0.075g sample with 3.0ml TPBS extract containing 2% SDS until homogenized, centrifuge at 12000g for 15min, transfer the supernatant to a new centrifuge tube, repeat centrifugation 1-2 times.

[0061] (2) Protein quantification: Follow the operation instructions of the BCA protein quantification kit to determine the concentration of the sample.

[0062] (3) SDS-PAGE gel electrophoresis: After mixing the extracted total protein with SDS-PAGE loading buffer, bathe in boiling water for 5 minutes, centrifuge at 7500g for 5 minutes, 1-2 times. Then, the samples were subjected to denaturing polyacrylamide discontinuous gel electrophoresis (the loading amount was not less than 20 μg). SDS-PAGE ...

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Abstract

The invention relates to a polyclonal antibody for detecting the residue and the degradation of a CP4-EPSPS protein in a processed food and application thereof. An animal is immunized by using a CP4-EPSPS protein peptide segment of a coupled keyhole limpet hemocyanin (KHL) to obtain antiserum, namely the antibody of the invention. The application comprises a Western blotting and a colloidal gold test strip and has the characteristics of simplicity, practicability, high sensitivity and the like, wherein the Western blotting is used for detecting the residue and the degradation of the CP4-EPSPS protein in food; and test strip technology is used for the rapid detection of the CP4-EPSPS protein in the food. The invention provides a high-practicability method for the safety detection of a genetically modified food, and has a very high practical application value.

Description

Technical field: [0001] The invention relates to a polyclonal antibody and its application, in particular to a polyclonal antibody which reacts with CP4-EPSPS protein with specific antigen-antibody and its application in detecting the residue and degradation of CP4-EPSPS protein in processed food. The application includes two aspects of an immunoblotting method and a colloidal gold test strip, and belongs to the field of food safety transgene detection. Background technique: [0002] When conducting safety evaluation or labeling management of genetically modified products, especially food, it is often necessary to conduct qualitative or semi-quantitative determination of the transferred exogenous ingredients. [0003] Herbicide-specific resistance cp4-epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene is a common exogenous gene in transgenic soybean. Introducing this gene can make plants resistant to the herbicide glyphosate. [0004] The existing CP4-EPSPS protein d...

Claims

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Application Information

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IPC IPC(8): C07K16/40C07K16/06G01N33/573G01N33/52
Inventor 黄明沈文飚吴洪洪肖笑徐晟周兴虎何健周光宏
Owner NANJING AGRICULTURAL UNIVERSITY
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