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Monoclonal antibody for detecting herbicide-resistant glyphosate protein and application of monoclonal antibody

A monoclonal antibody, application technology, applied in the field of agricultural biology, can solve problems such as difficulty in achieving high sensitivity and specificity

Active Publication Date: 2015-12-16
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key to these qualitative or semi-quantitative determinations is the use of monoclonal antibodies, however, the existing monoclonal antibodies against G2-EPSPS protein are difficult to achieve high sensitivity and specificity

Method used

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  • Monoclonal antibody for detecting herbicide-resistant glyphosate protein and application of monoclonal antibody
  • Monoclonal antibody for detecting herbicide-resistant glyphosate protein and application of monoclonal antibody
  • Monoclonal antibody for detecting herbicide-resistant glyphosate protein and application of monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Bacterial total DNA was extracted from glyphosate-contaminated soil samples, and the quality of the total DNA from sample No. 2 (G2) was better after electrophoresis detection. Carry out partial enzyme digestion (Sau3AI) on the total DNA of G2 bacteria, recover the 3-20kb size fragment, and carry out BamHI enzyme digestion on the pACYC184 plasmid, and then carry out the ligation reaction. The ligation product was transformed into aroA gene-deleted Escherichia coli ER2799, and colonies were selected for growth on M9 medium. After the plasmid was extracted from the growing colony, it was sequenced and identified, and the plasmid containing the EPSPS coding gene aroA was named pAC G2aroA . According to the results of NCBIblast analysis, a 1332bp long aroA gene encoding 444 amino acids was found, primers were designed according to the G2-aroA (ie G2-EPSPS) sequence, a BamHI restriction sequence was artificially added at the 5' end of the upstream primer, and at the 5' end ...

Embodiment 2

[0050] Preparation and Identification of G2-EPSPS Protein Monoclonal Antibody

[0051] 1. Experimental method

[0052] 1.1 Immunization of mice

[0053] (1) Choose 8W + BALB / c female mice were immunized 4 times in groups by dose: (eye blood was taken as a negative control before the initial immunization), and the immunization doses were 50, 100, 150, 200 μg / only (i.e. the G2-EPSPS protein obtained in Example 1 ). The same volume of Freund's complete adjuvant was added to the subcutaneous multi-point injection in the first immunization; the subsequent immunization was performed once every two weeks, and the same dose of recombinant antigen was added to the same volume of Freund's incomplete adjuvant subcutaneous multi-point injection, followed by two additional immunizations. About 10 days after the third immunization, the ELISA plate was coated with the recombinant antigen, and the antibody titer of the mouse serum was measured by indirect ELISA;

[0054] (2) Three days be...

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Abstract

The invention provides a hybridoma cell strain with the preservation serial number of CGMCCNO.10497, and further provides a monoclonal antibody secreted by hybridoma cells and application of the monoclonal antibody for detecting herbicide-resistant glyphosate protein. The monoclonal antibody secreted by the hybridoma cell strain can be used for detecting immunological hybridization of G2-EPSPS protein, and can detect recombined G2-EPSPS protein and G2-EPSPS transgenic plant seeds; no cross reaction happens to the CP4-EPSPS protein, and no cross reaction happens to four types of G2-EPSPS non-transgenic plants of different sources either.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a hybridoma cell line, a monoclonal antibody and their application in detecting herbicide-resistant glyphosate protein. Background technique [0002] Since the 1980s, transgenic herbicide-resistant crops have been promoted and planted on a large scale, creating a new situation in the development of herbicide-resistant crops. Due to the characteristics of glyphosate herbicide with broad spectrum, no soil residue and environmental friendliness, glyphosate has been registered in more than 100 countries, and its usage is increasing day by day. It has become the most widely used pesticide with the largest output in the world. The value has been ranking first among pesticides, and the annual sales value in the world has exceeded 2.93 billion US dollars. However, as a non-selective herbicide, it also has a killing effect on crops, which greatly limits the application range of ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/00G01N33/577C12R1/91
Inventor 陆伟张维刘奇郑健张君林敏
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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