Hybridoma cell strains and monoclonal antibodies generated by same and application of hybridoma cell strain and monoclonal antibody in detecting G2-EPSPS protein

A technology of G2-EPSPS and hybridoma cell lines, applied in the field of immune colloidal gold test paper, can solve the problems of large inter-batch variation coefficient, insufficient quality control ability, low sensitivity, etc., and achieve high sensitivity and specificity

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

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Problems solved by technology

However, the current immune-binding colloidal gold chromatography generally has defects such as low sensitivity, insufficient qua

Method used

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  • Hybridoma cell strains and monoclonal antibodies generated by same and application of hybridoma cell strain and monoclonal antibody in detecting G2-EPSPS protein
  • Hybridoma cell strains and monoclonal antibodies generated by same and application of hybridoma cell strain and monoclonal antibody in detecting G2-EPSPS protein
  • Hybridoma cell strains and monoclonal antibodies generated by same and application of hybridoma cell strain and monoclonal antibody in detecting G2-EPSPS protein

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Embodiment 1

[0035] 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

[0060] Preparation and Identification of G2-EPSPS Protein Monoclonal Antibody

[0061] 1. Experimental method

[0062] 1.1 Immunization of mice

[0063] (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;

[0064] (2) Three days be...

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Abstract

The invention provides a pair of hybridoma cell strains. The hybridoma cell strains comprise the first hybridoma cell strain and the second hybridoma cell strain which are stored independently. The preservation number of the first hybridoma cell strain is CGMCC NO.10495. The preservation number of the second hybridoma cell strain is CGMCC NO.10494. The invention further provides a pair of paired monoclonal antibodies excreted by the two hybridoma cell strains, an application of the paired monoclonal antibodies in detecting G2-EPSPS protein and immuno gold staining test paper. According to the technical scheme, the sensitivity of 1 ng/mL can be obtained through detection of the immuno gold staining test paper on G2-EPSPS protein, no cross reaction is carried out on CP4-EPSPS protein, no cross reaction is carried out on 27 non-GMO crops and 14 G2-EPSPS transgenic crops with different sources, and high sensitivity and specificity are achieved.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a pair of hybridoma cells, a pair of monoclonal antibodies, their use in detecting G2-EPSPS protein and an immune colloidal gold test paper. Background technique [0002] Glyphosate is a new type of herbicide developed by Monsanto and registered in 1974. The registered trade name is Glyphosate has stable physical and chemical properties, low synthesis cost, high efficiency, broad spectrum, low toxicity, and low residue. It is an excellent herbicide and one of the most widely used herbicides in the world. Studies have found that glyphosate blocks the metabolic pathway of shikimic acid by competitively binding to EPSPS (5'-enol acetonyl Misceloyl-3-phosphate synthase), which prevents plants from synthesizing aromatic amino acids and their derivatives, and shikimate Oxalic acid accumulates, causing plant death. Since most EPSP synthases in plants in nature are glyphosate...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/573G01N33/558C12R1/91
Inventor 张维陆伟郑健张君刘奇林敏
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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