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Monoclonal antibody of glyphosate-resistant protein G2-aroA and application thereof

A technology of monoclonal antibody and hybridoma cell line, applied in the direction of anti-enzyme immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of application limitation, glyphosate sensitivity, no monoclonal antibody, etc., and achieve cost Low, high affinity, strong specific effect

Active Publication Date: 2012-08-22
BEIJING ORIGIN SEED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its application is limited due to the sensitivity of grass crops such as corn to glyphosate
At present, there is no monoclonal antibody against G2-aroA protein, and there is no report on the use of monoclonal antibody to G2-aroA protein to identify G2-aroA transgenic plants

Method used

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  • Monoclonal antibody of glyphosate-resistant protein G2-aroA and application thereof
  • Monoclonal antibody of glyphosate-resistant protein G2-aroA and application thereof
  • Monoclonal antibody of glyphosate-resistant protein G2-aroA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the acquisition of hybridoma cell line and the preparation of anti-G2-aroA protein monoclonal antibody

[0047] Balb / C mice: Beijing Weitong Lihua Experimental Animal Co., Ltd.

[0048] Sp2 / 0: Beijing Kangwei Century Biotechnology Co., Ltd.

[0049] 1. Preparation of immunogen S780

[0050]The DNA fragment shown in Sequence 1 was connected to the multiple cloning site of the prokaryotic expression vector pET-28a(+) to obtain the prokaryotic expression vector pET-28a-G2-aroA expressing G2-aroA protein. Transform pET-28a-G2-aroA into Escherichia coli BL21, and use IPTG to induce its expression of G2-aroA protein. After collecting the bacteria, the bacteria are broken by ultrasonic waves, and the expressed protein is released into the extract, and then His tag is used to The protein purification kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0009A) was purified, and the G2-aroA protein was purified and then detected by...

Embodiment 2

[0104] Example 2, application of monoclonal antibody against G2-aroA protein

[0105] 1. Experimental materials

[0106] 1. Transgenic G2-aroA maize

[0107] Prepared according to the following method:

[0108] (1) Maize transformation starting material: maize variety Zong 31 (excellent maize inbred lines display Zong 3 and Zong 31. Maize Science, 2009 (5)) young ears 9 to 13 days after pollination, peel off the bract leaves, and carry out Disinfect surfaces. Peel the immature embryos from the sterilized young ears, put them into the infection culture medium (recipe reference: Methods in Molecular Biology, vol.343: Agrobacterium Protocols, 2 / e, volume 1) to wash once or twice, and set aside .

[0109] (2) Transformation of Agrobacterium with recombinant expression vector pS3300-UMG2: the recombinant expression vector pS3300-UMG2 carrying the mG2-aroA sequence (plasmid map as shown in image 3 As shown, the specific construction method is at the end of the article). The m...

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Abstract

The invention discloses a monoclonal antibody of glyphosate-resistant protein G2-aroA and application thereof. The monoclonal antibody provided by the invention is produced from the hybridoma cell strain AntiG2-5F11. The preservation number of the hybridoma cell strain AntiG2-5F11 in the China General Microbiological Culture Collection Center is CGMCC No.5772. The monoclonal antibody provided by the invention has high affinity and specificity on the glyphosate-resistant protein G2-aroA, can be used for testing the expression of the glyphosate-resistant protein G2-aroA in corn and other transgenic plants, and has the advantages of rapidness, sensitivity and high specificity.

Description

technical field [0001] The invention relates to a monoclonal antibody against glyphosate-resistant protein G2-aroA and application thereof. Background technique [0002] Glyphosate herbicide is a broad-spectrum, non-selective herbicide, which inhibits EPSPS (5-enolpyruvyl oxalate-3-phosphate synthase, which is an important pathway in the synthesis of aromatic amino acids in plants. enzyme) activity, blocks the biosynthesis of shikimate pathway in plants, strongly inhibits cell division, and has a strong inhibitory effect on many annual and perennial weeds. Since glyphosate is easily decomposed by microorganisms, has no residual toxicity in the soil, and is non-toxic to animals, it has been widely used since the successful development of Roundup in 1976. However, due to the sensitivity of grass crops such as corn to glyphosate, its application is limited. Therefore, transferring glyphosate-resistant genes into crops can not only expand the scope of glyphosate use, but also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/573
Inventor 韩庚辰刘素霞宋哲李雪峰李翔姜付坤
Owner BEIJING ORIGIN SEED TECH
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