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Method for detecting roundup ready protein G2-aroA and special enzyme linked immune kit

An enzyme-linked immunosorbent reagent and protein technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of glyphosate sensitivity, no monoclonal antibody identification, application limitations, etc., and achieve the effect of strong specificity

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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  • Method for detecting roundup ready protein G2-aroA and special enzyme linked immune kit
  • Method for detecting roundup ready protein G2-aroA and special enzyme linked immune kit
  • Method for detecting roundup ready protein G2-aroA and special enzyme linked immune kit

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Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1, the acquisition of hybridoma cell line and the preparation of anti-G2-aroA protein monoclonal antibody

[0051] Balb / C mice: Beijing Weitong Lihua Experimental Animal Co., Ltd.

[0052] Sp2 / 0: Beijing Kangwei Century Biotechnology Co., Ltd.

[0053] 1. Preparation of immunogen S780

[0054] 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 b...

Embodiment 2

[0108] Example 2. ELISA kit and application for detecting glyphosate-resistant protein G2-aroA

[0109] 1. Preparation of ELISA kit for detection of glyphosate-resistant protein G2-aroA

[0110] The ELISA kit of the glyphosate-resistant protein G2-aroA of the present invention consists of a polyclonal antibody against the G2-aroA protein and an alkaline phosphatase-labeled monoclonal antibody against the G2-aroA protein (enzyme-labeled antibody for short). , coating buffer, washing solution, G2-aroA protein standard, substrate buffer, stop buffer and blocking solution.

[0111] 1. Preparation methods of various buffer solutions

[0112] (1) Coating buffer (carbonate coating buffer): Take 1.5g Na 2 CO 3 , 2.93 g NaHCO 3 , and distilled water to 1000mL, pH9.6.

[0113] (2) Washing solution (PBST): take 1mL Tween 20, add phosphate buffer saline (PBS) to make the volume to 1000mL, pH7.5; phosphate buffer saline (PBS): take 8.0g NaCl, 0.2g KH 2 PO 4 , 2.96 g Na 2 HPO 4 12H...

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Abstract

The invention discloses a method for detecting a roundup ready protein G2-aroA and a special enzyme linked immune kit. The enzyme linked immune kit contains independently packaged anti-G2-aroA protein monoclonal antibody; the amino acid sequence of the G2-aroA protein refers to sequence 9 in a sequence list; and the monoclonal antibody is generated by a hybridoma cell strain AntiG2-5F11 CGMCC No.5772. The enzyme linked immune kit provided by the invention can be used for qualitatively or quantitatively detecting the expression of the roundup ready protein G2-aroA in transgenic plants such as corn, and has the advantages of rapidness, sensitiveness and strong specificity.

Description

technical field [0001] The invention relates to a method for detecting glyphosate-resistant protein G2-aroA and a special ELISA kit. 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 reduce ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/573
Inventor 韩庚辰刘素霞李雪峰宋哲徐晔颍郭旻
Owner BEIJING ORIGIN SEED TECH
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