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Immune nano-magnetic particle for detecting Cry1Ab/Cry1Ac insecticidal proteins and preparation method thereof

A technology of nano-magnetic particles and insecticidal proteins, applied in measuring devices, biological testing, material inspection products, etc., to achieve the effects of eliminating interference from impurity molecules, convenient operation, and simple materials

Inactive Publication Date: 2011-09-07
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few research reports and related patented technologies at home and abroad on Cry1Ab / Cry1Ac insecticidal protein immunoassay technology, and there is an urgent need to develop efficient, accurate and rapid new immunoassay technology and products

Method used

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  • Immune nano-magnetic particle for detecting Cry1Ab/Cry1Ac insecticidal proteins and preparation method thereof
  • Immune nano-magnetic particle for detecting Cry1Ab/Cry1Ac insecticidal proteins and preparation method thereof
  • Immune nano-magnetic particle for detecting Cry1Ab/Cry1Ac insecticidal proteins and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Preparation of water-soluble carboxyl nano-magnetic particles

[0035] 1. Add 0.85g of anhydrous FeCl3 and 0.6g FeCl 2 4H 2 O was dissolved in 100ml ultrapure water, heated and stirred until completely dissolved, and the solution was transparent yellow at this time;

[0036] 2. Prepare 0.05M PAA solution with ethylene glycol as solvent and prepare 5M NaOH aqueous solution. When the temperature is maintained at 80-85°C, add 15ml of 5M NaOH solution to the mixed solution obtained in step 1, the color of the solution turns black, after 2 minutes of reaction, add 20ml of 0.05M PAA solution, continue heating and stirring for 30 minutes to obtain water-soluble Carboxyl nanoparticles. The whole experiment process was carried out under constant temperature water bath and nitrogen protection;

[0037] 3. Wash the above-mentioned water-soluble carboxyl nano-magnetic particles with absolute ethanol 3 times under the action of an external magnetic field (constant c...

Embodiment 2

[0049] Example 2 Preparation of Anti-Cry1Ab / Cry1Ac Insecticidal Protein Polyclonal Antibody

[0050] 1. According to the consensus sequence of the Cry1Ab / Cry1Ac insecticidal protein (SEQ ID NO.1) and the gene (SEQ ID NO.2), the Cry1Ab / Cry1Ac insecticidal protein gene was constructed using the whole gene synthesis technology, with a total of 660 base pairs, cloned To the fusion expression vector pGEX-4T-2 (insertion sites BamH I and Sal I) and transformed into Escherichia coli BL21 (DE3) cells, through bacterial expression in vitro, Glutathione Sepharose 4B affinity chromatography and fusion protein thrombin in column cleavage and other operation steps to obtain the Cry1Ab / Cry1Ac insecticidal protein;

[0051] 2. Mix 5ml of Cry1Ab / Cry1Ac insecticidal protein (concentration: 1.1mg / ml) with 5ml of Freund's adjuvant, and fully emulsify. BALB / c mice were immunized by multi-point injection on the back. The amount of antigen in each immunization is about 10 μg, and the immunization...

Embodiment 3

[0056] Example 3 Preparation of immune nanomagnetic particles

[0057] 1. Wash the water-soluble carboxyl nano-magnetic particles prepared in Example 1 with MES buffer solution of 30 mM and pH=5 under the action of an external magnetic field, so that the water-soluble carboxyl nano-magnetic particles are dispersed in the MES buffer solution with a final concentration of 40 mg / ml ;

[0058] 2. Take 100 μl of water-soluble carboxyl nano-magnetic particle suspension, add 50 μl each of EDC (50 mM) and NHS (50 mM) solutions in sequence, and shake at room temperature for 30 minutes;

[0059] 3. Discard the supernatant under the action of a magnetic field, and wash with MES solution for 3 times;

[0060] 4. Take 100 μl of the anti-Cry1Ab / Cry1Ac insecticidal protein polyclonal antibody (1.09 mg / ml) prepared in Example 2, add activated water-soluble carboxyl nano-magnetic particles, and shake at room temperature for 30 minutes;

[0061] 5. Magnetic separation, collecting the supernat...

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Abstract

The invention discloses an immune nano-magnetic particle for detecting Cry1Ab / Cry1Ac insecticidal proteins and a preparation method thereof. The immune nano-magnetic particle for detecting Cry1Ab / Cry1Ac insecticidal proteins disclosed by the invention is coated by a polyclonal antibody resisting the Cry1Ab / Cry1Ac insecticidal proteins, and the size of the magnetic particle is 150-200nm. The immune nano-magnetic particle is prepared via the steps of: preparing a carboxyl nano-magnetic particle with high water solubility by an improved chemical precipitation method, and coupling the antibody resisting the Cry1Ab / Cry1Ac insecticidal proteins with the activated nano-magnetic particle by a chemical coupling method. The immune nano-magnetic particle is used for detecting the Cry1Ab / Cry1Ac insecticidal proteins via a chemiluminescence EIA (enzyme linked immuno assay) method; the sensitivity is high; the maximum detected protein concentration is 1000ng / ml, and the minimum detectable concentration is 1ng / ml; and magnetic enrichment is performed easily, so as to remove the interference of impurity molecules.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to an immune nano-magnetic particle for detecting Cry1Ab / Cry1Ac insecticidal protein and a preparation method thereof. Background technique [0002] Bt protein is a paraspore crystal protein produced by Bacillus thuringiensis during spore formation. Its molecular structure is complex, and it exists in the form of toxin protein in the paraspore crystal, and can be activated by proteolysis in the intestinal tract of insects; The activated Bt toxin protein can interact with specific receptors on the surface of insect intestinal epithelial cells, induce and cause cell swelling or even lysis, causing insects to stop eating and eventually die. Through transgenic technology, the gene of Bt toxin protein is introduced into crops, which provides a new idea for controlling pest damage to agriculture. [0003] At present, the Cry-like Bt toxin protein is the most widely used one, and the Cry...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
Inventor 陈法军潘卫东
Owner NANJING AGRICULTURAL UNIVERSITY
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