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Method of purifying Bt toxalbumin from insect-resistant cotton seeds

A Bt poisonous protein and insect-resistant cotton technology, applied in the field of biochemistry, can solve the problems reflecting the harm of Bt gene crops, changes in protein properties and functions, etc.

Inactive Publication Date: 2013-06-12
CHINA AGRI UNIV
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Problems solved by technology

[0004] Most of the Bt poisonous proteins used in current research come from prokaryotic expression. Since the expression products of foreign genes in plants may have complex post-translational modification processes, which may lead to changes in the properties and functions of proteins, prokaryotic proteins are used. The expressed Bt poisonous protein cannot fully reflect the harm of transgenic Bt crops

Method used

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  • Method of purifying Bt toxalbumin from insect-resistant cotton seeds
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  • Method of purifying Bt toxalbumin from insect-resistant cotton seeds

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preparation example Construction

[0065] Preparation method of cell culture medium: add calf serum (purchased from GIBCOBRL, catalog number 26170-043) and sodium bicarbonate to DMEM medium, the final concentration of calf serum is 20% (mass percentage content), The final concentration of sodium bicarbonate was 0.2% (mass percentage), and the pH was 7.4.

[0066] Bt Cry1Ac protein: Shanghai Youlong Biotechnology Co., Ltd., catalog number C010301.

[0067] Cotton variety Shiyuan 321: Hebei Shennong High-tech Co., Ltd.

[0068] The seeds of Guoxin Mian No. 6 (an insect-resistant variety transfected with the gene encoding the Bt Cry1Ac protein) were purchased from the Guoxin Agricultural Research Association in Hejian City, Hebei Province.

[0069] Unless otherwise specified, the PBS buffer used in this embodiment is as follows: the formula of PBS buffer (pH7.5): the solvent is water, containing 0.02M Na 2 HPO 4 、0.0015M KH 2 PO 4 and 0.14M NaCl.

Embodiment 1

[0070] Embodiment 1, purify Bt Cry1Ac protein from insect-resistant cotton seeds

[0071] 1. Extraction of total soluble protein

[0072] The seeds of Guoxin Cotton No. 6 were dehulled and ground into a fine powder. Take 10 g of the fine powder, add 100 ml of pH10.5, 0.01 MCAPS buffer, stir and extract with a magnetic stirrer at 4°C for 4 hours, and then centrifuge at 4°C and 10,000rpm for 20 Minutes, avoid the surface oil and take the upper liquid, which is the total soluble protein extract.

[0073] 2. Ammonium sulfate precipitation

[0074] Add ammonium sulfate powder to the soluble total protein extract obtained in step 1 to make its saturation in the extract reach 60%, and stir for 40 minutes in an ice bath using a magnetic stirrer.

[0075] 3. Reconstitution and dialysis of precipitate

[0076] Centrifuge the extract that completed step 2 at 4°C and 7000rpm for 20 minutes to collect the precipitate; add 2ml of deionized water to the precipitate, adjust the pH to 10.5 ...

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Abstract

The invention discloses a method of purifying Bt toxalbumin from insect-resistant cotton seeds. The invention provides a method of purifying Bt Cry1Ac protein from insect-resistant cotton seeds. The method comprises the following steps: (1) peeling the insect-resistant cotton seeds and then extracting total soluble protein to obtain a solution A; (2) conducting ammonium sulfate precipitation to the solution A, and centrifugally collecting the precipitate, wherein the saturation of the ammonium sulfate is 60%; (3) dissolving the precipitate obtained from the step (2), and dialyzing to obtain a solution B; and (4) conducting immuno-affinity chromatography to the solution B, collecting protein components, namely Bt toxalbumin, combined with the specificity of anti-Bt Cry1Ab / Ac protein monoclonal antibody, wherein the anti-Bt Cry1Ab / Ac protein monoclonal antibody is produced by secreting hybridoma Bt2F9 CGMCC No.5162. The method has the following advantages that the purification procedures are simplified, the purification cost is lowered, the purification time is shortened and reducing the loss of target proteins is reduced.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a method for purifying Bt toxin protein from insect-resistant cotton seeds. Background technique [0002] Bacillus thuringiensis (Bt for short) will produce a parasporal crystal during the formation of spores. The main component of this crystal is a protein with insecticidal activity, which is called insecticidal crystal protein (ICP, Insecticidal Crystal Protein) ), also known as Bt poisonous protein. [0003] Bt toxin can poison insects such as Lepidoptera, Diptera and Coleoptera, and is currently the most widely used biological insecticide in the world. Although humans and animals are not the natural target species of Bt toxic proteins, the debate on whether Bt genetically modified crops are harmful to humans, animals and the environment has never stopped. [0004] Most of the Bt poisonous proteins used in current research come from prokaryotic expression. Since the e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/325C07K1/36C07K1/34C07K1/30C07K1/22
Inventor 王保民张亮曹振郭素琴谭桂玉南铁贵李召虎
Owner CHINA AGRI UNIV
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