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Affinity chromatography medium for separating and purifying GABA receptor and preparation method thereof

A technology for separation, purification and chromatography medium, which is applied in the biological field to achieve the effect of stable and reliable purification method, great application prospect and low price.

Active Publication Date: 2015-10-14
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fipronil acts on GABA receptors and has high affinity for GABA receptors. So far, there has been no report of using fipronil as an affinity ligand to isolate GABA receptors

Method used

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  • Affinity chromatography medium for separating and purifying GABA receptor and preparation method thereof
  • Affinity chromatography medium for separating and purifying GABA receptor and preparation method thereof
  • Affinity chromatography medium for separating and purifying GABA receptor and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation method of fipronil affinity ligand

[0029] Synthesis of product A: Add 4.40 g (10 mmol) fipronil and 30 mL CH 2 Cl 2 , add 2 g of 4-dimethylaminopyridine (DMAP) and 4 mL of triethylamine under ice bath and nitrogen protection, stir for 30 min, add 4 mL of bromoacetyl bromide (about 46 mmol) dropwise, and react at room temperature for 8 h. The reaction solution was poured into 40 mL of ice water and washed with CH 2 Cl 2 (20 mL × 3) extraction, combined organic phase, anhydrous MgSO 4 Dry overnight, precipitate under reduced pressure, and separate by silica gel chromatography [V 石油醚 :V 乙酸乙酯 =8:1] to obtain a yellow solid A with a yield of 70%, m.p. 167-169°C.

[0030] Synthesis of product B: Add 2.32g (0.004mol) of product A, 7.4g (0.04mol) of potassium phthalimide, 0.8g of tetrabutylammonium bromide (TBAB) and 40mL of DMF into a 100mL three-necked flask , Reaction at 80°C for 6h. The reaction solution was poured into 40 mL of ice water and...

Embodiment 2

[0032] Example 2 Preparation method of fipronil affinity ligand

[0033] Synthesis of product A: Add 4.40 g (10 mmol) fipronil and 30 mL CH 2 Cl 2 , add 2 g of 4-dimethylaminopyridine (DMAP) and 4 mL of triethylamine under ice bath and nitrogen protection, stir for 30 min, add 2.6 mL of bromoacetyl bromide (about 30 mmol) dropwise, and react at room temperature for 12 h. The reaction solution was poured into 40 mL of ice water and washed with CH 2 Cl 2 (20 mL × 3) extraction, combined organic phase, anhydrous MgSO 4 Dry overnight, precipitate under reduced pressure, and separate by silica gel chromatography [V 石油醚 :V 乙酸乙酯 =8:1] to obtain a yellow solid A with a yield of 70%, m.p. 167-169°C.

[0034] Synthesis of product B: Add 2.32g (0.004mol) of product A, 3.7g (0.02mol) of potassium phthalimide, 0.8g of tetrabutylammonium bromide (TBAB) and 40mL of DMF into a 100mL three-necked flask , reacted at 80°C for 10 h. The reaction solution was poured into 40 mL of ice water...

Embodiment 3

[0036] Example 3 Preparation of affinity chromatography medium

[0037]Take 5 g of epoxy-activated Sepharose CL-6B gel, washed and drained, and add it to a 50 mL round bottom flask. Add 500 mg of fipronil affinity ligand, 20 mL of 0.1 mol / L sodium carbonate buffer (pH 9.0) and 5 mL of DMSO into the reaction flask, and shake in a water bath at 37 °C for 12 h (attached figure 1 ). Transfer the material liquid to a sand core funnel to drain, wash with deionized water, add 1 mol / L ethanolamine and shake at 37°C for 4 h; Sodium buffer solution, deionized water and 0.1 M pH 8.0 NaAc containing 0.5 M NaCl boric acid-sodium tetraborate buffer solution for washing and washing, and drying after washing to obtain the fipronil ligand affinity chromatography medium of the present invention. The prepared gel was stored in 20% ethanol at 4°C for future use.

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Abstract

The invention relates to an affinity chromatography medium for separating and purifying a GABA receptor and a preparation method thereof. The technological process comprises the following steps: carrying out chemical modification on fipronil to obtain an affinity ligand; by taking agarose gel as a matrix, after activating by an epoxy activating agent, coupling the fipronil affinity ligand to obtain an affinity medium; packing the affinity medium so as to be used for separating and purifying fish GABA receptor protein. The affinity chromatography medium synthesized according to the invention has the advantages of being good in selectivity, strong in specificity, high in mechanical strength and good in separation effect; in addition, the preparation process is simple and the magnifying is easy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an affinity chromatography medium for separating and purifying GABA receptor protein and a preparation method thereof. Background technique [0002] γ-aminobutyric acid (gama-aminobutyric acid, GABA) receptors are the main inhibitory neurotransmitter receptors in the nervous system of insects and mammals, not only for insecticides such as fipronil, avermectin and lindane The target of action is also closely related to many neurological diseases in humans, including epilepsy, schizophrenia, insomnia, Parkinson's syndrome, etc. Therefore, the isolation and purification of GABA receptor and the study of its three-dimensional structure have important guiding significance for the development of high-efficiency, low-toxicity, and safe pesticides and for overcoming the resistance of pests. [0003] Affinity chromatography is a liquid chromatography method that uses an affinity ...

Claims

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Application Information

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IPC IPC(8): B01J20/24B01J20/281B01J20/30C07K14/705C07K1/36C07K1/34C07K1/22C07K1/18
Inventor 任天瑞于广金张雷张博庞文文
Owner SHANGHAI NORMAL UNIVERSITY
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