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Active carrier and preparation method for directional immobilization of protein A and preparation method of protein A immunoadsorption material

An immunoadsorption material and active carrier technology, applied in the field of protein immobilization, can solve the problems of high price, high synthesis cost of succinimide-based carrier, loss of activity, etc., to reduce production cost and protein A immunoadsorption therapy Cost, effect of improving adsorption capacity

Active Publication Date: 2020-04-07
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the clinical promotion and application of Immunosorba adsorption column, there are two shortcomings: the preparation cost of protein A immunosorbent material is high, resulting in high price; the activity loss of protein A after coupling with the carrier leads to poor adsorption performance
However, due to the presence of sulfhydryl groups, protein A is easy to form disulfide bonds, and a reducing environment needs to be provided during the reaction, and the synthesis cost of succinimide-based carriers is high, which limits its wide application.

Method used

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  • Active carrier and preparation method for directional immobilization of protein A and preparation method of protein A immunoadsorption material
  • Active carrier and preparation method for directional immobilization of protein A and preparation method of protein A immunoadsorption material
  • Active carrier and preparation method for directional immobilization of protein A and preparation method of protein A immunoadsorption material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Preparation of active carrier

[0035](1) Epichlorohydrin activation: take 100mL of agarose gel microspheres (Sepharose 6FF), rinse with distilled water, drain, add to a 1000mL round bottom flask, add 150mL of 3mol / L sodium hydroxide aqueous solution , 0.3 g of sodium borohydride, 100 mL of epichlorohydrin, placed in a constant temperature shaker, reacted at 40 ° C for 2 hours, rinsed with a large amount of distilled water until neutral, and ended the reaction. The amount of epoxy groups on the carrier was detected by the sodium thiosulfate method, and it was measured that there were 55 μmol of epoxy groups per gram of the carrier.

[0036] (2) Bonded aspartic acid: Take 100 mL of the agarose gel obtained by the reaction in step (1), wash it with a large amount of distilled water, drain it and add it to a 1000 mL round bottom flask, add 500 mL of 1.5 mol / L The aspartic acid solution (adjusted to pH 12 with sodium carbonate) was placed in a constant temperature shake...

Embodiment 2

[0061] 1. Preparation of active carrier

[0062] (1) Epichlorohydrin activation: take 100mL cellulose microspheres (MT500, 80-100μm), rinse with distilled water, drain, add to a 1000mL round bottom flask, add 150mL 2mol / L sodium hydroxide Aqueous solution, 0.3 g of sodium borohydride, 100 mL of epichlorohydrin, placed in a constant temperature shaker, reacted at 30 ° C for 6 hours, washed with a large amount of distilled water until neutral, and ended the reaction. The amount of epoxy groups on the carrier was detected by the sodium thiosulfate method, and it was measured that there were 45 μmol of epoxy groups per gram of carrier.

[0063] (2) Bonded aspartic acid: take 100 mL of the cellulose microspheres obtained by the reaction in step (1), rinse them with a large amount of distilled water, drain them and add them to a 1000 mL round bottom flask, add 500 mL of 0.5 mol / L The aspartic acid solution (adjusted to pH 11 with sodium carbonate) was placed in a constant temperatu...

Embodiment 3

[0075] 1. Preparation of active carrier

[0076](1) Epichlorohydrin activation: Take 100mL of agarose gel microspheres (Sepharose 4FF), wash them with distilled water, drain them, add them to a 1000mL round bottom flask, and add 150mL of 2mol / L sodium hydroxide aqueous solution , 0.3 g of sodium borohydride, 100 mL of epichlorohydrin, placed in a constant temperature shaker, reacted at 20 ° C for 8 hours, rinsed with a large amount of distilled water until neutral, and ended the reaction. The amount of epoxy groups on the carrier was detected by the sodium thiosulfate method, and it was measured that there were 35 μmol of epoxy groups per gram of the carrier.

[0077] (2) Bonded aspartic acid: Take 100 mL of the agarose gel microspheres obtained by the reaction in step (1), rinse with a large amount of distilled water, drain and add to a 1000 mL round bottom flask, add 500 mL of 0.2 mol / L of aspartic acid solution (adjusted to pH 10 with sodium carbonate), placed in a consta...

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Abstract

The invention discloses an immunoadsorption material prepared by direct oriented immobilization of the protein A from cell disruption supernate and particularly discloses an active carrier for preparing the immunoadsorption material and a method for preparing a protein A immunoadsorption material by using the carrier. The active carrier is obtained by taking hydrophilic gel microspheres as a substrate and reacting the hydrophilic gel microspheres with epoxy chloropropane, aspartic acid, epoxy chloropropane and sodium hydroxide in sequence. The active carrier contains a metal chelating group and an epoxy group and can be used for direct oriented immobilization of target protein with a histidine tag from the cell disruption supernate under certain conditions. By using the active carrier disclosed by the invention, the protein A immunoadsorption material can be prepared by direct immobilization of the protein A with the histidine tag from the cell disruption supernate, and the purification process of the protein A is omitted, so that the immobilization cost is greatly reduced; immobilization conditions are mild; the prepared protein A immunoadsorption material has high adsorption efficiency and can be used for clinical immunoadsorption therapy.

Description

technical field [0001] The invention relates to the technical field of protein immobilization, in particular to an active carrier and preparation method for directional immobilization of protein A and a preparation method of protein A immunoadsorption material. Background technique [0002] Various autoimmune diseases and organ transplant rejection are a series of diseases caused by the production of autoantibodies in the human body against their own tissues and organs or transplanted organs, causing damage to tissues and organs. It is a difficult problem in the treatment of domestic and foreign medical circles one. Autoimmune diseases are caused by the disorder of the human immune system, and have a high disability rate or fatality rate. Common ones include systemic lupus erythematosus (SLE), hemophilia, rheumatoid arthritis (RA), systemic sclerosis (SSc), myasthenia gravis (MG), Sjogren's syndrome (SS), etc. Transplant rejection is the main cause of organ transplant fail...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/24B01J20/30C07K1/22
CPCB01J20/24B01J2220/4868C07K1/22
Inventor 张旭锋杨家梅邓瑶杨海艳郭仁玲王宇
Owner YUNNAN NORMAL UNIV
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