Method for measuring mpo halogenase activity

A technology of halogenase activity and halogenase, which is applied in the field of natural protease purification, can solve the problems of long time, long time required, high preparation cost, etc., and achieve the effect of strong specificity

Active Publication Date: 2019-01-25
CHANGCHUN HENGXIAO BIOTECH CO LTD
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Problems solved by technology

However, this method to determine the content of MPO in the blood indirectly indicates the activity of MPO enzyme in the blood. Sometimes the content of MPO is not related to the activity of MPO enzyme, such as the population with MPO gene variation and the population with MPO inhibitors in the blood; The ELISA kit requires several MPO monoclonal antibodies, and the preparation cost is high and the time is long; at the same time, the ELISA method has many steps, takes a long time, and has disadvantages such as poor accuracy

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  • Method for measuring mpo halogenase activity
  • Method for measuring mpo halogenase activity
  • Method for measuring mpo halogenase activity

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Embodiment Construction

[0069] The step that the MPO halogenase measured by the present invention is separated from HL-60 cells is:

[0070] a. Isolate MPO from HL-60 cells: culture HL-60 cells, collect the cells by centrifugation, lyse the cells with phosphate buffer (pH8.0), and then use Dounce homogenization to break the cells, centrifuge, and collect the cell sediments. Cell sediments contain MPO;

[0071] b. 1% CTAB (Cetyltrimethylammonium bromide)-phosphate buffer (pH8.0) dissolves MPO in the above cell deposits;

[0072] c. Preparation of Q-Sepharose chromatography column: Equilibrate the Q-Sepharose chromatography column with 0.5% CTAB-phosphate buffer (pH8.0), the above MPO solution passes through the Q-Sepharose chromatography column, and the binding protein (pI≤8.0 ) onto the Q-Sepharose chromatography column, and collect the filtrate (containing MPO, pI=9.2);

[0073] d. Preparation of SP-Sepharose chromatography column: Equilibrate the SP-Sepharose chromatography column with 0.2% CTAB-...

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Abstract

A method for measuring MPO halogenase activity, the purpose of the present invention is to separate MPO with natural protein structure and enzymatic activity from HL-60 cells by ion chromatography, establish a method for measuring MPO halogenase activity, and use purified MPO as calibration The product was used to measure MPO halogenase activity units in human serum / plasma. The MPO halogenase assayed in the present invention is isolated from HL-60 cells. The present invention establishes the NaI-TMB (3,3',5,5'-tetramethylbenzidine, tetramethylbenzidine) method to directly measure the MPO halogenase activity in serum / plasma, and is used to predict the possibility and disease occurrence of cardiovascular and cerebrovascular diseases Prognosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for purifying natural protease. Background technique [0002] Myeloperoxidase (Myeloperoxidase, MPO), also known as peroxidase, is a glycosylated heme protease secreted by neutrophils, monocytes and macrophages in some tissues , is a member of the heme peroxidase superfamily. Before human granulocytes enter the circulation, MPO is synthesized in the bone marrow and stored in azurophilic granules. External stimuli can cause neutrophils to gather and release MPO. In mature granulocytes (mainly neutrophils and monocytes), MPO is the most abundant glycoprotein, accounting for about 5% of the total protein content in peripheral blood polymorphonuclear neutrophils (PMNs), 95% of MPO in blood originates from PMNs [0003] Myeloperoxidase (MPO) is a dimer composed of two subunits, each subunit consists of a heavy chain (α chain, 466 amino acid residues) and a light chain (β chain...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31G01N1/28G01N1/30G01N1/34
CPCG01N1/28G01N1/30G01N1/34G01N21/31G01N2021/3185
Inventor 杜培革李宝民刘斌关大伟赵雪隋宝真王玉华史永丰王贺关恒安丽萍苑广信王曼力林林
Owner CHANGCHUN HENGXIAO BIOTECH CO LTD
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