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Method for separating and purifying natural protease 3 from azurophilic granules of human blood neutrophile granulocytes

A technology for separation and purification of neutrophils, applied in the field of medical testing products, can solve the problems of yield and purity impact, cumbersome separation steps of chromatographic columns, loss, etc., to improve the extraction process, increase the enrichment degree and relative purity , the effect obtained efficiently

Active Publication Date: 2018-10-02
武汉博百欧生物科技有限公司
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Problems solved by technology

Yield and purity are severely affected due to the crude whole cell extract containing a large amount of impurity proteins, the required chromatographic column separation steps are cumbersome, and there is a large amount of loss

Method used

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  • Method for separating and purifying natural protease 3 from azurophilic granules of human blood neutrophile granulocytes
  • Method for separating and purifying natural protease 3 from azurophilic granules of human blood neutrophile granulocytes
  • Method for separating and purifying natural protease 3 from azurophilic granules of human blood neutrophile granulocytes

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preparation example Construction

[0043] The existing preparation and purification process of natural protease 3 (PR3) has more impurity proteins in the blood neutrophil crude extract due to the unreasonable extraction process of primary white blood cell tissue fluid, and the insufficient chromatographic steps lead to a complete The impurity proteins in the crude cell extract cannot be separated well, resulting in the final yield, purity and specificity of native protease 3 being seriously affected. The present invention aims at the problems existing in the existing human blood protease 3 purification method, in order to obtain high-purity, high-activity and high-specificity natural protease 3 antigens in large quantities, through the research on the biochemical characteristics of natural protease 3 (PR3), design An improved method for isolating and purifying native proteinase 3 (Proteinase 3, PR3) from azurophilic granules of human blood neutrophils. The first purpose of this method is to improve the extracti...

Embodiment 1

[0049] In this example, 30.5 g of neutrophil azurophilic granules recovered and extracted from discarded medical supplies after human blood transfusion were used, ground, dissolved, and centrifuged to obtain a chromatographic initial protein solution containing protease 3, which was combined with A series of chromatographic methods such as cation exchange chromatography, dye ligand chromatography, molecular sieve chromatography and affinity chromatography finally obtained 35.0 mg of high-purity PR3 antigen. The specific implementation steps are as follows:

[0050] 1. Use PBS buffer solution to elute the white blood cells in the discarded medical supplies after human blood transfusion, centrifuge the eluate, discard the supernatant and keep the lower cell layer, add cell lysate to the cell layer and centrifuge, keep the lower layer of sediment, and then get astrocytosis Blue granule cell tissue. The azurophilic granulosa cell tissue is rich in proteinase 3. The centrifugatio...

Embodiment 2

[0059] In this example, the purity of the natural protease 3 antigen extracted and purified in Example 1 was verified, and the protease 3 antigen purified by India Yashraj Biotechnology Company was used for parallel comparison. The verification method is polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie blue staining, and then densitometry analysis (Densitometry Analysis) of the electrophoresis gel results after scanning with BioRad's ChemiDoc MP imager. The test results are attached Figure 11 shown. From attached Figure 11 It can be clearly seen from the protein glue shown that the proteinase 3 antigen extracted and purified from Example 1 has higher purity and thermal stability than the product of Yashraj Biotechnology Company in India.

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Abstract

The invention provides a method for separating and purifying natural protease 3 from azurophilic granules of human blood neutrophile granulocytes. The method comprises the following steps: using neutrophile granulocyte azurophilic granules which are recovered and extracted from waste medical supplies after human blood transfusion, grinding, dissolving and centrifuging to obtain a column chromatographic column initial protein solution containing the protease 3, and separating and purifying the natural protease 3 from the azurophilic granules of the human blood neutrophile granulocytes by combining purification methods such as a cation exchange chromatographic column, a dye ligand chromatographic column, a molecular sieve chromatographic column and an affinity chromatographic column. The yield, the purity, the activity, the specificity and the stability of the natural protease 3 prepared by the method are greatly improved, and the high quality is more suitable for scientific research andmedical detection.

Description

technical field [0001] The invention relates to the technical field of medical testing products, in particular to a method for separating and purifying natural protease 3 from azurophilic granules of human blood neutrophils. Background technique [0002] Antineutrophil cytoplasmic antibodies (ANCA) are found in necrotizing vasculitis. Most of these antibodies belong to the IgG type, and there are obvious positive reactions in the indirect immunofluorescence reaction of ethanol-fixed neutrophils and monocytes. One of their main targets is the proteinase 3 (PR3) antigen (the so-called cytoplasmic cANCA). Anti-proteinase 3 (PR3)-specific antibody is a sensitive and specific marker of neutrophil-specific autoantibody-associated vasculitic diseases, especially Wegener's granulomatosis (Wegener's granulomatosis), which can help Clinicians diagnose a variety of systemic inflammatory diseases. The antibody is more than 90% specific in patients with active Wegener's granulomatosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
CPCC12N9/6421
Inventor 孙鹏康雅明
Owner 武汉博百欧生物科技有限公司
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