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Protein cross-linked nano-silicon and preparation method, exosome separation and purification method and application

A protein cross-linking, separation and purification technology, applied in biochemical equipment and methods, cell dissociation methods, microorganisms, etc., can solve the problems of time-consuming and labor-consuming, low purity of exosomes, high cost, etc., to save time and The effect of cost, high purity, and guaranteed quality

Active Publication Date: 2021-06-25
XIAMEN ASSAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ultracentrifugation generally takes more than 7 hours, and is time-consuming and labor-intensive; the amount of exosomes collected by the molecular exclusion method, that is, the immune capture method, is small and expensive; although the PEG precipitation method can separate exosomes on a large scale, it is difficult to collect When a large amount of exosomes are obtained, lipoproteins are inevitably introduced, resulting in the problem of low purity of the isolated exosomes

Method used

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  • Protein cross-linked nano-silicon and preparation method, exosome separation and purification method and application
  • Protein cross-linked nano-silicon and preparation method, exosome separation and purification method and application
  • Protein cross-linked nano-silicon and preparation method, exosome separation and purification method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment a

[0062] Example A: 40ml of the outer bude liquid added to 10 6 10 7 10 8 10 9 10 10 Particles Number of Annexin V Crosslinked 10 μm Nanoicon (Set to Annexin V A, Annexin VD, Annexin V, Annexin VD, Annexin V E Series), add CACL 2 By 15 mm (millimoles per liter), the lower speed is mixed with 50 mL, and the shaker is mixed onto the shaker at a low speed of the shaker at 4 ° C; centrifuge at 2000g, abandon the supernatant and collect the precipitate; 1 ml CA- HEPESBuffer (including 10mm hepes, pH 7.5; 1mm MGCL 2 ; 5mm KCl; 2% BSA; 15mm CaCl 2 And 150 mMNacl was washed 3 times and naturally dried; then 500 ul eluent (containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA) was mixed with precipitate for 10 min, 4 ° C. Cartridge, centrifugation, The supernatant was collected, that is, the purified concentrated exosomer; the obtained exocial body was stored in the -80 ° C refrigerator.

Embodiment b

[0063] Example B: 40M Extracedrous Extraction of 10 6 10 7 10 8 10 9 10 10 Particles number of TIM 4 crosslinked 10 μm nano silicon (set to TIM 4A, TIM 4B, TIM 4C, TIM 4D, TIM 4E series), add CACL 2 To 2 mm, the shaker is mixed with 50 mL, and the lower speed is mixed with the shaker at low speed to the underside of 4 ° C. The post 4 ° C was centrifuged for 10 min, discarded the supernatant and collected precipitation; with washing disengagement (comprising 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.0005% Tween 20, 2mmcacl) 2 Centrifugation was washed 3 times and naturally air dried, 500 ul (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mMedta) was added to 500 ul eluent (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mm EDTA) and precipitation The mixture was mixed for 10 min, 4 ° C. Centrifuge at 4 ° C for 10 min, and the supernatant was collected, that is, the purified outer body; the obtained exocrid was prepared in the -80 ° C refrigerator.

[0064] Through NTA detection, the number of outer bo...

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Abstract

The present invention provides a protein cross-linked nano-silicon and its preparation method, exosome separation and purification method and application, wherein the protein cross-linked nano-silicon is prepared by modifying carboxylated nano-silicon spheres with Annexin V protein or Tim 4 protein. The protein cross-linked nano-silicon prepared by the present invention is used for the separation and purification of stem cell exosomes, which can quickly and large-scale separate the stem cell exosomes in the sample, and ensure that the isolated stem cell exosomes have a high purity. The field of preparation of stem cell exosomes is of great significance. However, the application of exosomes prepared by the exosome separation and purification method provided by the present invention in the preparation of exosome-related drugs, such as the application of drugs for nerve injury and heart repair, can save a lot of time and effort. The cost ensures the quality of the drug and has great application value.

Description

Technical field [0001] The present invention relates to the field of biomedical technology, and more particularly to a protein crosslinked nano silicon and a method of separation and purification method and application. Background technique [0002] Exosomes are cellular small sac saculations of a circular single layer film structure of 30 to 150 nm formed by a series of regulatory processes formed by * # * hemocytosis-fusion-outer row * # *. The exogenous body is released from many types of cells, and is widely distributed among saliva, plasma, milk, urine, etc., can carry protein, transport RNA, play an important role in cells and information transduction. The discovery of the foreign body enriching the understanding of the inter-cell communication method, deepening the understanding of the physiology and pathological process of the body, the 2013 Nobel Bio / Medical Award awarded three scientists who contributed in cell vesic To achieve a new height of the study of the foreign...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2509/00
Inventor 孙坪田云鹏李柱
Owner XIAMEN ASSAY BIOTECH CO LTD
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