Protein cross-linking nano silicon as well as preparation method and 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 high cost, low exosome purity, time-consuming and labor-intensive, saving time and energy. Cost, high purity, guaranteed quality effect

Active Publication Date: 2018-11-20
XIAMEN ASSAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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-linking nano silicon as well as preparation method and exosome separation and purification method and application
  • Protein cross-linking nano silicon as well as preparation method and exosome separation and purification method and application
  • Protein cross-linking nano silicon as well as preparation method and exosome separation and purification method and application

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Embodiment a

[0062] Example a: 10 6 , 10 7 , 10 8 ,10 9 , 10 10 The number of Annexin V cross-linked 10μm nano-silicon (set to Annexin V a, Annexin V b, Annexin V c, Annexin Vd, Annexin V e series), add CaCl 2 to 15mM (mmoles per liter), dilute to 50ml, mix on a sideways shaker at low speed at 4°C for 1h-12h; centrifuge at 2000g for 10min at 4°C, discard the supernatant and collect the precipitate; use 1ml Ca- HEPESbuffer (containing 10mM HEPES, pH 7.5; 1mM MgCl 2 ; 5mM KCl; 2% BSA; 15mM CaCl 2 ; and 150mMNaCl) and washed by centrifugation for 3 times and then air-dried; then add 500ul eluent (including 20mM Tris-HCl, pH 7.4, 150mM NaCl, 2mM EDTA) and mix with the precipitate and let it stand for 10min, then centrifuge at 500g for 10min at 4°C, The collected supernatant is the purified concentrated exosomes; the prepared exosomes are stored in a -80°C refrigerator for later use.

Embodiment b

[0063] Example b: Add 10 to 40m exosome crude extract 6 , 10 7 , 10 8 ,10 9 , 10 10 Tim 4 of the number of particles cross-links 10μm nano-silicon (set to Tim 4a, Tim 4b, Tim 4c, Tim 4d, Tim 4e series), add Cacl 2 to 2mM, dilute to 50ml, and mix on a sideways shaker at low speed for 1-12h at 4°C. Then centrifuge at 2000g for 10min at 4°C, discard the supernatant and collect the precipitate; 2 ) and washed by centrifugation for 3 times and air-dried, with 500ul (20mM Tris-HCl, pH 7.4, 150mM NaCl, 2mM EDTA); then add 500ul eluent (20mM Tris-HCl, pH 7.4, 150mM NaCl, 2mM EDTA) and precipitate Mix and let stand for 10 minutes, centrifuge at 500g for 10 minutes at 4°C, collect the supernatant, which is the purified exosomes; the prepared exosomes are stored in a -80°C refrigerator for later use.

[0064] The number of exosomes prepared by the above method was detected by NTA, and the yield of exosomes was analyzed. The test results were as follows: figure 2 shown by figure...

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Abstract

The invention provides protein cross-linking nano silicon as well as a preparation method and an exosome separation and purification method and application. The protein cross-linking nano silicon is prepared from carboxylated nanometer silicon spheres modified with an Annexin V protein or Tim 4 protein. The protein cross-linking nano silicon provided by the invention is applied to separation and purification of cell exosome, stem cell exosome in a sample can be rapidly separated on a large scale, high purity can be ensured for the separated stem cell exosome, and the silicon has great significances in the field of preparation of the stem cell exosome. Due to application of the exosome prepared by using the exosome separation and purification method provided by the invention in preparing medicines related to the exosome, such as application in preparing medicines for nerve injury and heart repairing, a great deal of time can be saved, a lot of cost can be reduced, the quality of the medicines can be ensured, and the application values are great.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a protein cross-linked nano-silicon and its preparation method, exosome separation and purification method and application. Background technique [0002] Exosomes are small extracellular vesicles with a diameter of 30-150 nm and a circular single-layer membrane structure formed by cells through a series of regulatory processes such as "endocytosis-fusion-efflux". Exosomes are released by many types of cells in the body and are widely distributed in body fluids such as saliva, plasma, milk, and urine. They can carry proteins and transport RNA, and play an important role in intercellular material and information transduction. The discovery of exosomes has enriched researchers' understanding of intercellular communication and deepened their understanding of the body's physiological and pathological processes. The 2013 Nobel Prize in Biology / Medicine was awarded to three scientis...

Claims

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Application Information

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Patent Type & Authority Applications(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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