Extracellular vesicle surface protein specific aptamer screening technology based on magnetic-activated cell sorting

Abstract

The invention discloses an extracellular vesicle surface protein specific aptamer screening technology based on magnetic-activated cell sorting. The extracellular vesicle surface protein specific aptamer screening technology includes the following steps of: 1) synthesizing upstream primers, downstream primers and a random library; 2) culturing target cells and obtaining a cell culture supernatant;3) using a grain diameter selection method for extracting extracellular vesicles; 4) using immunomagnetic beads of which the surfaces are modified with CD63 antibodies for capturing the extracellularvesicles; 5) conducting positive screening to acquire library sequences capable of being combined with target extracellular vesicles; 6) conducting positive screening and negative screening alternatively to acquire specific target nucleic acid aptamers; and 7) adopting a flow cytometry technology to detect the enrichment condition of the library. According to the extracellular vesicle surface protein specific aptamer screening technology based on magnetic-activated cell sorting, the grain diameter selection method is adopted to extract the extracellular vesicles, the extracellular vesicles are extracted easily and rapidly, and the recovery rate of the extracellular vesicles is high; the immunomagnetic beads of which the surfaces are modified with the CD63 antibodies are used for capturingexosomes, and the extracellular vesicles can be separated rapidly; and furthermore, positive screening and negative screening are conducted alternatively, thus, the screening efficiency of the targetnucleic acid aptamers can be improved, and by utilizing the screened aptamers, disease liquid biopsies and treatment which take extracellular vesicle surface proteins as targets can be conducted.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an extracellular vesicle surface protein-specific aptamer screening technology based on an immunomagnetic bead method. Background technique [0002] Extracellular Vesicles (EVs) are vesicle-like bodies with a double-membrane structure that are shed from the cell membrane or secreted by the cell, with a diameter ranging from 40nm to 1000nm. They are released from the cell to the extracellular environment and exist in the blood. , saliva, amniotic fluid, breast milk and urine and other body fluids. EVs contain specific substances in their source cells, including proteins (such as cytokines, membrane receptor proteins, cytoskeletal proteins, heat shock proteins, etc.), nucleic acids (such as DNA, mRNA, small interfering RNA, long non-coding RNA, etc.) and lipids, and when different physiological conditions and diseases occur, proteins and nucleic acids in EVs are differ...

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Based on Cell-SELEX technology, a variety of tumor cell-specific aptamers have been screened, but...

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