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A method for detecting PD-L1 protein on the surface of extracellular vesicles by nucleic acid aptamer of programmed death receptor-ligand 1

A technology of PD-L1 and programmed death, which is applied in the field of detecting PD-L1 protein on the surface of extracellular vesicles by nucleic acid aptamers of programmed death receptor-ligand 1, which can solve the cumbersome detection method steps and large sample consumption , Complicated and time-consuming operations and other issues, to achieve the effect of affordable price, high sensitivity, fast and efficient detection

Active Publication Date: 2020-10-09
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The main purpose of the present invention is to overcome the uncertainty and incompleteness of detecting the expression level of tumor cell PD-L1 in the prior art, as well as the problems of cumbersome detection method steps, complex and time-consuming operation, large sample consumption, and high price. Provides a method for detecting PD-L1 protein on the surface of extracellular vesicles with a nucleic acid aptamer for programmed death receptor-ligand 1, using a nucleic acid aptamer that recognizes PD-L1 with high specificity and high affinity to detect extracellular vesicles The PD-L1 expressed on the surface can be detected by micro-thermophoresis equipment. This method is simple, fast, efficient, affordable, sensitive and sensitive, and consumes a small amount of detection samples.

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  • A method for detecting PD-L1 protein on the surface of extracellular vesicles by nucleic acid aptamer of programmed death receptor-ligand 1
  • A method for detecting PD-L1 protein on the surface of extracellular vesicles by nucleic acid aptamer of programmed death receptor-ligand 1
  • A method for detecting PD-L1 protein on the surface of extracellular vesicles by nucleic acid aptamer of programmed death receptor-ligand 1

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Effect test

Embodiment 1

[0035] Example 1 Extraction of exosomes secreted by melanoma cell line A375 by differential centrifugation

[0036] For exosomes extracted from the culture supernatant of the melanoma cell line A375, avoiding interference from exosomes in fetal bovine serum, bovine exosomes were obtained by centrifuging fetal bovine serum overnight at 100,000 × g to remove exosomes. Human fetal bovine serum (FBS). Then, cells were cultured at 37°C in medium supplemented with 10% exosome-depleted FBS and 1% (v / v) penicillin-streptomycin. After 60 h of incubation, the cell culture supernatant was collected and centrifuged at 2000 x g for 20 min at 4 °C (Eppendorf, 5424R) to remove cells and cell debris, and the supernatant was collected and centrifuged at 16500 x g for 45 min at 4 °C To remove a large number of microvesicles (Eppendorf, 5424R). The microvesicle pellet was resuspended in PBS and stored in a refrigerator at 4°C. The supernatant was centrifuged at 100,000g for 2 hours at 4°C to ...

Embodiment 2

[0037] Example 2 Morphological characterization of exosomes secreted by melanoma cell line A375

[0038] To identify exosomes, morphological analysis of exosomes is required. Then, in order to characterize the purified vesicles as exosomes, it can be characterized by transmission electron microscopy and nanoparticle tracking analyzer. First, the shape and size of the exosomes were characterized by transmission electron microscopy. The specific operation: the exosomes were dropped on the carbon-coated copper grid and allowed to stand for 1 minute (use tweezers to clamp the copper grid lightly to prevent the copper grid from After staining with 2% uranyl acetate at room temperature for 1 minute, blot the dye solution along the edge with filter paper, put the copper grid under the lamp and bake it for 10 minutes, and use a transmission electron microscope (Tecnai, G2spirit FEI, USA) observed and photographed. As for the concentration and particle size distribution of exosomes, ...

Embodiment 3

[0040] Example 3 Verification that exosomes secreted by the melanoma cell line A375 express PD-L1

[0041] After successful extraction of exosomes, it is important to characterize the proteins expressed by the exosomes, such as CD63 and PD-L1, which can be identified by western blotting. Use 12% SDS-PAGE electrophoresis analysis, load cell lysate (W), microvesicles (M) and exosomes (E) respectively, the total amount of protein loaded is equal, and the three gels run in parallel; then transfer to nitric acid On the cellulose membrane, the blot was blocked with blocking buffer (Beyotime Biotechnology) for 1 hour at room temperature, and after washing, it was incubated with PD-L1, CD63, and Actin primary antibodies at 4°C overnight. After washing, HRP-conjugated The secondary antibody (Cell Signaling Technology) was incubated at room temperature for 2 hours; after washing, the blot on the color membrane was developed with ECL detection reagent (Pierce), and the chemiluminescence ...

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Abstract

The invention discloses a method of detecting an extracellular vesicle surface PD-L1 protein by aptamer of a programmed death receptor-ligand 1 (PD-L1). The method comprises the following steps: 1) asample mixture of a PD-L1 aptamer with a constant concentration and extracellular vesicles with different concentrations is prepared; 2) the sample mixture is placed in a microscale thermophoresis (MST) device for measurement; and 3) the fluorescence of each sample mixture with a different extracellular vesicle concentration is measured, drawing is carried out on the obtained normalized fluorescence in relative to time, an MST trajectory is fit, drawing is then carried out on the normalized fluorescence of the MST trajectory in relative to the extracellular vesicles, a binding curve associatedwith the extracellular vesicle concentration and the normalized fluorescence is fit, and thus, extracellular vesicle surface PD-L1 protein detection is realized. Compared with the prior art, the method disclosed in the invention has the advantages that the sensitivity is high, the operation is simple and convenient, the detection is quick and efficient, few samples are consumed, an economic and affordable price is realized, and the potential for wide promotion is realized.

Description

technical field [0001] In recent years, precision medical liquid biopsy technology has been widely used in early cancer screening, auxiliary diagnosis, drug efficacy prediction and monitoring and other fields. Accurate prediction and dynamic monitoring of the efficacy of PD-1 / PD-L1 inhibitors has become the most concerned issue in the clinical community. The present invention relates to a method for detecting PD-L1 protein on the surface of extracellular vesicles by a nucleic acid aptamer of programmed death receptor-ligand 1, which will contribute to the advancement of a new method for detecting the expression level of PD-L1. Background technique [0002] With the development of science and technology, as a new revolutionary technology for tumor treatment, immunotherapy, especially immune checkpoint therapy, has developed rapidly and has become one of the important directions in the field of cancer research. Among them, immunotherapy based on PD-1 / PD-L1 antibody is a resea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115
CPCG01N33/532G01N33/533G01N33/6803
Inventor 杨朝勇黄梦娇宋彦龄王腾陈小锋朱志
Owner XIAMEN UNIV
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