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Nucleic acid aptamer of programmed death receptor-ligand 1 (PD-L1) and application of nucleic acid aptamer

A technology of programmed death and nucleic acid aptamer, which is applied in the field of molecular recognition tools to detect the expression level of PD-L1, can solve the problems of ununiform pathological evaluation standards, easy inactivation, high cost, etc., and achieve broad application prospects and low synthesis cost , not easy to inactivate the effect

Active Publication Date: 2019-07-12
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some problems in the detection of PD-L1 expression level at present, such as the current detection antibodies are different, the staining techniques and conditions are not uniform, and the pathological evaluation standards are not yet unified, resulting in the inability to effectively detect responders and non-responders. identification
In addition, antibodies have the disadvantages of instability, easy inactivation, and high cost

Method used

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  • Nucleic acid aptamer of programmed death receptor-ligand 1 (PD-L1) and application of nucleic acid aptamer
  • Nucleic acid aptamer of programmed death receptor-ligand 1 (PD-L1) and application of nucleic acid aptamer
  • Nucleic acid aptamer of programmed death receptor-ligand 1 (PD-L1) and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Screening of nucleic acid aptamers for programmed death receptor-ligand 1

[0068] 1) chemically synthesized DNA initial library, as shown in SEQ ID No.8, wherein each DNA strand consists of 80 bases, including fixed sequences of 20 bases at both ends and random sequences of 40 bases in the middle, Specifically: 5'-AGC GTC GAATAC CAC TAC AG-40nt-CTA ATG GAG CTC GTG GTC AG-3', the library capacity is 10 15 above. Take 3nmol DNA initial library dissolved in binding buffer (12mmol / L PBS, pH 7.4, 150mmol / L NaCl, 5mmol / L KCl, 0.55mmol / L MgCl 2 ), perform denaturation treatment: 95°C for 10 minutes, then place on ice for 5 minutes, and finally place at room temperature for 10 minutes;

[0069] 2) Prokaryotic expression of the fusion protein His-PD-L1, through the affinity interaction between His and Ni, the PD-L1 protein was immobilized on Ni agarose microspheres (beads) to obtain PD-L1 protein agarose microspheres PD- L1 beads; incubate the pretreated initial li...

Embodiment 2

[0074] Example 2 Investigating the binding ability of single-stranded DNA enrichment library to PD-L1 protein by flow cytometry

[0075]First, use PCR to amplify the 0, 8, 12, 18, 20, 22, 24, and 26 rounds of DNA enrichment libraries with fluorescent labels, respectively, using the forward primer (SEQ ID No.9): 5'-FAM-AGC GTC GAA TAC CAC TAC AG-3' and reverse primer (SEQ IDNo.10): 5'-Biotin-CTG ACC ACG AGC TCC ATT AG-3', the PCR product has FAM at the 5' end and a band at the 3' end For double-stranded DNA with biotin, add streptavidin agarose microspheres, react at room temperature for 20 minutes, and then use 0.1mol / L NaOH to carry out alkali denaturation and single-strandization, desalt and filter through NAP-5 column, and purify to obtain No. 0, 8, 12, 18, 20, 22, 24, 26 rounds of single-stranded DNA enrichment library;

[0076] Use 200 μl of binding buffer to prepare single-stranded DNA enrichment library solutions for rounds 0, 8, 12, 18, 20, 22, 24, and 26 at a concent...

Embodiment 3

[0078] Example 3 Determination of the dissociation constant between the 26th round single-stranded DNA enrichment library and PD-L1 protein by flow cytometry

[0079] First PCR amplifies the 26th round DNA enrichment library with fluorescent labeling, using the forward primer 5'-FAM-AGC GTCGAA TAC CAC TAC AG-3' (SEQ ID No.9): with the reverse primer (SEQ ID No. .10): 5'-Biotin-CTG ACCACG AGC TCC ATT AG-3', the PCR product is double-stranded DNA with FAM at the 5' end and biotin at the 3' end, add streptavidin agarose micro spheres, reacted at room temperature for 20 minutes, and then used 0.1mol / L NaOH to perform alkali denaturation and single-strandization, desalted and filtered through NAP-5column, and purified to obtain the 26th round of single-stranded DNA enrichment library;

[0080] The dissociation constant (K d ). Use 200 μl of binding buffer to configure the 26th round of single-stranded DNA enrichment library solutions of the above concentrations, and add about 10 ...

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Abstract

The invention discloses a nucleic acid aptamer of a programmed death receptor-ligand 1 (PD-L1) and application of the nucleic acid aptamer, and relates to a nucleic acid. The nucleic acid aptamer withhigh specific recognition on PD-L1 and high affinity combination force on PD-L1 is provided. The nucleic acid aptamer can be screened and prepared by means of a protein SELEX method based on agarosemicrobead separation-flow cytometry analysis. The nucleic acid aptamer has the advantages that G basic groups are abundant, a stem-loop structure is provided, synthesis and marking are easy, the costis low, the stability is high, and non-immunogenicity is excellent; the nucleic acid aptamer is utilized to achieve the specific recognition of PD-L1 proteins, tumor cell lines of PD-L1 expression proteins and exudates of the PD-L1 expression proteins excreted by the tumor cell lines, the application of the nucleic acid aptamer in the field of biochemical analysis and detection is verified, the nucleic acid aptamer is expected to be used as a molecular recognition tool for detecting the expression level of PD-L1 and applied to precise prediction and dynamic monitoring of the treatment effect of PD-1 / PD-L1 inhibitors in the field of clinical medical tumor immunity research.

Description

technical field [0001] The present invention relates to a nucleic acid aptamer capable of highly specific recognition and high affinity binding to programmed death receptor-ligand 1 and its application in biochemical analysis and clinical medicine, such as the field of tumor immunity research, and is expected to be used as an aptamer for detecting PD-L1 Molecular recognition tools for expression levels. Background technique [0002] With the advancement of science and the development of cancer-related disciplines, tumor immunity research and immunotherapy have developed rapidly. At present, it is believed that immune escape is an important mechanism of tumor development, and T cells play an important role in immune escape. Programmed death receptor (PD-1, Programmed Death 1) is expressed on the surface of T cells, while programmed death receptor-ligand 1 (PD-L1, Programmed Death-Ligand 1) is expressed on the surface of tumor cells, which is associated with T The PD-1 prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/574A61K47/26A61P35/00
CPCA61K47/26A61P35/00C12N15/115C12N2310/16G01N33/57492G01N33/68G01N2333/70532
Inventor 杨朝勇黄梦娇宋彦龄王腾陈小锋朱志
Owner XIAMEN UNIV
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