Nucleic acid aptamer for detecting human pdl1 protein and its application in preparation of detection preparation

A nucleic acid aptamer and human detection technology, applied in the field of molecular biology, can solve the problems of nucleic acid aptamer reports without human PDL1 protein, and achieve the effects of easy in vitro chemical synthesis, stable sequence and good repeatability

Active Publication Date: 2021-03-26
HUNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] No aptamer targeting human PDL1 protein has been reported so far

Method used

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  • Nucleic acid aptamer for detecting human pdl1 protein and its application in preparation of detection preparation
  • Nucleic acid aptamer for detecting human pdl1 protein and its application in preparation of detection preparation
  • Nucleic acid aptamer for detecting human pdl1 protein and its application in preparation of detection preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the screening of the aptamer of human PDL1 protein specific binding

[0043] The PDL1 plasmid was constructed, transfected into the non-small cell lung cancer cell line H1299, and the cell line with stable and high expression of PDL1 was screened by sorting flow cytometry and designated as H1299-PDL1(H-P). Using the aptamer library related to non-small cell lung cancer reported so far, we investigated its binding ability to H1299 cells and H1299 cells (H-P) overexpressing PDL1 protein, and finally selected an aptamer with obvious differences and named it Ap3.

Embodiment 2

[0044] Example 2: Defining the binding of nucleic acid aptamer Ap3 to PDL1 protein

[0045] Incubate H-P cell lysate with aptamer Ap3 or library at 4°C for 1h, then incubate the mixture with streptavidin-labeled agarose beads at 4°C for 1h, wash with 2X protein loading buffer The protein bound to the beads was eluted and analyzed by Western Blotting.

[0046] Human pure PDL1 protein (Shenzhou Biological Technology Co., Ltd.) and Ap3 or library were incubated at 4 degrees for 1 hour, then the mixture was incubated with streptavidin-labeled agarose beads at 4 degrees for 1 hour, washed with 2X The proteins bound to the beads were eluted with protein loading buffer and analyzed by Western Blotting.

Embodiment 3

[0047] Example 3: Nucleic acid aptamer Ap3 can compete with PDL1 antibody for binding to H-P cells

[0048] The nuclear PDL1 antibody was incubated with H-P cells alone or Ap3 and PDL1 antibody 20 times higher than the PDL1 antibody were incubated with H-P cells at the same time. After washing, the fluorescence intensity (PE) of the PDL1 antibody bound to the cells was compared.

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Abstract

The invention discloses a nucleic acid aptamer for detecting a human PDL1 protein. The sequence of the nucleic acid aptamer contains a sequence shown in the formula of SEQ ID NO. 1. Compared with theexisting nucleic acid aptamer, the nucleic acid aptamer provided by the invention has affinity and specificity similar to those of a protein, a small molecular weight and low immunogenicity, can be easily synthesized in vitro and modified, has good stability and is easy to store. The nucleic acid aptamer can fast and simply detect a PDL1 protein level, has good repeatability and greatly reduces acost.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a nucleic acid aptamer that can be used for detecting human PDL1 at cell and tissue levels and an application method for preparing a detection reagent. [0002] technical background [0003] Traditional tumor treatment methods are mainly surgical treatment combined with chemotherapy and radiotherapy. But there are many deficiencies in traditional therapy, such as large side effects, patients are often tortured; in addition, it is easy to relapse. In recent years, more and more attention has been focused on new treatment methods such as targeted therapy and immunotherapy. [0004] The human immune system maintains a dynamic balance through immune activation and immunosuppression receptors. Taking T cells as an example, immunosuppression is mainly achieved through the immunosuppressive receptors on their surface, including two clinically used drug targets CTLA...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/535G01N33/533
CPCC12N15/115C12N2310/16G01N33/533G01N33/535G01N33/68C12N2310/3521C12N2310/51
Inventor 谭蔚泓孙洋叶茂方晓红莫柳婷赵子龙
Owner HUNAN UNIV
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