Nucleic acid aptamer specifically binding to P-glycoprotein, and preparation method and application of nucleic acid aptamer

A nucleic acid aptamer and glycoprotein technology, applied in biochemical equipment and methods, DNA preparation, material inspection products, etc., can solve the problem of no related reports on nucleic acid aptamers, and achieve high specificity and high affinity

Active Publication Date: 2020-06-19
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no relevant report on nucleic a

Method used

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  • Nucleic acid aptamer specifically binding to P-glycoprotein, and preparation method and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically binding to P-glycoprotein, and preparation method and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically binding to P-glycoprotein, and preparation method and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A method for preparing a nucleic acid aptamer specifically binding to P-glycoprotein:

[0044] The initial random oligonucleotide (ssDNA) library and primer sequences used for SELEX screening in this example were synthesized by Shanghai Sangon Bioengineering Co., Ltd., wherein 45 bases in the initial random oligonucleotide library were randomly generated. 36 fixed base sequences, the sequence is as follows:

[0045] 5'-ATCCAGAGTGACGCAGCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGGACACGGTGGCTTAGT-3'.

[0046] The upstream primer sequence is: 5'-ATCCAGAGTGACGCAGCA-3'.

[0047] The downstream primer sequence is: 5'-ACTAAGCCACCGTGTCCA-3'.

[0048]Biotin-modified upstream primer: 5′-biotin-C6-ATCCAGAGTGACGCAGCA-3′.

[0049] Biotin-modified downstream primer: 5′-biotin-C6-ACTAAGCCACCGTGTCCA-3′.

[0050] Tosyl-modified magnetic beads (TMB magnetic beads) and streptavidin-modified magnetic beads (SMB magnetic beads) were purchased from Dynal, Norway Company, the PCR kit w...

Embodiment 2

[0114] Application of a P-gp nucleic acid aptamer to fluorescence visualization of P-gp in human brain microtubule endothelial cells (HBMECs):

[0115] (1) Cultivate HBMECs: The HBMECs used in the experiment were purchased from Sebakon Shanghai Biotechnology Co., Ltd., and prepared according to 1% double antibody (penicillin and streptomycin mixture (100×); Beijing Suo Laibao Technology Co., Ltd.), 10% serum (Sijiqing fetal bovine serum; Zhejiang Tianhang Biotechnology Co., Ltd.) and RPMI-1640 medium (Gibco, USA) were used to prepare cell culture medium. Cells were divided into 1×10 5 The density of cells / mL was inoculated into confocal small dishes (Corning, USA) and placed at 37°C, 5% CO 2 The cells were cultured in a cell culture box with a concentration of 1 to 2 days, so that 80% of the cells covered the bottom of the dish were observed under an inverted microscope.

[0116] (2) HBMECs with good adherent growth were treated with 2 mL of 4% paraformaldehyde histological ...

Embodiment 3

[0119] Visualization and quantification of P-gp on frozen sections of rat brain tissue by a fluorescent nucleic acid aptamer:

[0120] (1) A total of 6 11-week-old healthy SPF male SD rats were selected (purchased from the Experimental Animal Resource Center of China Institute of Pharmaceutical and Biological Products), divided into two groups and numbered, 3 in each group, and one group was the control group , and the other group is the simulated loss group. One week of adaptive feeding was carried out before the start of the experiment, and then the rats with simulated weight loss were tail-suspended, and the control group was fed normally. After 21 days, the two groups of rats were anesthetized with 3% pentobarbital sodium, and brain samples were collected and cryosections of brain tissue were made.

[0121] (2) Fluorescence staining was performed on rat brain tissue sections, and the staining steps were as follows:

[0122] ①Dilute factor VIII-related antigen antibody (B...

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Abstract

The invention relates to a nucleic acid aptamer specifically binding to P-glycoprotein (P-gp), and a preparation method and application of the nucleic acid aptamer, and belongs to the technical fieldof biology. The nucleotide sequence of the nucleic acid aptamer is selected from SEQ ID No.1 to SEQ ID No.14 in sequence tables; the nucleic acid aptamer is prepared by screening, amplifying and sequencing an original random oligonucleotide library through an SELEX technology in combination with surface modified magnetic beads; the nucleic acid aptamer is small in molecular weight, can be chemically synthesized, and is low in cost; the affinity and the specificity are high; marking is facilitated; and the repeatability and the stability are high, and storage is easy. The nucleic acid aptamer can be made into a fluorescent molecular probe, and fluorescent visualization and quantification of P-gp on in-vitro tissues and cells of an organism are realized; and the nucleic acid aptamer can be specifically bound with P-gp, inhibits the function of P-gp, and becomes a potential nucleic acid type inhibitor of the function of P-gp.

Description

technical field [0001] The invention relates to a nucleic acid aptamer specifically binding to P-glycoprotein, a preparation method and application thereof, and belongs to the technical field of nucleic acid aptamers. Background technique [0002] P-glycoprotein (P-gp) is a drug efflux protein encoded by the mdr 1 gene, which is widely distributed in the capillary endothelial cells in the brain, columnar epithelial cells in the intestinal tract, and the luminal membrane of the liver cells facing the bile canaliculus Surface, etc., after combining with the substrate, it is expelled from the cytoplasm, which plays an important role in the absorption, distribution, metabolism and excretion of drugs in the body. It has a wide range of substrates, including flavonoids, chalcones, biological Natural products such as alkalis and saponins. Studies have shown that the efflux function of P-gp is related to the multidrug resistance of tumor cells, and finding low-toxicity and high-eff...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10G01N33/53
CPCC12N15/1048C12N15/115C12N2310/16G01N33/53
Inventor 李玉娟梁敏武广霞郭晶晶邓玉林
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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