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Nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patient and application of nucleic acid aptamer

A nucleic acid aptamer, drug-resistant cell technology, applied in the biological field, can solve the problems of low toxicity and immunogenicity, and cannot be used to detect drug resistance in ovarian cancer patients, and achieves strong affinity and specificity, low cost, good recognition effect

Active Publication Date: 2022-06-03
ZHEJIANG CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on nucleic acid aptamers is gradually increasing. Chinese patent CN111944819A discloses a rapidly screened ovarian cancer nucleic acid aptamer and its application in the preparation and detection of ovarian cancer preparations. Compared with traditional antibodies, the disclosed nucleic acid aptamers, It has the characteristics of a wide range of recognized targets, strong specificity, strong affinity, low toxicity and immunogenicity, but it cannot be used to detect drug resistance in ovarian cancer patients
Some researches on nucleic acid aptamers targeting cancer drug resistance, for example, Chinese patent CN109337909A discloses a nucleic acid aptamer for detecting drug-resistant cell lines of liver cancer and its application, but there is little research on nucleic acid aptamers for identifying drug resistance in ovarian cancer

Method used

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  • Nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patient and application of nucleic acid aptamer
  • Nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patient and application of nucleic acid aptamer
  • Nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patient and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

Cell-SELEX screening process

(1) Design of nucleic acid library and primers:

Random nucleic acid library:

5'-AAGGAGCAGCGTGGAGGATA(N)45TTAGGGTGTGTCGTCGTGGT-3';

Upstream primer: 5'-fluorescein isothiocyanate-AAGGAGCAGCGTGGAGGATA-3';

Downstream primer: 5'-biotin-ACCACGACGACACACCCTAA-3';

(2) Positive sieve:

①Library processing:

The above random nucleic acid library was placed in a centrifuge at 10,000 rpm, centrifuged for 10 min, fully dissolved in DPBS, denatured at 95°C for 5 min, and quickly placed in ice for 10 min.

[0037] ②Incubate:

The treated random library was incubated with the cultured and pretreated ovarian cancer paclitaxel-resistant cell line A2780 / PTX at 37°C for 60 min.

[0038] ③Separation:

After the incubation, remove the solution in the incubation dish, wash the cells in the incubation dish with washing buffer (PBS, containing 0.45% glucose, 5mM magnesium chloride); scrape the cells in the incubation dish with 600 μL of pre-cooled sterile water...

Embodiment 2

Determination of optimal nucleic acid aptamers

According to the nucleic acid library screened in Example 1, 6 candidate nucleic acid sequences were selected through computer analysis and comparison, and were named ZH-A2, ZH-A4, ZH-A6, ZH-A9, ZH-A10, ZH- A11 and determine the optimal nucleic acid aptamer.

[0046] Specific steps:

(1) Streaming inspection

Adherent state A2780 / PTX cells were digested from culture dishes with 0.2% EDTA, cells were collected into centrifuge tubes, and washed twice with DPBS; were added to A2780 / PTX cells soaked in binding buffer (D-PBS, containing 0.45% glucose, 5mM magnesium chloride, 100mg / L tRNA, 1g / L BSA); then placed in a shaker at 37°C and incubated for 45min; the incubation was completed After that, it was centrifuged and washed twice with washing buffer (PBS, containing 0.45% glucose, 5mM magnesium chloride), and finally the fluorescence was detected by flow cytometry;

The same method was used to detect the binding of candidate seque...

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Abstract

The invention discloses a nucleic acid aptamer for recognizing extracellular vesicles of an ovarian cancer drug-resistant patient and application of the nucleic acid aptamer, and belongs to the technical field of biology. The nucleic acid aptamer obtained through cell screening has high affinity and specificity and low immunogenicity, can be chemically synthesized in vitro, is low in cost and small in molecular weight, can modify and replace different parts of the nucleic acid aptamer, and is stable in sequence and easy to store. The nucleic acid aptamer can be used for preparing anti-tumor drugs, ovarian cancer drug-resistant cell strains or ovarian cancer drug-resistant patient serum exosome detection kits or ovarian cancer drug-resistant patient serum exosome detection probes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients and its application. Background technique [0002] Ovarian cancer is one of the three most common malignant tumors in women, and it is also the most deadly malignant gynecological tumor in the world. At present, the main clinical treatment methods for ovarian cancer are surgery and chemotherapy, among which cisplatin and paclitaxel are commonly used chemotherapy drugs for the clinical treatment of ovarian cancer. Ovarian cancer lacks typical symptoms in the early stage, and the anatomical position of the ovary is deep, so it is mostly advanced when diagnosed. In addition, most patients will have varying degrees of drug resistance to chemotherapy, resulting in a very low long-term survival rate for ovarian cancer patients. [0003] Aptamers are single-stranded DNA or RNA molecules ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/574A61K31/7088A61P35/00
CPCC12N15/115C12N15/1048G01N33/57449G01N33/56966A61K31/7088A61P35/00C12N2310/16C12N2310/315
Inventor 谭蔚泓张慧肖亚婷甘绍举
Owner ZHEJIANG CANCER HOSPITAL
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