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A nucleic acid aptamer targeting CD133 protein and its screening method and use

A nucleic acid aptamer and targeted combination technology, which is applied in the fields of pharmaceutical formulations, biochemical equipment and methods, medical preparations with non-active ingredients, etc., can solve the problems of undifferentiated thyroid cancer lacking aptamers, etc., and achieve easy Long-term storage, reversible denaturation and renaturation, and easy transportation at room temperature

Active Publication Date: 2021-03-02
ZHEJIANG CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, studies have also reported that CD133 protein is expressed in undifferentiated thyroid cancer (Anaplastic thyroid carcinoma, ATC), but there is currently a lack of suitable aptamers for the treatment of anaplastic thyroid cancer

Method used

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  • A nucleic acid aptamer targeting CD133 protein and its screening method and use
  • A nucleic acid aptamer targeting CD133 protein and its screening method and use
  • A nucleic acid aptamer targeting CD133 protein and its screening method and use

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Screening of nucleic acid aptamers targeting CD133 protein

[0034] a. The aptamer targeting CD133 protein was screened from the ssDNA library by cell screening method;

[0035] b. Detect the ssDNA screened in step a by high-throughput sequencing, and analyze the stability and affinity of the sequence, and screen out a nucleic acid aptamer that targets and binds to the CD133 protein;

[0036] c. Verify that the aptamer targeting CD133 protein selected in step b can specifically target and bind CD133 protein by immunofluorescence method, and express CD133 protein ATC cells combined.

[0037] The sequence of one of the screened nucleic acid aptamers targeting and binding to the CD133 protein is shown as SEQ ID NO.1 in the sequence listing, ie AP-1-M.

Embodiment 2

[0038] Example 2: Expression and localization of CD133 protein in different cell lines

[0039] Such as figure 1 Shown, Caco-2: colon adenocarcinoma cell; FRO: undifferentiated thyroid cancer cell; NTH: normal thyroid cell; 293T: human embryonic kidney cell line.

[0040] Confocal Microscopy was used to observe the expression of CD133 protein in different cell lines ( figure 1 ), red fluorescence (ie figure 1The membrane-like distribution of bright spots in the four photomicrographs in the lower left corner of the center) represents the expression distribution of the membrane protein CD133 protein, and the blue fluorescence (ie figure 1 Nucleated bright spots) in each micrograph represent DAPI, indicating cell nuclei. The results showed that CD133 protein was expressed on the cell membrane in undifferentiated thyroid cancer FRO cells and colon adenocarcinoma Caco-2 cells as a positive control group, while HEK-293T cells as a negative control group and normal thyroid cells N...

Embodiment 3

[0041] Example 3: Aptamer AP-1-M binds to CD133 positive cells and negative cells.

[0042] Such as figure 2 As shown, by using FITC to label the AP-1-M nucleic acid aptamer, HEK-293T cells overexpressing CD133 protein were regarded as positive cells, and wild-type HEK-293T cells were regarded as negative cells. The 1-M nucleic acid aptamer was incubated with positive cells and negative cells, and then observed under a laser confocal microscope to find that AP-1-M was combined with positive cells and located on the cell membrane ( figure 2 -A), while not binding to negative cells ( figure 2 -B), which also shows that AP-1-M is a targeting nucleic acid aptamer for CD133 protein.

[0043] The results show that AP-1-M is a nucleic acid aptamer that specifically binds to CD133 protein, and the affinity curve of the nucleic acid aptamer is measured by flow cytometry, and the Kd value of AP-1-M is 101.4nM .

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Abstract

The invention provides a nucleic acid aptamer targeting CD133 protein and its screening method and application. The sequence of the nucleic acid aptamer is as SEQ ID NO.1. Combine the aptamer of CD133 protein, and detect the screened ssDNA by high-throughput sequencing, and finally analyze the stability and affinity of the obtained sequence by flow cytometry and other methods, and select the aptamer AP‑1‑M. Then, it was proved by immunofluorescence that the aptamer AP‑1‑M can specifically target and bind to CD133 protein, and bind to ATC cells expressing CD133 protein to play the role of targeted delivery, providing a new method for the targeted therapy of ATC ideas and methods.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a nucleic acid aptamer targeting and binding to CD133 protein. Background technique [0002] Thyroid cancer (thyroid carcinoma) is the most common thyroid malignancy, accounting for about 1% of systemic malignancies. According to the degree of malignancy, it can be divided into differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC). Because ATC can rapidly expand to the neck, it will cause respiratory distress and esophageal obstruction, and it is very prone to lymph node and distant metastasis. The median survival time is less than 6 months, and even the survival rate within 1 year is only 10%. However, the molecular mechanism of ATC is still unclear, and the diagnosis and treatment methods are limited. Therefore, better diagnosis and treatment methods are urgently needed. [0003] CD133 protein is a member of the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10A61K47/54A61K31/704A61P35/00
CPCA61K31/704A61K47/549A61P35/00C12N15/111C12N15/115C12N2310/16C12N2330/31
Inventor 朱栩杭葛明华那仁满都拉黄萍杨畅李清林兰霞斌曹君温庆良黄煜庆周菁楠
Owner ZHEJIANG CANCER HOSPITAL
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