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Aptamer for performing target combination on CD133 albumen as well as screening method and application thereof

A technology of nucleic acid aptamer and targeted combination, which is applied in the direction of medical formula, 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, easy transportation at room temperature, and good chemical stability

Active Publication Date: 2019-03-29
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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  • Aptamer for performing target combination on CD133 albumen as well as screening method and application thereof
  • Aptamer for performing target combination on CD133 albumen as well as screening method and application thereof
  • Aptamer for performing target combination on CD133 albumen as well as screening method and application thereof

Examples

Experimental program
Comparison scheme
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] like 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 Nthy...

Embodiment 3

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

[0042] like 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 an aptamer for performing target combination on CD133 albumen as well as a screening method and application thereof. A sequence of the aptamer is shown in SEQ ID NO.1. Accordingto the aptamer disclosed by the invention, by adopting a cell screening method, the aptamer for performing target combination on CD133 albumen is screened out from an ssDNA library, the screened ssDNA is detected through a high-throughput sequencing method, and finally, the stability and the compatibility of the obtained sequence can be analyzed through a method of flow cytometry, and then the aptamer AP-1-M is picked out. An immunofluorescence method proves that the aptamer AP-1-M can specially perform targeted combination on the CD133 albumen, and with the combination of the aptamer AP-1-Mand ATC cells for expressing the CD133 albumen, a targeted delivery effect can be achieved, and a new thinking and method can be provided for the targeted treatment on the ATC.

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 Applications(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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