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DNA aptamer specifically combined with human breast cancer cells MCF-7 and application thereof

A technology of human breast cancer cells and nucleic acid aptamers, which is applied in the fields of biomedicine and clinical medicine, can solve the problem that the sensitivity speed cannot meet the requirements of rapid diagnosis of breast cancer, and achieve non-immunogenicity, small molecular weight, easy retouching effect

Active Publication Date: 2018-07-10
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to reasons such as sensitivity and speed, these two technologies cannot well meet the requirements of early and rapid diagnosis of breast cancer.

Method used

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  • DNA aptamer specifically combined with human breast cancer cells MCF-7 and application thereof
  • DNA aptamer specifically combined with human breast cancer cells MCF-7 and application thereof
  • DNA aptamer specifically combined with human breast cancer cells MCF-7 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Screening of human breast cancer cell line MCF-7 specific nucleic acid aptamers

[0032] Design and synthesize the following single-stranded DNA libraries and primer sequences:

[0033] Random ssDNA library: (N40 represents 40 random bases)

[0034] 5'-AGCAGAGTTCACGACCCGATAAG-N40-GAGTTACATACCAATCGTCGCAG-3'

[0035] Upstream primer: 5'-FAM-AGCAGAGTTCACGACCCGATAAG-3'

[0036] Downstream primer: 5'-biotin-CTGCGACGATTGGTATGTAACTC-3'

[0037] 1. Obtain the specific nucleic acid aptamer of human breast cancer cell line MCF-7 by Cell-SELEX:

[0038] 2.1 Cell culture: use 1640 incomplete medium plus 10% fetal bovine serum to cultivate human breast cancer cells MCF-7 and SK-BR-3; DMEN plus 10% fetal bovine serum to cultivate human breast cancer cells MDA-MB-231; MEGM Add 100ng / ml cholera toxin to culture human normal breast epithelial cells MCF-10A. All cells were kept at 37°C, 5% CO2 cultivated under conditions.

[0039] 2.2 Cell pretreatment before screening: ...

Embodiment 2

[0048] Example 2: Investigation of the secondary structure, specificity, affinity and dissociation constant of four nucleic acid aptamers

[0049] A human breast cancer cell line MCF-7-specific nucleic acid aptamer, in this embodiment, the nucleic acid aptamer mainly includes any one DNA sequence in the following four nucleotide sequences modified by FAM at the 5' end:

[0050] Sequence 1:

[0051] 5'-AGCAGAGTTCACGACCCGATAAGCGGCGATCGCATTCTTCTAGACGGTAGCGACTTGAGACATGAGTTACATACCAATCGTCGCAG-3'

[0052] Sequence 2:

[0053] 5'-AGCAGAGTTCACGACCCGATAAGCCAGCACTGTGAGGGGAATGCGGCCCGAAGTGGTCTAGACGAGTTACATACCAATCGTCGCAG-3'

[0054] Sequence 3:

[0055] 5'-AGCAGAGTTCACGACCCGATAAGTGCATTAGCACGTCCGAGAAAGGCCAGACGAGGTCACACAGAGTTACATACCAATCGTCGCAG-3'

[0056] Sequence 4:

[0057] 5'-AGCAGAGTTCACGACCCGATAAGTCAGTTCGAAATCTGGTACTGCACGGAAATCAGGGGTAGGAGTTACATACCAATCGTCGCAG-3'

[0058] 1. Use DNAMAN software to simulate the secondary structure of the above four nucleic acid aptamers, the results a...

Embodiment 3

[0068] Example 3: Application of nucleic acid aptamer-based fluorescent sensor in the detection of human breast cancer cell MCF-7

[0069] Since sequence 3 has the best specificity among the four nucleic acid aptamer sequences (such as image 3), so this embodiment takes Sequence 3 as an example to construct a fluorescent sensor based on nucleic acid aptamer for in vitro detection of human breast cancer cell MCF-7. First, modify the Biotin molecule and the TAMRA quencher group at the 3' end and 5' end of sequence 3, respectively, and design a 16-base-long modified FAM fluorescent group that is complementary to the 5' end of the nucleic acid aptamer. The nucleic acid sequence was used as a reporter probe (Probe: 5'-FAM-GGTCGTGAACTCTGCT-3'). Before detection, the nucleic acid aptamer is hybridized with the reporter probe to form a complex Aptamer-Probe. At this time, the fluorescent signal cannot be generated because the FAM fluorescent group and the TAMRA quencher group are cl...

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Abstract

The invention discloses a DNA aptamer specifically combined with human breast cancer cells MCF-7. The nucleotide sequence of the DNA aptamer is shown by any one or several sequences in SEQ ID NO:1 toSEQ ID NO:4, or is obtained through chemical modification, chemical labeling or basic group change on any one sequence in SEQ ID NO:1 to SEQ ID NO:4. The invention also discloses application of the DNA aptamer or the nucleotide sequence to diagnosis reagents, molecular imaging probes or target media. The invention also relates to a human breast cancer detection and diagnosis kit, which has wide application prospects in breast cancer detection, diagnosis and treatment. The DNA aptamer related by the invention has the advantages of high affinity, high specificity, small molecular weight, high stability, synthesis simplicity, low cost, simplicity in modification, no immunogenicity and the like, and can meet the requirement of human breast cancer fast diagnosis at present.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and clinical medicine, and relates to a DNA nucleic acid aptamer specifically binding to human breast cancer cell MCF-7 and its application, and specifically relates to the detection and diagnosis reagent of the nucleic acid aptamer in the preparation of human breast cancer And the application in the therapeutic drug or preparation of human breast cancer. Background technique [0002] In recent years, the incidence of breast cancer in my country has gradually increased, and the situation of its prevention and treatment is not optimistic. In my country, especially in remote and impoverished areas, the incidence of breast cancer among women has reached 24.20 per 100,000 people. In the world, female breast cancer has become the second largest tumor disease after lung cancer, posing a huge threat to people's lives and property. As a highly heterogeneous tumor, breast cancer has four typical mole...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11G01N33/574A61K31/704A61K47/54A61K49/00A61P35/00A61P15/14
CPCA61K31/704A61K49/0034A61K49/0052C12N15/113C12N2310/10G01N33/57415
Inventor 何农跃刘梅王志飞杨通
Owner SOUTHEAST UNIV
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