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Nucleic acid aptamers for detecting extracellular vesicles of human lung cancer cell strain A549 and application of nucleic acid aptamers

A nucleic acid aptamer and lung cancer cell technology, applied in the fields of biomedicine and clinical medicine, can solve problems such as poor treatment effect, complicated pathogenesis, and difficulty in discovery, and achieve the effects of easy modification, simple preparation, and low cost

Pending Publication Date: 2021-10-22
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Non-small cell lung cancer is the most common type of lung cancer, accounting for about 80% of all lung cancers. It has the characteristics of high incidence, complex pathogenesis, difficulty in early detection, and poor treatment effect.

Method used

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  • Nucleic acid aptamers for detecting extracellular vesicles of human lung cancer cell strain A549 and application of nucleic acid aptamers
  • Nucleic acid aptamers for detecting extracellular vesicles of human lung cancer cell strain A549 and application of nucleic acid aptamers
  • Nucleic acid aptamers for detecting extracellular vesicles of human lung cancer cell strain A549 and application of nucleic acid aptamers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening of human lung cancer cell line A549 cell line extracellular vesicle surface protein-specific nucleic acid aptamer based on immunomagnetic bead method

[0033] 1. Design and synthesize upstream and downstream primers and random libraries

[0034] The specific primer pair F / R and the random library sequence were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequence of the upstream primer F is shown in SEQ ID NO: 1, and the sequence of the downstream primer R is shown in SEQ ID NO: 2. The random library The sequence of is shown in SEQ ID NO: 3:

[0035] Upstream primer F (SEQ ID NO: 1): 5'-FAM-GTTGGTGAGGTAACGGCTCA-3'

[0036] Downstream primer R (SEQ ID NO: 2): 5'-biotin-CGGATAACGCTTGCCACCTA-3'

[0037] Random library: (SEQ ID NO: 3): 5'-GTTGGTGAGGTAACGGCTCA-40nt-TAGGTGGCAAGCGTTATCCG-3' (40nt means 40 random bases)

[0038] 2. Acquisition of human lung cancer cell line extracellular vesicle A549-EVs surface protein-specific nucleic acid...

Embodiment 2

[0059] Example 2 Screening of human lung cancer cell line A549 cell line extracellular vesicle surface protein-specific nucleic acid aptamer based on particle size selection method

[0060] 1. Design and synthesize upstream and downstream primers and random libraries

[0061] The specific primer pair F / R and the random library sequence were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequence of the upstream primer F is shown in SEQ ID NO: 1, and the sequence of the downstream primer R is shown in SEQ ID NO: 2. The random library The sequence of is shown in SEQ ID NO: 3:

[0062] Upstream primer F (SEQ ID NO: 1): 5'-FAM-GTTGGTGAGGTAACGGCTCA-3'

[0063] Downstream primer R (SEQ ID NO: 2): 5'-biotin-CGGATAACGCTTGCCACCTA-3'

[0064] Random library: (SEQ ID NO: 3): 5'-GTTGGTGAGGTAACGGCTCA-40nt-TAGGTGGCAAGCGTTATCCG-3' (40nt means 40 random bases)

[0065] 2. Obtaining human lung cancer cell line extracellular vesicle A549-EVs surface protein-specific nucleic acid ...

Embodiment 3

[0084] Example 3 The investigation of the secondary structure, specificity, affinity and dissociation constant of two nucleic acid aptamers

[0085] A human lung cancer cell line A549 extracellular vesicle surface protein-specific nucleic acid aptamer, mainly including the following 5'-end FAM-modified DNA sequence:

[0086] Sequence SEQ ID NO: 4 (Ap3)

[0087] 5'-FAM-GTTGGTGAGGTAACGGCTCACCAGGGGTTCGGTAGTCGTGTGGTTTCGGTCGGGTGTAGTTAGGTGGCAAGCGTTATCCG-3'

[0088] Sequence SEQ ID NO:5(Ap6)

[0089] 5'-FAM-GTTGGTGAGGTAACGGCTCAACAGGGGTTCGGTAGTCGTGTGGTTTCGGTTGGGTGTAGTTAGGTGGCAAGCGTTATCCG-3'

[0090] The above-mentioned nucleic acid aptamer that can specifically recognize and bind to the extracellular vesicles of the human lung cancer cell line A549, its nucleotide sequence can intercept part of the active fragment or increase part of the sequence based on it, and the homology with the above sequence is 60% above.

[0091] The nucleotide sequence of the above-mentioned nucleic acid...

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Abstract

The invention discloses nucleic acid aptamers for detecting extracellular vesicles of a human lung cancer cell strain A549. Nucleotide sequences of the nucleic acid aptamers are shown as SEQ ID NO: 4 (Ap3) and SEQ ID NO: 5 (Ap6). Or the nucleic acid aptamers are a series of derivatives which are obtained through chemical modification, chemical labeling or basic group change on the basis of SEQ ID NO: 4 (Ap3) and SEQ ID NO: 5 (Ap6) sequences. The nucleic acid aptamers disclosed by the invention can be applied to the recognition of extracellular vesicles of the human lung cancer cell strain or the preparation of a kit for detecting the extracellular vesicles of the human lung cancer cell strain or a combination of extracellular vesicle drug delivery and specific recognition of targets. The nucleic acid aptamers can be applied to identification of human lung cancer or preparation of the kit for detecting human lung cancer. The nucleic acid aptamers have very wide application prospects in detection, diagnosis and treatment of human lung cancer.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and clinical medicine, and specifically relates to a nucleic acid aptamer for detecting extracellular vesicles of human lung cancer cell line A549 and an application thereof. Background technique [0002] Lung cancer is the leading cause of cancer-related death. Globally, it is divided into two histological types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Non-small cell lung cancer is the most common type of lung cancer, accounting for about 80% of all lung cancers. It has the characteristics of high incidence, complex pathogenesis, difficulty in early detection, and poor treatment effect. Moreover, the prognosis of NSCLC patients largely depends on the stage of the disease. For example, the 5-year survival rate drops from 60% in clinical stage I to 1% in clinical stage IV. Early diagnosis of lung cancer is urgently needed. Therefore, the development of efficient,...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/574
CPCC12N15/115G01N33/57423C12N2310/16
Inventor 何农跃何磊李智洋
Owner SOUTHEAST UNIV
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