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A nucleic acid aptamer specifically binding to ischemic brain tissue and its application and kit

A nucleic acid aptamer and specific binding technology, applied in the biological field, can solve the problems of unreported screening, the inability of screening research to be carried out effectively, and the inability of subculture of neuronal cells to achieve the effect of strong binding ability.

Active Publication Date: 2020-01-14
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing nucleic acid aptamer screening technology mainly screens through cultured cell lines. However, the composition of ischemic brain tissue is complex, a variety of cells interact, and neuron cells cannot be subcultured. Therefore, using cells as targets to treat cerebral ischemic diseases The research on nucleic acid aptamer screening cannot be carried out effectively, and there has been no report on the screening of nucleic acid aptamers that specifically bind to the target of cerebral ischemic disease

Method used

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  • A nucleic acid aptamer specifically binding to ischemic brain tissue and its application and kit
  • A nucleic acid aptamer specifically binding to ischemic brain tissue and its application and kit
  • A nucleic acid aptamer specifically binding to ischemic brain tissue and its application and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Screening of nucleic acid aptamers specifically binding to ischemic brain tissue

[0039] 1. Synthesize the DNA library sequence and primer sequence shown below

[0040] The initial library DNA sequence is:

[0041] 5'-AGCGTCGAATACCACTACAG-30-nt-CTAATGGAGCTCGTGGTCAG-3'

[0042] The sequences at both ends are used to combine with primers for polymerase chain reaction (PCR) amplification, the middle 30 bases are random sequences, and the sequences of the primers are:

[0043]5'-FAM-AGCGTCGAATACCACTACAG-3'

[0044] 5'-Biotin-CTGACCACGAGCTCCATTAG-3'.

[0045] 2. Acquisition of ischemic brain tissue slices

[0046] By blocking the left middle cerebral artery (MCAO) of the mouse with a thread embolism, the left hemicerebral ischemic state of the mouse is caused, and the ischemic time is 4 hours. Subsequently, the mice with cerebral ischemia behavior were perfused with paraformaldehyde, and after dehydration, the left and right brains were made into frozen secti...

Embodiment 2

[0063] Example 2 Verification test of nucleic acid aptamer LCW17 of the present invention targeting ischemic brain tissue

[0064] 1. Fluorescence imaging verification of nucleic acid aptamer LCW17 targeting ischemic brain tissue

[0065] Dissolve 30 pmol of 6-carboxyfluorescein at the 5-terminal of the nucleic acid aptamer LCW17 in 200 microliters of DPBS buffer A, denature at 95 degrees for 5 minutes, and cool on ice for 10 minutes. Normal brain slices were incubated for 40 minutes, washed three times, and imaged under a confocal fluorescence microscope. At the same time, the single-stranded DNA of the initial library labeled with 6-carboxyfluorescein at the same concentration was treated in the same way, incubated with the previously blocked brain slices for imaging, and used for control experiments.

[0066] The result is as Figure 5 As shown, the initial library basically did not bind to the two brain slices, and the number of fluorophores bound to the ischemic brain s...

Embodiment 3

[0082] The preparation of embodiment 3 kits

[0083] The nucleic acid aptamer shown in SEQ ID NO: 5 was synthesized and prepared into a kit.

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Abstract

The invention relates to the technical field of biology, in particular to an aptamer specially combined with cerebral ischemia tissues as well as application and a kit thereof. A secondary structure of the aptamer has a stem-loop structure, and a nucleotide sequence of the aptamer is shown in SEQ ID NO:5. According to the aptamer provided by the invention, the cerebral ischemia tissues and targetproteins Vigilin for expressing secretion can be specially identified, and the combination capability is strong, and thus, a new method and a molecular tool are provided for identification of nerve injury of cerebral ischemia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid aptamer specifically binding to ischemic brain tissue and its application and kit. Background technique [0002] Stroke is the second biggest killer next to cardiovascular disease. There are about 33 million stroke patients worldwide, of which the incidence of ischemic stroke accounts for 60%-80%. Stroke not only has a high fatality rate, but more neurons are damaged after stroke, and cannot be effectively repaired and treated, thus losing their ability to act and being paralyzed in bed, bringing heavy economic and psychological burdens to families and society . [0003] Nucleic acid aptamers form a stable three-dimensional space structure through intermolecular interactions such as hydrogen bonds, van der Waals forces, and hydrophobic stacking, and can specifically recognize proteins or other small molecular substances. High, stable chemical properties, easy chemica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/68
CPCC12N15/111C12N15/115C12N2310/16C12N2330/31G01N33/6893G01N2333/47G01N2800/2871C12N2310/531
Inventor 谭蔚泓涂海军刘超蒋伟田西彬
Owner HUNAN UNIV