PEG modified nucleic acid aptamer as well as preparation method and application thereof

A nucleic acid aptamer and reaction technology, applied in biochemical equipment and methods, medical preparations containing active ingredients, pharmaceutical formulations, etc., to achieve good stability

Inactive Publication Date: 2018-11-23
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, unmodified aptamers degrade quickly in vivo, which has a great impact on their performance in experiments

Method used

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  • PEG modified nucleic acid aptamer as well as preparation method and application thereof
  • PEG modified nucleic acid aptamer as well as preparation method and application thereof
  • PEG modified nucleic acid aptamer as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1) Combine PEG5000, 0.400M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (also known as EDC·HCl) and 0.100M N-hydroxysuccinimide The (NHS) molar ratio was 4:4:1 (50 μL each) after mixing, and shaking at 80 rpm for 15 min.

[0041] 2) Add FT6 aptamer with a volume ratio of 1:30 to PEG to 50μL of 2-(N-morpholine)ethanesulfonic acid buffer (also known as MES buffer) with a pH of 6 and then add it to Step 1) The prepared mixture was shaken at 80 rpm for 4 hours, and gel electrophoresis was performed after the reaction was completed.

Embodiment 2

[0043] 1) Combine PEG5000, 0.400M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (also known as EDC·HCl) and 0.100M N-hydroxysuccinimide The (NHS) molar ratio is 5:5:1 (50 μL each) after mixing, and the reaction is shaken at 80 rpm for 10 min.

[0044] 2) Add FT7 aptamer with a volume ratio of 1:10 to PEG to 50μL of 2-(N-morpholine) ethanesulfonic acid buffer (also known as MES buffer) with a concentration of 20ml / L and a pH of 4 Solution), then add to the mixed solution prepared in step 1), shake for 2h at a rotation speed of 60 rpm, and perform gel electrophoresis detection after the reaction.

Embodiment 3

[0046] A PEG-modified nucleic acid aptamer preparation method, the steps are as follows:

[0047] 1) Combine PEG5000, 0.400M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (also known as EDC·HCl) and 0.100M N-hydroxysuccinimide The (NHS) molar ratio is 2:2:1 (50 μL each) after mixing, and shaking at 100 rpm for 20 min.

[0048] 2) Add FT8 aptamer with a volume ratio of 1:50 to PEG to 50μL of 2-(N-morpholine)ethanesulfonic acid buffer (also known as MES buffer) with a pH of 7 and then add it to Step 1) The prepared mixture was shaken at 80 rpm for 5 hours, and gel electrophoresis detection was performed after the reaction.

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Abstract

The invention discloses a PEG modified nucleic acid aptamer as well as a preparation method and application thereof. PEG modification is performed by aiming at a GCRV (grass carp reovirus) nucleic acid aptamer; modification is performed by using different molecular weights of PEG at different reaction time and different reaction proportions; the optimal reaction conditions are screened; the cell viability is not influenced; the inhibition effect is achieved on the GCRV infection. The modified GCRV nucleic acid aptamer has application prospects for preparing viral disease treatment medicine, and also has application prospects for preparing GCRV molecular probes and detection reagents.

Description

Technical field [0001] The invention belongs to the field of biological detection, and specifically relates to a preparation method and effect of a PEG-modified grass carp reovirus nucleic acid aptamer. Background technique [0002] Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) caused a large number of fish deaths, and the mortality rate was as high as 85%, resulting in serious losses in the grass carp breeding industry. Once the grass carp hemorrhagic disease breaks out, there is currently no effective antiviral treatment. Therefore, hemorrhagic disease of grass carp has become a bottleneck in grass carp breeding, which severely restricts the breeding of grass carp. [0003] Aptamer is an oligonucleotide sequence (RNA or DNA) obtained by in vitro screening technology, which has strict recognition ability and high affinity with the corresponding ligand. Aptamers have the advantages of high specificity, wide target molecules, and easy synthesis in vitro, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115A61K31/711A61P31/14
CPCA61K31/711A61P31/14C12N15/115C12N2310/16
Inventor 梁红茹李宁求付小哲林强刘礼辉黄志斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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