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Nucleic acid aptamer specifically bound with ethyl carbamate as well as preparation method and application of nucleic acid aptamer

A urethane and nucleic acid aptamer technology, applied in the field of biomedicine, can solve the problems of expensive instruments, high requirements for testers, and high maintenance costs in the later period, and achieve the effect of broad clinical application prospects and basic application prospects, and the effect of convenient combination.

Active Publication Date: 2022-05-10
WULIANGYE
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] At present, the determination of ethyl carbamate at home and abroad mainly relies on large-scale instruments such as GC-MS (GB 5009.223-2014), HPLC-MS (SN / T 0285-2012), although these instruments can be used to sensitively measure lower concentrations of Urethane, but there are disadvantages such as complicated sample pretreatment, expensive instruments, high requirements for testers, and high maintenance costs in the later period
However, there is still no report on the screening of urethane aptamers

Method used

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  • Nucleic acid aptamer specifically bound with ethyl carbamate as well as preparation method and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically bound with ethyl carbamate as well as preparation method and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically bound with ethyl carbamate as well as preparation method and application of nucleic acid aptamer

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Embodiment 1

[0058] 1. In vitro screening steps of nucleic acid aptamers

[0059] (1) Immobilization of random ssDNA library. Take a certain amount of random ssDNA library (SEQ ID NO.3: 5′-ACCGACCGTGCTGGACTCT-N30-AGTATGAGCGAGCGTTGCG-3′, where N represents any base in A, T, C, and G, and N30 refers to 30 arbitrary nuclei A sequence of nucleotide bases joined together.

[0060] Mixed with P3 (5′-CGCAACGCTCGC-3′(SEQ ID NO.4) and modified Biotin at the 5′ end of CGCAACGCTCGC) at a molar ratio of 1:2, annealed at 95°C for 5min, and then incubated at 30°C for 60min to make random The 12 nucleotide sequences at the 3' end of the library are complementary to P3 through hydrogen bonding. Transfer the paired mixture into a centrifuge tube containing magnetic beads and incubate at 30°C for 60 min. Since streptavidin is attached to the surface of the magnetic beads, the random library is immobilized on the magnetic beads through the interaction of biotin-avidin . Finally, use Binding Buffer (BB bu...

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a nucleic acid aptamer specifically bound with ethyl carbamate as well as a preparation method and application of the nucleic acid aptamer. In order to overcome the defects in the prior art, the invention provides a nucleic acid aptamer specifically bound with ethyl carbamate as well as a preparation method and application of the nucleic acid aptamer. A nucleic acid aptamer EC1 of ethyl carbamate is obtained through screening by virtue of a Capture-SELEX technology, and a sequence is truncated and optimized by predicting possible binding sites and structural characteristics, so that an aptamer EC1-34 with relatively good binding performance is obtained. The nucleic acid aptamers EC1 and EC1-34 provided by the invention have important application prospects in online monitoring, reduction of the level of ethyl carbamate in Baijiu and prevention of carcinogenic risks.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a nucleic acid aptamer specifically combined with ethyl carbamate, a preparation method and application thereof. Background technique [0002] Nucleic acid aptamer refers to a single oligonucleotide with 10 to 100 bases and a specific complex three-dimensional structure obtained through the screening of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, and can ssDNA or RNA molecules that specifically bind to harmful targets such as metal ions, pesticides, and biotoxins. Nucleic acid aptamers have the recognition characteristics of protein antibodies, a wide range of target molecules, small molecular weight, low immunogenicity, easy chemical synthesis, transformation and labeling and other advantages. In recent years, nucleic acid aptamers have been widely used in biomedical fields such as therapeutic new drugs, drug delivery, cancer cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/574G01N33/53A61K31/7088A61P35/00
CPCC12N15/115C12N15/1048G01N33/57438G01N33/57423G01N33/57419G01N33/57407G01N33/57484G01N33/5308A61K31/7088A61P35/00C12N2310/16C12N2310/531C12Q2531/113C12Q2563/157C12Q2563/149C12Q2563/131C12Q2565/125Y02A50/30
Inventor 吴远根夏恋安明哲周韩玲
Owner WULIANGYE
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