A chemically modified base-containing single-stranded DNA aptamer that specifically recognizes the anthrax protective antigen pa83 and its application

A nucleic acid aptamer and DNA molecule technology, applied in the biological field, can solve the problems of being inferior to antibodies, the random base sequence should not be too long, limiting the amount of information in the initial library, etc., and achieve the effect of broad application prospects.

Active Publication Date: 2022-02-11
中国人民解放军疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the initial library based on the natural base system only has four variables of ACT(U)G, which is not as good as the combination of more than 20 amino acids of the antibody, and in order to ensure the specificity of the aptamer, the random base sequence should not be too long. The length is generally 25-42 bases, which further limits the amount of initial library information
[0004] Many studies at home and abroad have shown that the application of chemically modified nucleotides to library construction can greatly increase the amount of library information, thereby screening aptamers with better performance, but different modification methods have a significant impact on the function of aptamers

Method used

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  • A chemically modified base-containing single-stranded DNA aptamer that specifically recognizes the anthrax protective antigen pa83 and its application
  • A chemically modified base-containing single-stranded DNA aptamer that specifically recognizes the anthrax protective antigen pa83 and its application
  • A chemically modified base-containing single-stranded DNA aptamer that specifically recognizes the anthrax protective antigen pa83 and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1 Preparation of a single-stranded DNA adapter containing a chemically modified base of anthracnose protective antigen PA83

[0047] First, design and custom chemical modified nucleic acid library

[0048] The RNA adapter is easily degraded by the RNA adapter, and the present invention selects a single-stranded DNA adapter that screens the structural stable structure in the environment. The deoxy uracil (e.g., e.g., e.g., e.g., e.g., e.g., figure 1 The preparation of the nucleic acid library is introduced, respectively, respectively, respectively, respectively, respectively, respectively, respectively. The indole group, the phenol group and the amine group have nanopolarity, polarular and no charge and polar positive charge, respectively. The nucleic acid library full length 80bp, cagggacgccaagg-n40-ccatgacccggctgctgctcg, the front and rear ends are fixed sequences for amplification, including only natural nucleotide ATCG; 40 medium random sequences are designed to ...

Embodiment 2

[0096] Example 2, nucleic acid adapter AP5 in detecting the application of PA83 protein

[0097] First, biotin labeled nucleic acid fitting AP5

[0098] Synthetic biotin-labeled nucleic acid fitting AP5, with a sequence of 10 DA, 5'-Biotin-aaaaaaaaa-AP5-3 '(5'-Biotin-aaaaaaaaaaa-ap5-3' (5'-Biotin-aaaaaaaaaaaaa-ap5-3 '), App RNASTRUCTURE Software Predicting the secondary structure of the nucleic acid fit Figure 4 .

[0099] Second, the nucleic acid adapter AP5 constructs an adapter biosensor based on surface plasmon resonance technology

[0100] Aptamer construct resonance biosensor based on surface plasmon can refer to the following literature Wang S, Dong Y, Liang X.Development of a SPR aptasenxor containing orientedaptamer for direct capture and detection of tetracycline in multiple honeysamples [J] .Biosensors & Bioelectronics, 2018, 109: 1. The construction process is as follows:

[0101] 1. Secure the glucan nanopenic chip to the fluid chamber of the SPR instrument to adjust ...

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Abstract

The invention discloses a chemically modified base-containing single-stranded DNA aptamer specifically recognizing anthrax protective antigen PA83 and its application. The present invention takes the anthrax protective antigen (PA83) as the target, uses the nucleic acid library containing chemically modified nucleotides, and uses the magnetic bead method SELEX to screen out the high-affinity aptamer AP5, which can be used in the environment of various interfering proteins Under this method, it can specifically recognize PA83 protein; it can be applied to aptamer biosensor based on surface plasmon resonance technology and fluorescence polarization technology to detect PA83 protein, thereby detecting Bacillus anthracis. Potential of novel detection techniques for Bacillus protective antigens.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and more particularly to a specific identification of anthracnose protective antigen PA83 containing chemically modified base single-stranded DNA adapter and its application thereof. Background technique [0002] The infection of Aprona is spores, which can be entered into the body by skin wounds, digestive tract or lungs, in which aerosol propagation is the main transmission pathway of biological terrorist attacks. The protective antigen Pa is an important part of the anthrax toxin complex. The anthrax protected antigen refers to the PA83 and PA63, the US Army Infectious Disease Research Institute and the US CDC have shown that there is a large amount of PA83 in the body fluid in the medium supernatant supernatant and patient infection in anthrax and patient infection. Therefore, the recognition component of the research and development recognition of the PA83 protein is of great significance for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/569G01N33/68G01N21/552G01N21/64
CPCC12N15/115C12N15/1048G01N33/56911G01N33/68G01N21/553G01N21/554G01N21/6445G01N21/6486C12N2310/16C12N2310/315C12N2310/3181C12N2310/3517G01N2333/32
Inventor 杨益郝荣章宋宏彬赵荣涛李瑾慧唐玥王玉乐郭旭东
Owner 中国人民解放军疾病预防控制中心
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