Chemically modified basic group-containing single-stranded DNA aptamer capable of specifically recognizing anthrax protective antigen PA83 and application thereof

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 initial library information, etc., and achieve the effect of broad application prospects

Active Publication Date: 2020-09-22
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

Method used

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  • Chemically modified basic group-containing single-stranded DNA aptamer capable of specifically recognizing anthrax protective antigen PA83 and application thereof
  • Chemically modified basic group-containing single-stranded DNA aptamer capable of specifically recognizing anthrax protective antigen PA83 and application thereof
  • Chemically modified basic group-containing single-stranded DNA aptamer capable of specifically recognizing anthrax protective antigen PA83 and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1. Preparation of single-stranded DNA aptamers containing chemically modified bases that specifically recognize the anthrax protective antigen PA83

[0047] 1. Design and customize nucleic acid libraries of chemically modified nucleotides

[0048] Because RNA aptamers are easily degraded by nucleases, the present invention selects and screens single-stranded DNA aptamers with stable structures in the detection environment. Select deoxyuracil (such as figure 1 shown) into the preparation of the nucleic acid library, respectively expressed as Trp-dU, Tyr-dU, Lys-dU. Indole groups, phenol groups and amine groups have the characteristics of nonpolar, polar uncharged and polar positively charged, respectively. The full-length nucleic acid library is 80bp, CAGGGGACGCACCAAGG-N40-CCATGACCCGCGTGCTGACATCG, the front and rear ends are fixed sequences for amplification, including only the natural nucleotide ATCG; the middle random sequences are designed to be 40, and synt...

Embodiment 2

[0096] Example 2, the application of nucleic acid aptamer AP5 in the detection of PA83 protein

[0097] 1. Biotin-labeled nucleic acid aptamer AP5

[0098] Synthesize biotin-labeled aptamer AP5, add a 10 dA sequence to the 5' end, 5'-Biotin-AAAAAAAAAA-AP5-3' (5'-Biotin-AAAAAAAAAAGCCCACGGCGGWYCGCCGGCCACAGTYAWCWGWGGTGGGC-3'), using RNAStructure software The secondary structure of the nucleic acid aptamer is predicted as Figure 4 .

[0099] 2. Construction of aptamer biosensor based on surface plasmon resonance technology by nucleic acid aptamer AP5

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

[0101] 1. Fix the dextran nano-gold chip on the...

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Abstract

The invention discloses a chemically modified base-containing single-stranded DNA aptamer capable of specifically recognizing anthrax protective antigen PA83 and application thereof. According to thechemically modified base-containing single-stranded DNA aptamer, the anthrax protective antigen (PA83) is taken as a target, a nucleic acid library containing chemically modified nucleotides is utilized, a high-affinity aptamer AP5 is screened out by a paramagnetic particle method SELEX, and the aptamer can specifically recognize PA83 protein in the environment of various interference proteins; the aptamer can be applied to an aptamer biosensor based on a surface plasmon resonance technology and a fluorescence polarization technology and used for detecting PA83 protein so as to detect bacillusanthracis, and the aptamer has the potential of being applied to construction of a novel bacillus anthracis protective antigen detection technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chemically modified base-containing single-stranded DNA aptamer specifically recognizing anthrax protective antigen PA83 and its application. Background technique [0002] The infection form of Bacillus anthracis is spores, which can enter the body through skin wounds, digestive tract or lungs, among which aerosol transmission is the main transmission route of bioterrorism attacks. The protective antigen PA is an important part of the anthrax toxin complex. The anthrax protective antigens commonly referred to are PA83 and PA63. Studies by the U.S. Army Institute of Infectious Diseases and the U.S. Centers for Disease Control and Prevention have shown that a large amount of PA83 exists in the culture supernatant of anthrax spore germination and in the body fluids of patients before infection. Therefore, the development of recognition elements that recognize PA83 protein...

Claims

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Application Information

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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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