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Improved penicillin antibiotic aptamer without fixed point target substance and application thereof

A technology of penicillin and antibiotics, applied in the direction of DNA/RNA fragments, recombinant DNA technology, color/spectral characteristic measurement, etc., can solve the problems of ssDNA loss and introduction of other substances, and achieve convenient operation, strong applicability, and good fixation effect Effect

Active Publication Date: 2013-11-13
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process may cause the loss of ssDNA with high affinity for cadmium ions or the introduction of other substances

Method used

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  • Improved penicillin antibiotic aptamer without fixed point target substance and application thereof
  • Improved penicillin antibiotic aptamer without fixed point target substance and application thereof
  • Improved penicillin antibiotic aptamer without fixed point target substance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] like figure 1 As shown, this embodiment includes the following steps:

[0033] 1) Construct a random oligonucleotide library with a total length of 68bp. The sequence of the oligonucleotide library is: 5'-ACCGACCGTGCTGGACTCT-N30-AGTATGAGCGAGCGTTGCG-3', wherein both ends are fixed sequences of 19bp, and the middle 30 bases is a random sequence.

[0034] 2) Design three primers, wherein: P1 (5'-ACCGACCGTGCTGGACTCT-3') and P2 (5'-CGCAACGCTCGCTCATACT-3') are used as the upstream primer and downstream primer of PCR amplification oligonucleotide respectively, and the upstream primer (5 '-Biotin-CGCAACGCTCGCT CATACT-3') was used to immobilize the oligonucleotide library on streptavidin-labeled agarose particles.

[0035] 3) Take a certain amount of random oligonucleotide library, and mix it evenly with the fixed sequence of agarose particles in a certain ratio (1:2~3), and anneal the mixture. The annealing conditions are: 94°C for 60 seconds, 59°C Anneal for 60 minutes, ext...

Embodiment 2

[0045] like figure 2 Shown is the specific description of the aptamer prepared in Example 1.

[0046] In this example, through cloning and sequencing, the base composition of the prepared aptamer is analyzed as shown in Seq ID No.1, specifically:

[0047] 5'-ACCGACCGTGCTGGACTCTGGCACGGGCGGGAGATAAGTTTGCTGGACCAGTATGAGCGAGCGTTGCG-3'

[0048] In this example, Mfold software was used to simulate the secondary structure of penicillin aptamers. And by calculation, the free energy of the penicillin antibiotic aptamer is -14.92, and the four bases A, T, C, and G are relatively evenly distributed. The number of T+G accounts for 43.6% of the total bases.

Embodiment 3

[0050] like figure 2 As shown, the specific application of the aptamer prepared in Example 1, that is, the penicillin aptamer sensor, its structure, composition and preparation method are

[0051] Optimizing the concentration of aptamers in the system: add penicillin antibiotic aptamers in the concentration range of 1-20nM to the nanogold solution, and finally add 0.1365μM PDDA, and use a microplate reader to measure the ratio of light absorption at 650nm and 520nm The curve was drawn for the difference, and the peak corresponding to the penicillin antibiotic aptamer concentration was 6nM.

[0052] Optimizing the PDDA concentration in the system: After incubating 6nM penicillin antibiotic aptamer with 0.2 and 2μM 6-APA solution for 15 minutes, add 100μL nano-gold, and finally add PDDA with a concentration range of 0.084-0.158nM for 5 minutes, then use The difference between the ratio of light absorption values ​​at 650nm and 520nm measured by the microplate reader was drawn ...

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Abstract

The invention discloses a penicillin antibiotic aptamer obtained by the screening technology for an improved aptamer without a fixed point target substance in the field of screening a penicillin antibiotic aptamer without a fixed point target substance, and an application thereof. The unfixed nucleic acid is eluted through a fixed oligonucleotide library; the parent nucleus 6-APA (6-aminopenicillanic acid) of the penicillin antibiotic is added in positive screening to act on the fixed nucleic acid; after eluting the nucleic acid molecules which can be combined with 6-APA, PCR (polymerase chain reaction) amplification is directly performed for next screening; and other antibiotics are added in negative screening, and the nucleic acid molecules in non-specific binding with the penicillin antibiotics are removed by elution. Through multiple rounds of positive and negative screening, 10 nucleic acid aptamers with high specificity and strong affinity with 6-APA are obtained; and the nucleic acid aptamer with a stable secondary structure is selected for developing a nucleic acid aptamer sensor for detecting 6-APA.

Description

technical field [0001] The present invention relates to the field of aptamer screening of penicillin antibiotics without fixed point target substances, in particular to the use of streptavidin-labeled agarose particles to immobilize an oligonucleotide library, and to screen out penicillins by SELEX technology Antibiotic aptamers. Background technique [0002] An aptamer (aptamer, or nucleic acid aptamer) is a short oligonucleotide chain obtained through in vitro screening by SELEX technology (systematic evolution of ligands by exponential enrichment, that is, systematic evolution of ligands enriched exponentially). The word Aptamer comes from the Latin "aptus", which means pair adaptation. It can be folded into a clear three-dimensional structure, and binds to target molecules with high affinity and specificity through complementary spatial configuration. In 1990, Ellington and Szostak first reported in "Nature" that this large-capacity random single-stranded oligonucleoti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N21/31
Inventor 周培贺兰智文婷詹深山罗艳芳刘乐
Owner SHANGHAI JIAO TONG UNIV
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