ssDNA aptamer for recognizing ofloxacin with specificity and application of ssDNA aptamer

A technology of ofloxacin and aptamer, applied in recombinant DNA technology, DNA/RNA fragments, material testing products, etc., can solve problems such as enhanced phototoxicity, liver cell damage, and drug efficacy reduction. Quick and easy to operate, easy to modify and mark, stable results

Active Publication Date: 2015-08-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the long-term accumulation of quinolone antibiotics in animals reaches a certain level, the resistance to such antibiotics in the organism will increase, and the number of drug-resistant bacteria will gradually increase, which will eventually lead to the reduction of the efficacy of such drugs; in addition, also impairs cartilage growth in young animals
Large doses of such antibiotics entering the human body can cause adverse reactions such as gastrointestinal discomfort, damage to the central nervous system and liver cells
Ofloxacin belongs to fluoroquinolone drugs, because the 8-position is replaced by fluorine element to enhance phototoxicity, long-term consumption of animal source food exceeding the standard of veterinary drugs will not only cause direct or indirect damage to human health, but also cause human Drug resistance is not conducive to the prevention and treatment of bacterial diseases

Method used

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  • ssDNA aptamer for recognizing ofloxacin with specificity and application of ssDNA aptamer
  • ssDNA aptamer for recognizing ofloxacin with specificity and application of ssDNA aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of random ssDNA library and primers thereof

[0034] (a) Construction of a random ssDNA library with a length of 79 bases

[0035] 5′-TAGGGAATTC GTCGACGGAT CC-N35-CTGCAGGTCG ACGCATGCGC CG-3′ where N represents any one of bases A, T, C, and G, and N35 represents a random fragment length of 35 bases.

[0036] (b) Synthesis of upstream primers

[0037] Upstream primer 1: 5′-TAGGGAATTC GTCGACGGAT-3′

[0038] (c) Synthesis of downstream primers

[0039] Downstream primer 1: 5′-CGGCGCATGC GTCGACCTG-3′

[0040] Downstream primer 2: 5′-biotin-CGGCGCATGC GTCGACCTG-3′

Embodiment 2

[0041] Example 2 In vitro screening of nucleic acid aptamers

[0042] In order to screen ssDNA aptamers with high affinity and specificity to ofloxacin, a total of 7 rounds of nucleic acid aptamer screening were carried out. In order to improve the specificity of screening, a round of negative screening experiments was carried out after the sixth round of screening. In each round of screening, the recovery rate of ssDNA after the combination of the aptamer and ofloxacin was as follows: figure 1 shown.

[0043] (a) Take 1 μL of 100 μmol L -1 (0.1nmol) of the initial library ssDNA, add 199 μL of binding buffer, mix well, denature at 90°C for 10 minutes, rapidly ice-bath for 10 minutes, place at room temperature for 10 minutes, add the same amount of ofloxacin solution, mix well, and store at room temperature Slightly oscillating for 1 hour, ssDNA self-adaptively forms a three-dimensional structure, and a part of the three-dimensional structure formed by ssDNA can combine with ...

Embodiment 3

[0056] Example 3 Screened ssDNA cloning, sequencing, and structural analysis

[0057] (a) ssDNA clone sequencing

[0058] The ssDNA obtained after the final round of screening was amplified by PCR with upstream primer 1 and downstream primer 1, and the entire amount of the amplified product was loaded on 3% agarose, electrophoresed and the target band was excised, and the ssDNA was recovered by DNA gel recovery kit . Mix 7 μL of the purified PCR product with 1 μL of the pMD-19T vector, ligate overnight at 16°C under the action of T4 ligase, transform into Escherichia coli JM109 competent cells, and culture at 37°C overnight. After colony PCR verification, 21 positive clones were randomly selected and transferred to liquid LB medium, cultured for 12 hours, plasmids were extracted with a plasmid extraction kit, and 12 aptamers of different sequences from A1 to A12 were obtained by sequencing.

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Abstract

The invention discloses an ssDNA aptamer for recognizing ofloxacin with the specificity and application of the ssDNA aptamer and belongs to the field of biochemistry, molecular biology, analytical chemistry and combinatorial chemistry. According to the ssDNA aptamer and the application thereof disclosed by the invention, a method of screening an ofloxacin aptamer by utilizing a modified graphene oxide-SELEX technology (GO-SELEX) is provided to obtain four ssDNA aptamer sequences A1, A3, A4 and A5 with high affinity with the ofloxacin, so that a detection and recognition element which is good in stability, high in affinity and specificity, low in cost and easy for modification and marking is provided to detect residual ofloxacin.

Description

technical field [0001] The invention relates to a ssDNA aptamer specifically recognizing ofloxacin and application thereof, belonging to the fields of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry. Background technique [0002] Fluoroquinolones such as ofloxacin, sarafloxacin, pefloxacin, etc., are effective against Escherichia coli, Salmonella, Staphylococcus, Klebsiella, Shigella, Proteus, Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus and other bacteria have strong antibacterial activity, and they also have good antibacterial effects on Bacillus pyogenes, Streptococcus pneumoniae, Chlamydia pneumoniae, and Mycoplasma pneumoniae. The mechanism of action of ofloxacin is the same as that of other quinolone antibiotics, by inhibiting the synthesis of bacterial DNA gyrase (gyrase), blocking the normal synthesis and replication of DNA, leading to the death of bacteria. Therefore, quinolone drugs are one of the main anti-infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53
Inventor 周楠迪游元丁田亚平
Owner JIANGNAN UNIV
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