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Nucleic acid aptamer eta01 of Pseudomonas aeruginosa exotoxin a and its application

A technology of Pseudomonas aeruginosa and nucleic acid aptamers, applied in the field of biomedicine, can solve problems that have not yet been seen, and achieve the effects of wide application value, low preparation cost, and broad market prospects.

Active Publication Date: 2022-06-28
丽水君弘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it is more convenient and faster to directly detect Pseudomonas aeruginosa exotoxin A, due to the specificity of the antibody, only a few immunological techniques are used in this area
There is no report on the screening and application of nucleic acid aptamers targeting Pseudomonas aeruginosa exotoxin A

Method used

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  • Nucleic acid aptamer eta01 of Pseudomonas aeruginosa exotoxin a and its application
  • Nucleic acid aptamer eta01 of Pseudomonas aeruginosa exotoxin a and its application
  • Nucleic acid aptamer eta01 of Pseudomonas aeruginosa exotoxin a and its application

Examples

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example 1

[0040] Example 1: Detection of Pseudomonas aeruginosa Exotoxin A by Colorimetric Biosensor Using Nucleic Acid Aptamer ETA01 as Recognition Element

[0041] (1) Synthesize the nucleic acid aptamer ETA01 (synthesized by Shanghai Sangong Co., Ltd.), the sequence is: 5'-GAGCGGCACGAACCAGTAAAGTCTTCCCGACCGC-3'.

[0042](2) The nucleic acid aptamer ETA01 was dissolved in a suitable volume of selection buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl 2 , 5 mM KCl, pH 7.4); then heat-activated. The method of heat activation treatment is as follows: after denaturation at 95°C for 5 min, immediately placed in an ice-water bath for 10 min, and then placed at room temperature for 10 min.

[0043] (3) The heat-activated nucleic acid aptamer ETA01 (final concentration of 2 μM) was treated with exotoxin A (ETA), extracellular enzyme S (ExoS), and extracellular enzyme T (ExoT) of Pseudomonas aeruginosa, respectively. , one or more of extracellular enzyme Y (ExoY), and extracellular enzyme U (Exo...

Embodiment 2

[0049] Example 2 Using the nucleic acid aptamer ETA01 as the recognition element, the gel migration retardation experiment was used to detect the ETA of Pseudomonas aeruginosa

[0050] (1) Synthesize the ETA01 sequence, and label the fluorophore FITC at its 5' end (synthesized by Shanghai Sangong Co., Ltd.), the sequence is: 5'-TGCAACTCCAACTAACCATGTGTTCATCTGAGGGTGGTCTTCGCA-3'.

[0051] (2) Dissolve the FITC-labeled nucleic acid aptamer ETA01 in an appropriate volume of selection buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl) 2 , 5 mM KCl, pH 7.4); then thermally activated. The method of heat activation treatment is as follows: after denaturation at 95°C for 5 min, immediately placed in an ice-water bath for 10 min, and then placed at room temperature for 10 min.

[0052] (3) Incubate the heat-activated FITC-labeled ETA01 with two mixtures of Pseudomonas aeruginosa exotoxin containing and not containing ETA for 1 h at room temperature in a dark box, respectively, and the exot...

Embodiment 3

[0056] Example 3: Using the biotin-labeled nucleic acid aptamer ETA01 as the recognition element, the enzyme-linked aptamer adsorption method was used to detect the ETA of Pseudomonas aeruginosa

[0057] (1) Synthesize the sequence of nucleic acid aptamer ETA01, label the 5' end with biotin (synthesized by Shanghai Sangong Company), and the sequence is: 5'-TGCAACTCCAACTAACCATGTGTTCATCTGAGGGTGGTCTTCGCA-3'.

[0058] (2) Dissolve Pseudomonas aeruginosa ETA, ExoS, ExoT, ExoY, and ExoU expressed in E. coli in carbonate buffer at pH 9.6, the molar concentration is 100 mM, and add 100 μL / well to the enzyme-linked strip. in a humidified chamber overnight at 4°C.

[0059] (3) Discard the coating solution, add 100 μL of maleic acid blocking solution containing 1% casein to each well, and block at room temperature for 1 hour.

[0060] (4) Dissolve biotin-labeled ETA01 in an appropriate volume of selection buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl) 2 , 5 mM KCl, pH 7.4), followed b...

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Abstract

The present invention relates to a nucleic acid aptamer ETA01 of Pseudomonas aeruginosa exotoxin A and its application. The sequence of the nucleic acid aptamer ETA01 includes: 5'-TGCAACTCCAACTAACCATGTGTTCAT CTGAGGGTGGTCTTCGCA-3'. The nucleic acid aptamer ETA01 of the present invention can bind to Pseudomonas aeruginosa exotoxin A with high affinity and high specificity, and use the nucleic acid aptamer ETA01 as the recognition element of Pseudomonas aeruginosa exotoxin A to establish Pseudomonas aeruginosa The detection method of bacterial exotoxin A, the preparation of detection reagents, and the development of exotoxin A antagonistic drugs can be used in the diagnosis and treatment of Pseudomonas aeruginosa infection and the research on the biological function of Pseudomonas aeruginosa exotoxin A. The method has the advantages of simplicity, rapidity and good characteristics.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a nucleic acid aptamer ETA01 of Pseudomonas aeruginosa exotoxin A and its application. Background technique [0002] Pseudomonas aeruginosa is a common gram-negative rod-shaped bacterium that is optimally adapted to a variety of environmental conditions. Although Pseudomonas aeruginosa is an obligate aerobe, it can also perform anaerobic respiration through nitrate or other electron acceptors. Therefore, Pseudomonas aeruginosa is ubiquitous in soil, water or sewage, as well as in human, animal or plant hosts, and is widespread throughout the world. Pseudomonas aeruginosa infection in healthy individuals is very rare, but as an opportunistic pathogen, it is frequently parasitized in immunocompromised patients with cystic fibrosis, burns, or AIDS. Infections range from endocarditis, endocarditis, meningitis and sepsis to chronic lung infections. Since Pseudomonas aeruginosa ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/569G01N33/53A61K31/7088A61P31/04
CPCC12N15/115G01N33/56911G01N33/5308A61K31/7088A61P31/04C12N2310/16C12N2310/531
Inventor 吴冬
Owner 丽水君弘生物科技有限公司
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