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Nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A and application thereof

A nucleic acid aptamer, staphylococcal intestinal technology, applied in the biological field, can solve the problems of false negative, high cost, false positive, etc., and achieve the effect of inhibiting the proliferation of mononuclear cells, easy to synthesize and label, and small molecular weight

Inactive Publication Date: 2016-02-24
FUZHOU GENERAL HOSPITAL OF NANJING MILITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The detection of SEA mainly adopts immunological methods, including precipitation reaction, agglutination reaction, ELISA, solid-phase RIA, biosensor, etc. These methods use antibodies as the recognition elements of SEA detection, and the preparation of antibodies has a long cycle, cumbersome steps, and high cost. Inadequacies
In recent years, molecular biology methods targeting the detection of SEA genes have developed rapidly, but molecular biology methods such as PCR still have technical problems such as false positives, false negatives, and gene sinking expression

Method used

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  • Nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A and application thereof
  • Nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A and application thereof
  • Nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: In vitro screening of nucleic acid aptamers that specifically bind to SEA

[0027] (1) Design and synthesis of random single-stranded DNA (ssDNA) library

[0028] Design and synthesize a random ssDNA library with fixed regions of 18 nucleotides at both ends and a random region of 40 nucleotides in the middle as follows: 5’-ATACCAGCTTTATTCAATT–N40–AGATAGTAAGTGCAATCT-3’, with a library capacity of 4 40 . The primer sequences used are:

[0029] P1: 5'-ATACCAGCTTATTCAATT-3';

[0030] P2: 5'-AGATTGCACTTACTATCT-3';

[0031] P3: 5'-FAM-ATACCAGCTTTATTCAATT-3';

[0032] P4: 5'-triple Biotin-AGATTGCACTTACTATCT-3'.

[0033] The above nucleic acid sequences were synthesized by a biotechnology company and purified by HPLC.

[0034] (2) Screening of nucleic acid aptamers

[0035] 1) Carboxyl magnetic beads are used as the carrier of the target molecule SEA, which is convenient for the separation operation of the binding sequence. Using a commercial EDC kit, refer t...

Embodiment 2

[0043] Example 2: Fluorescence binding rate experiment monitoring the enrichment of ssDNA library

[0044] (1) In Example 1, the FAM-labeled ssDNA library obtained in each round of screening was used in the fluorescence binding rate experiment;

[0045] (2) Take 200 μl of ssDNA-containing library and dissolve it in selection buffer, heat denature at 95°C for about 5 minutes, put it in ice bath for 10 minutes, and place it at room temperature for 10 minutes; then mix it with SEA-beads (SEA load is 100 ng) Incubate at 37°C for 1 hour in a dark box;

[0046] (3) Collect supernatant (containing ssDNA not bound to SEA); ssDNA bound to SEA-beads was eluted with 200 μl selection buffer at 100°C for 5 min;

[0047] (4) Measure the fluorescence intensity of the initial, non-SEA-bound, and SEA-bound ssDNA libraries using a fluorescence quantitator, and calculate the fluorescence binding rate=(initial fluorescence intensity-eluent fluorescence intensity) / initial fluorescence intensity×1...

Embodiment 3

[0049] Example 3: Cloning, sequencing and biological analysis of aptamers

[0050] (1) Using the enriched ssDNA library as a template, PCR amplification is performed with primers P1 and P2;

[0051] (2) Take the purified PCR product and carry out T-A cloning;

[0052] (3) pick 50 positive clones and carry out nucleotide sequence analysis;

[0053] (4) Use ClustalXSoftware to analyze the homology of nucleic acid sequences, select the most 4 kinds of sequences as candidate aptamers, the candidate aptamers are respectively S3, S6, S12, S23; use the network tool mfold according to the lowest free energy In principle, the secondary structure of candidate aptamers is modeled.

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Abstract

The invention relates to a nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A and application thereof. The nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A has a unique stem loop structure and can realize structural reconstruction like chemical modification of FITC, an amino group, biotin and digoxin and truncation, prolongation or partial base substitution. The nucleic acid aptamer S3 can realize high-affinity and high-specificity bonding with Staphylococcus aureus enterotoxin A, inhibits superantigen activity of Staphylococcus aureus enterotoxin A in in-vitro experiments and has the advantages of no toxicity, small molecular weight, good permeability and easiness in synthesis and marking. The nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A can be applied to non-disease diagnosis and treatment methods for separation and enrichment or analysis and detection of Staphylococcus aureus enterotoxin A and to treatment of infection with Staphylococcus aureus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel nucleic acid aptamer S3 of Staphylococcus aureus enterotoxin A (SEA) and its application. Background technique [0002] Staphylococcus aureus is one of the most common pathogens causing hospital infection and social infection. It can not only cause suppuration, necrosis, and abscess formation of infected tissues, but also cause food poisoning and toxins by secreting various exotoxins, including Staphylococcus aureus enterotoxins (SEs), hemolysin, toxin shock syndrome toxin 1, etc. Serious diseases such as shock syndrome. In the United States, food poisoning caused by SEs accounts for 33% of bacterial food poisoning; in Canada, it is more, accounting for 45%. Such poisoning occurs more often in my country every year, and SEA is the most common toxin that causes food poisoning. As a superantigen, SEA can directly stimulate the activation and proliferation of T lymphocytes wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68A61K31/7088A61P31/04
Inventor 王开宇兰小鹏陈庄杨湘越张鲜惠
Owner FUZHOU GENERAL HOSPITAL OF NANJING MILITARY COMMAND P L A
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