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Aptamer C202 of staphylococcus aureus enterotoxin C2 as well as screening method and applications thereof

A staphylococcal gut and nucleic acid aptamer technology, which is applied in the screening process, pharmaceutical formulations, biochemical equipment and methods, etc., can solve the problems of poor specificity of adsorption selection and high cost of reagents, and achieve good reproducibility, low cost, Good permeability effect

Active Publication Date: 2017-04-26
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

However, the purification step in this method often adopts ion exchange method or tagged affinity purification method, the former has poor adsorption selection specificity, and the latter has high reagent cost

Method used

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  • Aptamer C202 of staphylococcus aureus enterotoxin C2 as well as screening method and applications thereof
  • Aptamer C202 of staphylococcus aureus enterotoxin C2 as well as screening method and applications thereof
  • Aptamer C202 of staphylococcus aureus enterotoxin C2 as well as screening method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Screening of nucleic acid aptamer C202

[0048] The screening method of the nucleic acid aptamer C202 of the Staphylococcus aureus enterotoxin C2, it comprises the following steps:

[0049] (1) Preparation of the screening library: design a random ssDNA library (5'-AGGGCCGAGCTCACTTGT-N 35 -CCGCCGGCACAATTGTGC-3'), and commissioned Sangon Bioengineering Co., Ltd. to synthesize.

[0050] (2) Coupling of Staphylococcus aureus enterotoxin C2 with carboxyl magnetic beads: the Staphylococcus aureus enterotoxin C2 protein was purchased from Toxin Technology Company of the United States, and the carboxyl magnetic beads and their coupling reagents were purchased from Bangs Laboratories Company of the United States. The operation refers to the instructions provided by the manufacturer; the change of the protein concentration in the Staphylococcus aureus enterotoxin C2 solution before and after coupling is measured by the BCA method for protein concentration, and the co...

Embodiment 2

[0058] Example 2: Analysis of the nucleic acid aptamer C202 sequence:

[0059] (1) After 9 rounds of screening, the enriched ssDNA library was collected, and Shanghai Passino Biotechnology Co., Ltd. was entrusted to use high-throughput sequencing technology to analyze the library sequence. The analysis process was: PCR amplification of the enriched library, And add the sequencing adapter and Index part; select and purify the library by gel electrophoresis; use the Agilent 2100Bioanalyzer to control the quality of the library through the Agilent High Sensitivity DNA Kit; use the Quant-iT PicogGreen dsDNAAssay Kit to quantify the library; use the IlluminateNextSeq 500 platform to single The chain library is used as a template for bridge PCR amplification, sequencing primer annealing, and sequencing while synthesizing; and performing comparison and enrichment analysis on the sequencing results.

[0060] (2) Using the UNAFold network platform to analyze at 25°C, 100mM Na + , 1 mM...

Embodiment 3

[0061] Example 3: Specificity analysis of the nucleic acid aptamer C202:

[0062] (1) FAM-labeled aptamer C202 was chemically synthesized in vitro and dissolved in selection buffer.

[0063] (2) Referring to step (2) in Example 1, BSA, Staphylococcus aureus enterotoxin A, Staphylococcus aureus enterotoxin B and Staphylococcus aureus enterotoxin C1 were respectively coupled with carboxyl magnetic beads to prepare BSA magnetic beads , Staphylococcus aureus enterotoxin A magnetic beads, Staphylococcus aureus enterotoxin B magnetic beads, Staphylococcus aureus enterotoxin C1 magnetic beads and Staphylococcus aureus enterotoxin C2 magnetic beads. Wherein, the BSA was purchased from Sigma Company, and the Staphylococcus aureus enterotoxin A, Staphylococcus aureus enterotoxin B and Staphylococcus aureus enterotoxin C1 were all purchased from Toxin Technology Company of the United States.

[0064] (3) Take 200 μL of the nucleic acid aptamer C202 solution obtained in step (1) and resp...

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Abstract

The invention relates to aptamer C202 of staphylococcus aureus enterotoxin C2 as well as a screening method and applications of the aptamer C202. The aptamer C202 has the sequence as follows: AGGGCCGAGCTCACTTGTCACGGGCACCCTAACTGGGAGGAGGCAGGATAACACCGCCGGCACAATTGTGC. The screening method comprises the step of carrying out screening from an ssDNA library through staphylococcus aureus enterotoxin C2 magnetic beads based on the in-vitro SELEX screening technology of the aptamer and by taking carboxylic magnetic beads as the solid-phase medium and staphylococcus aureus enterotoxin C2 as the target, and thus the aptamer in specific binding with the staphylococcus aureus enterotoxin C2 is obtained. The aptamer C202 provided by the invention can be bond to the staphylococcus aureus enterotoxin C2 with high affinity and high specificity.

Description

technical field [0001] The invention relates to a nucleic acid aptamer C202 of Staphylococcus aureus enterotoxin C2 and its screening method and application. Background technique [0002] Staphylococcus aureus enterotoxin is an exotoxin with superantigen activity secreted by Staphylococcus aureus. Staphylococcus aureus enterotoxin can be directly combined with MHC class Ⅱ molecules without restriction without being processed by antigen-presenting cells, and the complex formed can bind to the β-chain V region of T lymphocyte antigen receptors to activate T lymphocytes in large quantities, making It activates, proliferates, and releases a large amount of inflammatory cytokines to cause a strong immune response, which eventually leads to uncontrolled inflammation and damage to multiple organs, causing toxic shock and other diseases. In addition, because Staphylococcus aureus enterotoxin is more resistant to digestion, heat resistance and can trigger T cell responses with only ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10C12Q1/68G01N33/569A61K31/7088A61P35/00
CPCC12N15/1048C12N15/115C12N2310/16C12N2310/531C12N2320/13C12N2320/30G01N33/56938C12Q2565/519
Inventor 王开宇兰小鹏廖剑洪笑迁闫慧慧杨湘越
Owner FUZHOU GENERAL HOSPITAL OF NANJING MILITARY COMMAND P L A
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