利用 SELEX 方法筛选和鉴定用于检测 Epsilon 毒素的 DNA 纳米结构适配体

IF 1.8 4区 医学 Q3 PHARMACOLOGY & PHARMACY Iranian Journal of Pharmaceutical Research Pub Date : 2023-12-08 DOI:10.5812/ijpr-140505
Nafiseh Shafiei, H. Mahmoodzadeh Hosseini, Jafar Amani, Ali Mirhosseini, H. Jafary
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引用次数: 0

摘要

背景:产气荚膜梭菌(Clostridium perfringens)产生的Epsilon毒素(ETX)是已知最“有效”的毒素之一,其致命效力接近肉毒杆菌神经毒素。Epsilon毒素是导致肠炎的原因。因此,开发快速、简便的方法来检测ETX势在必行。适配体是一种单链寡核苷酸,可以与特异性靶分子紧密结合,其亲和力与单克隆抗体(mab)相当。DNA适体可以作为生物分子鉴定的工具,如病原体亚种。目的:本研究旨在分离抗ETX的高亲和力单链DNA (ssDNA)适配体。方法:本研究采用配体系统进化法(SELEX)、酶联apta吸附法(ELASA)和表面等离子体共振法(SPR)鉴定适配体,以确定新获得的适配体靶向ETX的亲和力和特异性。结果:对通过SELEX工艺获得的几个适体进行了研究。其中适配体2个,ETX克隆3号(ETX3;解离常数[Kd] = 8.4±2.4E-9M)和ETX11 (Kd = 6.3±1.3E-9M)对ETX具有良好的特异性。ETX3和ETX11的检出限分别为0.21和0.08 μg/mL。结论:所发现的适配体可用于各种基于适配体的快速诊断检测ETX。
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Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for ‎Detection of Epsilon Toxin
Background: Epsilon toxin (ETX), produced by Clostridium perfringens, is one of the most ‎potent toxins ‎known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin ‎is responsible ‎for enteritis. Therefore, the development of rapid and simple methods to ‎detect ETX ‎is imperative. Aptamers are single-stranded oligonucleotides that can bind ‎tightly to specific ‎target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). ‎DNA aptamers ‎can serve as tools for the molecular identification of organisms, such as ‎pathogen subspecies.‎ Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) ‎aptamers against ETX.‎ Methods: This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine ‎the affinity and ‎specificity of the newly obtained aptamers targeting ETX. ‎ Results: Several aptamers obtained through the ‎SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant [Kd] = 8.4 ± 2.4E-9M) ‎and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection ‎were 0.21 and 0.08 μg/mL for ETX3 and ETX11, respectively.‎ Conclusions: The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.
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来源期刊
CiteScore
3.40
自引率
6.20%
发文量
52
审稿时长
2 months
期刊介绍: The Iranian Journal of Pharmaceutical Research (IJPR) is a peer-reviewed multi-disciplinary pharmaceutical publication, scheduled to appear quarterly and serve as a means for scientific information exchange in the international pharmaceutical forum. Specific scientific topics of interest to the journal include, but are not limited to: pharmaceutics, industrial pharmacy, pharmacognosy, toxicology, medicinal chemistry, novel analytical methods for drug characterization, computational and modeling approaches to drug design, bio-medical experience, clinical investigation, rational drug prescribing, pharmacoeconomics, biotechnology, nanotechnology, biopharmaceutics and physical pharmacy.
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