Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes
Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes
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DOI：
10.1007/s00216-012-6523-4
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发表时间：
2013-02
影响因子：
4.3
通讯作者：
Y. Olguín;P. Villalobos;L. Carrascosa;M. Young;E. Valdez;L. Lechuga;R. Galindo
中科院分区：
文献类型：
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作者：
Y. Olguín;P. Villalobos;L. Carrascosa;M. Young;E. Valdez;L. Lechuga;R. Galindo
鞭毛状细菌引起的消化系统疾病是世界范围内一个巨大的公共卫生问题，需要对受污染的环境进行快速检测。在这项研究中，我们提出了一种基于先天性免疫系统的属性来检测与病原体相关的模式的方法。具体地说，我们使用Toll样受体5(TLR5)，这是一种专门识别鞭毛蛋白(细菌鞭毛的结构蛋白)的跨膜蛋白。在昆虫细胞中重组获得TLR5，将其固定化到脂质体中，形成TLR5-蛋白脂质体。通过表面等离子共振(SPR)和竞争流式细胞仪分析，评价蛋白脂质体对大肠埃希菌和鼠伤寒沙门氏菌鞭毛蛋白的敏感性。此外，我们还比较了将抗鞭毛蛋白抗体固定到脂质体中所获得的结果。鞭毛亲和力试验结果表明，TLR5蛋白脂质体和抗鞭毛抗体对鼠伤寒沙门氏菌的亲和力分别为13.8 × 10−9和7.73 × 10−9M。抗鞭毛蛋白亲和力KD分别为84.1 × 10−8M和3.5 × 10−8M，用于竞争流式细胞术。这项研究证明了将蛋白脂质体作为识别元件用于鞭毛细菌快速检测系统的实际可能性，这有助于避免人类食用受污染的食物，从而预防肠道感染。
Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognizeEscherichia coliandSalmonella typhimuriumflagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equationKD=kd/ka, showed values of 13.8 × 10−9and 7.73 × 10−9M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, againstS.typhimurium. The anti-flagellin affinity results forE.colishowedKDof 84.1 × 10−8M for SPR assays andKDof 3.5 × 10−8M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.