利用大肠杆菌外膜上的自发FV抗体检测SARS-CoV-2的表面等离子共振(SPR)生物传感器

IF 5.5 3区 工程技术 Q1 BIOCHEMICAL RESEARCH METHODS
Ji-Hong Bong, Soo Jeong Lee, Jaeyong Jung, Jeong Soo Sung, Min-Jung Kang, Misu Lee, Joachim Jose, Jae-Chul Pyun
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引用次数: 0

摘要

严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)核头状蛋白(NP)参与病毒基因组的包装,并在感染时大量产生。本研究利用与 SARS-CoV-2 核头蛋白(NP)具有结合亲和力的 Fv 抗体,开发了用于检测病毒液中 SARS-CoV-2 的 SPR 生物传感器。利用在大肠杆菌外膜上表达的 FV 抗体库筛选出了与 SARS-CoV-2 NP 具有特异性结合活性的 FV 抗体。FV 抗体包括位于 IgG 结合袋的重链的三个互补性决定区(CDR)和四个框架区(FR)。通过定点突变和自显影技术制备了 FV 抗体库;使用 NP 固定磁珠筛选了与 SARS-CoV-2 的核壳蛋白(NP)具有特异性结合活性的 FV 抗体。首先,筛选带有目标 FV 抗体的大肠杆菌分离物,并利用 FACS 分析估算筛选出的大肠杆菌克隆的结合亲和力(KD)。然后,获得筛选出的带有自动显示的 Fv 抗体的大肠杆菌克隆的外膜(OM),并将其分层置于 SPR 生物传感器上,比较了四种不同冠状病毒(CoV)培养液(SARS-CoV-2、SARS-CoV、MERS-CoV 和 CoV 株 229E)的结合曲线。最后,将筛选出的大肠杆菌克隆的 FV 抗体合成为多肽(11 个氨基酸残基),并估算了与 NP 的结合常数(KD)以及培养液中 CoV 株系的结合曲线。通过对接模拟,研究了NP与每种合成肽的结合位点和相互作用类型。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Surface Plasmon Resonance (SPR) Biosensor for the Detection of SARS-CoV-2 Using Autodisplyaed FV-antibodies on Outer Membrane of E. coli

Surface Plasmon Resonance (SPR) Biosensor for the Detection of SARS-CoV-2 Using Autodisplyaed FV-antibodies on Outer Membrane of E. coli

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) participates in viral genome packaging and abundantly produced when infected. In this work, SPR biosensor for the detection of SARS-CoV-2 in viral fluid using Fv-antibodies with the binding affinity to nucleocapsid protein (NP) of SARS-CoV-2. The FV-antibodies with a specific binding activity to the SARS-CoV-2 NP were screened using the FV-antibody library, which was expressed on the outer membrane of E. coli. FV-antibodies comprised three complementarity-determining regions (CDRs) and four frame regions (FRs) of the heavy chain at the binding pocket of IgG. The FV-antibody library was prepared by performing site-directed mutagenesis and by using the autodisplay technology; FV-antibodies with specific binding activities to the nucleocapsid protein (NP) of SARS-CoV-2 were screened using NP-immobilized magnetic beads. First, E. coli isolates with the target FV-antibody were screened, and the binding affinity (KD) was estimated for the screened E. coli clones using FACS analysis. Then, the outer membrane (OM) of the screened E. coli clones with autodisplayed Fv-antibodies was obtained and layered on an SPR biosensor, and the binding curves of four different coronavirus (CoV) culture fluids, SARS-CoV-2, SARS-CoV, MERS-CoV, and CoV strain 229E, were compared. Finally, the FV-antibodies of the screened E. coli clones were synthesized as peptides (11 amino acid residues), and the binding constants (KD) to NP as well as the binding curves of the CoV strains in culture fluids were estimated. Using docking simulation, binding sites and interaction types between NP and each synthetic peptide were investigated.

Graphical Abstract

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来源期刊
BioChip Journal
BioChip Journal 生物-生化研究方法
CiteScore
7.70
自引率
16.30%
发文量
47
审稿时长
6-12 weeks
期刊介绍: BioChip Journal publishes original research and reviews in all areas of the biochip technology in the following disciplines, including protein chip, DNA chip, cell chip, lab-on-a-chip, bio-MEMS, biosensor, micro/nano mechanics, microfluidics, high-throughput screening technology, medical science, genomics, proteomics, bioinformatics, medical diagnostics, environmental monitoring and micro/nanotechnology. The Journal is committed to rapid peer review to ensure the publication of highest quality original research and timely news and review articles.
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