新型纳米抗体的开发与鉴定及其对 SRMV 的中和活性。

Development and characterization of a novel nanobody with SRMV neutralizing activity.

机构信息

Department of Viral Biologics, China Institute of Veterinary Drug Control, Beijing, China.

Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou, China.

出版信息

Microb Cell Fact. 2024 Feb 10;23(1):45. doi: 10.1186/s12934-024-02311-6.

Abstract

Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 10 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.

摘要

小反刍兽疫(PPR)是一种由小反刍兽疫病毒(SRMV)引起的急性、接触性传染病,其在山羊和绵羊中的发病率可达 100%,死亡率很高。与传统抗体相比,来自羊驼等骆驼科动物的纳米抗体因其独特的优势而引起了广泛关注。本研究的主要目的是制备针对 SRMV 的特异性纳米抗体并鉴定其特性。为了获得针对 SRMV 的特异性纳米抗体的编码基因,我们首先从小反刍动物的 VHH 库中构建了一个免疫噬菌体展示文库,该文库是用 SRMV-F 和 -H 蛋白免疫的。通过噬菌体展示技术,经过四轮生物淘选后可获得针对靶抗原的特异性 VHH。结果表明,该 VHH 文库的大小为 2.26×10 CFU/mL,经过四轮生物淘选后,SRMV-F 和 -H 特异性噬菌体颗粒得到了极大的富集。选择并测序阳性噬菌体克隆,共鉴定出 5 个不同的针对 SRMV 的特异性纳米抗体。随后,将这 5 个纳米抗体的 DNA 片段分别克隆到大肠杆菌 BL21(DE3)中,其中 3 个成功表达和纯化。然后使用 ELISA 法评估了这些纯化的纳米抗体对灭活的 SRMV 的特异性和亲和力。结果表明,NbSRMV-1-1、NbSRMV-2-10 和 NbSRMV-1-21 与其他抗原(如灭活的 BTV、灭活的 FMDV、His 标记蛋白和 BSA)无交叉反应。这三种纳米抗体对灭活的 SRMV 的 ELISA 滴度高达 1:1000。然而,只有 NbSRMV-1-21 在最大稀释度为 1:4 时显示出 SRMV 中和活性。结果表明,本研究中产生的针对 SRMV 的纳米抗体在未来的应用中可能很有用。本研究提供了一种新的抗体工具,并为 SRMV 的治疗和检测奠定了基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/88b9/10858559/ec1c868b710d/12934_2024_2311_Figa_HTML.jpg

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索