Development and characterization of a novel nanobody with SRMV neutralizing activity

文献类型： 外文期刊

第一作者： Sun, Miao

作者： Sun, Miao;Chen, Yanfei;Chen, Jian;Li, Ling;Xue, Qinghong;Wang, Changjiang;Qu, Guanggang;Luo, Huaye;Zhang, Min

作者机构： China Inst Vet Drug Control, Dept Viral Biol, Beijing, Peoples R China;Shandong Binzhou Anim Sci & Vet Med Acad, Binzhou, Peoples R China;Chinese Acad Agr Sci, Shanghai Vet Res Inst, Shanghai, Peoples R China;Tech Bank Food Corp Ltd, Nanjing, Peoples R China

关键词： Small ruminant morbillivirus (SRMV); Nanobody; Fusion protein; Hemagglutinin protein

期刊名称：MICROBIAL CELL FACTORIES （ 2022影响因子：6.4； 五年影响因子：6.3 ）

ISSN：

年卷期： 2024 年 23 卷 1 期

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收录情况： SCI

摘要： 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 x 10(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.

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