Development and characterization of a novel nanobody with SRMV neutralizing activity

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 × 10¹⁰ 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.

Keywords: Fusion protein; Hemagglutinin protein; Nanobody; Small ruminant morbillivirus (SRMV).

Fig. 1

Serum antibody titer after immunization and biopanning process monitoring. (A) Serum antibody titer against SRMV-F and –H after immunization procedure, which were both up to 1:50000. (B) Enrichment evaluation of phage-displayed nanobodies by biopanning. Phage-displayed nanobodies were selected against SRMV-F and –H protein respectively, and E. coli TG1 was infected with bound phage. The number of phages eluted after each round of biopanning was counted based on the number of plaques (PFU/mL) formed after infection of the host bacteria with the eluted phage particles. (C) ELISA assay for selection of individual phage particles against SRMV-F and -H protein, respectively

Fig. 2

Amino acid sequence of anti-SRMV nanobodies after four rounds of biopanning. The frameworks and complementary determining regions were determined according to IMGT

Fig. 3

Expression and identification of nanobodies against SRMV. (A) Prokaryotic expression of nanobodies against SRMV (M standard protein marker, 1–2 supernatant and precipitation of negative bacteria respectively, 3–4 supernatant and precipitation of NbSRMV-1-1 expression bacteria respectively, 5–6 supernatant and precipitation of NbSRMV-2-10 expression bacteria respectively, 7–8 supernatant and precipitation of NbSRMV-1-11 expression bacteria respectively, 9–10 supernatant and precipitation of NbSRMV-1-21 expression bacteria respectively, 11–12 supernatant and precipitation of NbSRMV-2-34 expression bacteria respectively). (B) Purification of nanobodies against SRMV (M standard protein marker, 1 purified NbSRMV-1-1, 2 purified NbSRMV-2-10, 3 purified NbSRMV-1-21). (C) Western blotting analysis of nanobodies against SRMV (M standard protein marker, 1 NbSRMV-1-1, 2 NbSRMV-2-10, 3 NbSRMV-1-21, Nc BSA negative control). (D) Specificity analysis of nanobodies against inactivated SRMV. (E) Binding affinity analysis of the nanobodies against inactivated SRMV using ELISA