Differentiation of virulent and attenuated strains of novel duck reovirus by anti-p18 polyclonal antibodies

Abstract:

[Background] In our previous study, we passaged the virulent strain NP03 of novel duck reovirus (NDRV) on muscovy duck embryo fibroblast (MDEF) cells and obtained an attenuated strain NDRV S. Compared with that of the virulent strain, the p18 protein of NDRV S misses 35 residues at the C-terminus. [Objective] To prepare anti-p18 polyclonal antibodies and identify the differences in p18 between the virulent and attenuated strains of NDRV. [Methods] RT-PCR was employed to amplify the p18 gene sequences of the virulent and attenuated strains of NDRV. The obtained sequences were then cloned into the prokaryotic expression vector pET-28a for induced expression. The target proteins were purified by nickel columns and identified by SDS-PAGE and Western Blot. The polyclonal antibodies were prepared by immunizing the BALB/c mice with the purified proteins, and the antibodies were detected and identified by indirect ELISA, Western Blot, and indirect immunofluorescence assay (IFA). [Results] The SDS-PAGE and Western Blot results showed that the recombinant p18 proteins of the virulent and attenuated strains of NDRV were obtained. The indirect ELISA result showed that the serum titer of the polyclonal antibodies was 1:51 200. The result of Western Blot demonstrated that the prepared polyclonal antibodies could specifically detect the p18 proteins expressed by cells infected with virulent and attenuated strains. The molecular weights of the p18 proteins of virulent and attenuated strains varied, being 18 kDa and 14 kDa, respectively. The IFA results confirmed that the prepared polyclonal antibodies could recognize p18 proteins expressed in cells infected with virulent and attenuated strains. [Conclusion] We successfully prepared the anti-p18 polyclonal antibodies and identified the significant differences in its composition between virulent and attenuated strains of NDRV. The findings lay a solid foundation for investigating the biological function of p18 as well as the pathogenic mechanism of NDRV.

Key words:novel duck reovirus;p18 protein;polyclonal antibodies;virulence