Preparation and identification of novel duck parvovirus virus-like particles

Abstract:

[Background] Beak atrophy and dwarfism syndrome (BADS) caused by novel duck parvovirus (NDPV) infection leads to stunted growth and atrophy of the upper and lower beaks of ducklings. The outbreak of BADS has caused huge economic losses to the duck industry in China. [Objective] To construct NDPV virus-like particles (VLPs) in Escherichia coli, laying the foundation for the development of NDPV-related vaccines. [Methods] The full-length NDPV VP2 sequence was codon-optimized, synthesized, and then ligated into the pColdTF vector to obtain the pColdTF-NDPV-VP2 recombinant plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21(DE3) for expression. SDS-PAGE was employed to analyze the solubility of the recombinant protein. The TF tag was removed by thrombin protease, and then recombinant protein was purified by Ni-NTA affinity chromatography. Western blotting was employed to examine the reactogenicity of purified VP2 protein and dynamic light scattering and transmission electron microscopy to observe the morphology of the recombinant protein and the formation of VLPs. [Results] The recombinant plasmid pColdTF-NDPV-VP2 was constructed successfully and expressed in E. coli mainly in the soluble form. The TF-VP2 fusion protein had a molecular weight of 115 kDa, and the molecular weight was 67 kDa after removal of the TF tag. Western blotting showed that the VP2 protein specifically bound to NDPV-positive duck serum. The VP2 VLPs with diameters of 20 nm to 25 nm could be observed by transmission electron microscopy. [Conclusion] We used the E. coli expression system to design NDPV VLPs, which can provide a basis for the development of subunit vaccines and biological-related products against BADS.

Key words:novel duck parvovirus;virus-like particles;Escherichia coli expression system