Viral nervous necrosis (VNN) virus-like particle (VLP) 제작 및 그 응용

Abstract

Viral nervous necrosis virus (VNNV) is one of the most harmful viral diseases in aquaculture, usually inducing high mortalities at the beginning of seeding production of fry and larva. Thus the development of effective control measures is highly required. Several types of preventive vaccines against VNNV, such as inactivated vaccines, DNA vaccines and virus-like particles (VLP), have been reported. VLPs produced by recombinant molecular technology are equipped with structural and antigenic properties that are comparable to wild-type viral particles, which allows the induction of effective adaptive immune responses. The purpose of this study was to produce red-spotted grouper nervous necrosis virus (RGNNV) VLPs using an Escherichia coli expression system for application as a prophylactic vaccine and as a delivery vehicle for other heterologous antigens. The recombinant RGNNV capsid protein was expressed both in the cytoplasm and the inclusion body of E. coli, which was confirmed by western blot. A monomer form (37 kDa) and a trimeric form (120 kDa) of the capsid protein were shown in the western blot. Purified recombinant capsid protein from the cytoplasm was scanned by electron microscopy in order to check the formation of VLP particles, and demonstrated the presence of small sized particles (approximately 40 nm) that was corresponding to the size of RGNNV. To utilize this RGNNV VLPs as a vehicle for a foreign antigen, a partial fragment (68-384 bp) of viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene was inserted into the loop region of RGNNV capsid protein gene, and found the expression of the fused protein in the cytoplasm and the inclusion body of E. coli, which was confirmed by western blot using both G and VLP antibodies. The VLP expressing VHSV’s partial G protein had irregular shapes and sized approximatively 20-50 nm. To know the possibility of the VLP as a vehicle for DNA vaccine plasmids, the encapsulation of EGFP expressing plasmids in the VLP was conducted by simultaneous transformation of E. coli with both the VLP vector and the DNA vaccine vector. When E11 cells were infected with the VLPs, cells showed green-fluorescence, indicating the successful encapsulation of the DNA vaccine plasmids in the RGNNV VLPs. To increase the practical application of this VLP constructs, the RGNNV VLPs and the VLPs fused with VHSV’s partial G gene were expressed in previously generated auxotrophic Edwardsiella tarda mutant, which allows the development of combined vaccines.