Preparation Of Virus-Like Particle For Dhav-1Detection

Duck Viral Hepatitis (DVH) caused by Duck Hepatitis virus (DHV) is a cute and fatal disease in young ducks which is often associated with liver necrosis, hemorrhage and high mortality. DHAV-1is prevalent in China and lead large economic loss for duckery. Because they are small, DHAV-1particles are hard to purify. Through using baculovirus expression system to form DHAV-1virus-like particle (VLP), we can obtain DHAV-1antigens easily. DHAV-1VLP can be used for DHAV-1detection and served as a potential vaccine and immunogen of VLPs-specific monoclonal antibody for rapid diagnosis and immune level measure in the future.The capsid assembly pathways of picornaviruses are similar. It reported that the cleavage of P1by3CD is required for capsid assembly. In our study, according to the genome sequence of DHAV-1JX strain, we further designed two pairs of primers to amplify PI and3CD genes. A BamH I site was inserted before the coding sequence of P1in the forward primer, and the reverse primer included a Sal I site; A Xho I site was inserted before the coding sequence of3CD in the forward primer, and the reverse primer included a Kpn I site. P1and3CD genes were amplified from the extracted RNA by RT-PCR. Then we cloned the two genes into one baculovirus (vBac-P1-3CD) and expressed P1and3CD proteins by single-infection. The formation and aggregation of VLP within the infected insect cells were visualized by ultrathin-sectioning and transmission electron microscope (TEM). These particles measured35-40nm in diameter and appeared icosahedral. The VLPs were purified by sucrose gradient ultracentrifugation, there were two particle forms:spherical particles40-60nm in diameter and spherical particles27nm in diameter. One of the structure protein VPO was detected by western blot. This study showed that we obtained DHAV-1virus-like particles successfully.Briefly, our study demonstrated that it was a feasible way to form DHAV-1virus-like particle through baculovirus expression system. DHAV-1virus-like particle showed specific reaction with PcAb to VPO. It proves that this antigen is useful for DHAV-1detection.