Construction and identification of genotype Ⅱ grass carp reovirus virus-like particles

In order to construct genotype II grass carp reovirus (GCRV) virus-like particulars (VLPs) based vaccines, the immunogenic GCRV-VP3/VP4/VP35 protein was assembled into GCRV-VLPs using baculovirus expression system (BEVS). The structural protein VP35 encoded by the segment S11 of GCRV-II was inserted into baculovirus vector pFastBacHTA^TM using BEVS, and the identified recombinant vector was transformed into DH10Bac competent cells to screen recombinant plasmid Bacmid-VP35. Recombinant baculovirus pFHB-VP35 was obtained by transfecting recombinant plasmid Bacmid-VP35, recombinant baculovirus pFHB-VP3 and pFHB-VP4 was obtained by transfecting recombinant plasmid Bacmid-VP3 and Bacmid-VP4 which have been successfully constructed in our laboratory. Then the titers of recombinant baculovirus were determined by Bac-PAK rapid titer kit and the expression of proteins was identified by IFA and Western blot. The results showed that high titers of pFHB-VP35, pFHB-VP3, and pFHB-VP4 were obtained, and the corresponding proteins were successfully expressed in infected Sf9 cells. GCRV-VLPs were assembled co-infection of Sf9 cells with pFBH-VP35, pFBH-VP3, and pFBH-VP4, and the GCRV-VLPs were identified by electron microscopy (EM). The results showed that the three proteins could self-assemble in Sf9 cells and formed VLPs with diameter of 65-72 nm similar with natural viruses. This study lays a foundation for development of novel vaccines for preventing the disease caused by GCRV genotype II.