Production Of EV71VLPs Using The Multibac Baculovirus Expression System

The Enterovirus71(EV71) causes hand, foot and mouth disease (HFMD) in infants and children. Currently, EV71has become the cause of major and regular epidemics across the Asia-Pacific region. Since no effective antiviral agents are available, developing vaccines for primary prevention is considered to be the best choice among control strategies against EV71.EV71is a non-enveloped, single-stranded RNA virus within the Picornaviridae family, and its genome is composed of a single open reading frame that contains P1, P2and P3regions. P2and P3regions encode nonstructural proteins (e.g.3CD) responsible for virus replication and virulence while P1region encodes P1precursor that can be cleaved by3CD protease into VPO, VP1and VP3.The virus-like particles (VLPs) are empty particles comprised of viral capsid proteins yet devoid of viral nucleic acids, thus mitigating the potential side effects. Previous studies have demonstrated that EV71VLPs induced Thl and Th2immune responses in mice. The EV71VLPs is generally producted by a recombinant baculovirus (Bac-P1-3CD) co-expressing PI and3CD proteins. Infection of insect cells with a Bac-P1-3CD leads to the cleavage of P1by3CD protease into individual proteins (VP1, VP3and VPO) and self-assembly of VLPs within the cells. However, the cleavage efficiency of the3CD proteins is low and the PI is incompletely cleaved into mature proteins.In this study, based on the baculovirus Multibac system, four strategies were developed to improve EV71VLPs production and the efficiency of VLPs production was compared. In the first strategy, coding regions of the EV71viral structure proteins (VP1, VP3and VPO proteins) were recombinanted into the baculovirus genome, and then the Sf9cells were infected with the recombinant baculovirus to produce the VLPs.In the second strategy, recombinant baculoviruses containing different copies of P1and3CD genes were constructed. The level of the mature protein expression and the EV71VLPs production were tested by Western blotting. The highest production of EV71VLPs was obtained from recombinant baculovirus containing two copies of P1and one copy of3CD gene.In EV71maturation, VPO is further processed into VP2and VP4, which is critical for the infectivity of the EV71. However, the function of VP2and VP4during vrial package is still unknown. In the third strategy, four structural proteins VP4, VP2, VP3and VP1were co-expressed. Self-assembly of EV71VLPs in the cells was analysed and the VP2protein was detected in the VLPs.The fourth strategy was to co-express five individual structural proteins (VPO? VP4?VP2?VP3and VP1) of EV71in recombinant baculovirus. Data showed that the VP2or VPO protein was not simultaneously packed into the VLPs.To optimize the foreign protein expression efficiency of the Multibac system, the recombinant baculoviruses containing1to3copy luciferase genes were constructed and the expression level of luciferase was tested using the Dual Luciferase reports system. The results showed that the expression level of foreign protein was improved with optimized copies of the foreign genes.In addition, the interaction protein of the baculovirus killing gene P13encoded product was identified from phage library screening. Using pull down assay, P13protein is further confirmed to interact with GBPBP protein in the silkworm (Bombyx mori).