Construction of a highly efficient display system for baculovirus and its application on multigene co-display

Abstract

The classical baculovirus display system (BDS) has often recruited fields including gene delivery, gene therapy, and the genetic engineering of vaccines, as it is capable of presenting foreign polypeptides on the membranes of recombinant baculovirus through a transmembrane protein. However, classical BDS's high cost, complicated operation, low display efficiency and its inability to simultaneously display multiple gene products impede its practicality. In this study, we present a novel and highly efficient display system based on ires-dependent gp64 for rescuing gp64-null Bacmid of baculovirus construction without affecting the viral replication cycle, which we name the baculovirus multigene display system (BMDS). Laser scanning confocal microscopy demonstrated that eGFP, eYFP, and mCherry were translocated on the membrane of Spodoptera frugiperda 9 cell successfully as expected. Western blot analysis further confirmed the presence of the fluorescent proteins on the budded, mature viral particles. The results showed the display efficiency of target gene on cell surface is fourfold that of classical BDS. In addition, a recombinant baculovirus displaying three kinds of fluorescent proteins simultaneously was constructed, thereby demonstrating the effectiveness of BMDS as a co-display system.

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles

“…The traditional baculovirus expression system often relies on the transfection of recombinant baculovirus DNA isolated from bacteria [31, 32]. In contrast, the Ac-MultiBac system used in this study does not need transfection but employs DAP-deficient bacteria that infect insect cells to produce recombinant baculovirus () [13, 20, 23–25, 33]. When supernatants of infected Sf9 cells were centrifuged at 80 000 g and the pellets were resuspended in PBS (pH 7.4), observations with TEM showed a complete baculovirus structure, indicating successful production of recombinant baculovirus ().…”

“…In our previous studies, it has been described that the recombinant baculovirus produced by the Ac-MultiBac system has the highest infection efficiency at 96 hpi [24, 25]. Strong red fluorescence was indeed observed at 96 hpi (), and western blotting was used to check protein expression in cells and supernatants.…”

“…Recombinant viruses were generated as previously described [20,24,25]. Sw106 cells with recombinant Ac-MultiBac were cultured to an OD 600 (absorbance at 600 nm) of 1.0 and collected by centrifugation.…”

“…Sf9 cells infected with recombinant baculoviruses were harvested by centrifugation at 400 g for 10 min after 96 hpi (hours post-infection) [24,25]. After three washes with PBS, cells were resuspended in PBS (pH 7.4, Gibco) and sonicated (10 times, 30 W) [26].…”

Baculovirus Surface Display of Zika Virus Envelope Protein Protects against Virus Challenge in Mouse Model

“…This system exhibits several advantages: (1) it has a good biosafety profile in mice, (2) it can be readily manipulated for insertion of exogenous DNA fragments, (3) it has the capacity for rapid development and potential for low cost production, (4) high titres ([ 10 9 PFU/mL) of virus can be easily reached in Sf9 cells, and (5) the recombinant protein expressed can be readily purified (Xu et al 2011;Yamaji 2014;Premanand et al 2018;Gupta et al 2019). Due to its characteristic advantages of security, economy and convenience, the baculovirus expression system is regarded as the most versatile surface display system in eukaryotes (Zheng et al 2018). To date, it has been commonly exploited to display proteins or peptides such as HA of influenza virus, VP1 of EV71, N and S proteins of SARS-like coronavirus (Bai et al 2008;Baxter et al 2011;Premanand et al 2018).…”