Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell

Abstract

Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses' proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD) and varicella zoster glycoprotein E (VZV gE). Recombinant plasmids were generated, transfected into insect cells (SF9), and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.

Keywords: Baculovirus expression system; HSV; InsectDirect system; VZV.

Figure 1

This figure represents the details of the 3C/LIC cloning strategy, PCR product containing the 5LIC extension were treated with LIC-qualified T4 DNA polymerase in the presence of dATB, and then annealed to the 3C/LIC vector.

Figure 2

Expressions of HSV-1 gD and VZV gE in insect cells, cells were infected with recombinant plasmid. At 72 h p.i, transfected cells were analysed for synthesis and localisation of the viral glycoproteins within the cell and at the cell surface. Expressed glycoproteins gD and gE were detected in the cytoplasms of the acetone-fixed transfected cells (A and C) and on cells membrane of the unfixed cells (B and D). Neither antibody was reacted with the negative control (E and F).

Figure 3

(A) Time course of HSV-1 gD expression analysed by SDS–PAGE. Lane 1: molecular weight marker; Lane 2–5: 2–5 day p.i.

Figure 4

Fig. 3 dot blot analyses of HSV-1 gD (A) and VZV gE (B) recombinant protein, samples were transferred to dot blot manifold and vacuum-absorbed onto a 0.45 μ nitrocellulose membrane, incubated with the selected antibody and then incubated with peroxidase-labelled antibody. Lane 1 transfected cells; Lane 2 solubilised protein; Lane 3 purification flow; Lane 4 first wash; Lane 5 negative control (non-infected cells were subjected to the same procedure of the purification process); Lane 6 second wash; Lane 7 eluted recombinant protein.