Expression of equine herpesvirus type 1 glycoprotein gp14 in Escherichia coli and in insect cells: a comparative study on protein processing and humoral immune responses

Abstract

The extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gpl4, the homologue of gB of herpes simplex virus, was expressed in Escherichia coli and in insect cells using a recombinant baculovirus. Immunoblot analysis revealed that the recombinant E. coli expressed a fusion protein ofM r 135K which was composed of the truncated gpl4 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety. Both proteins were sequestered within the cells in form of inclusion bodies. Infection of insect cells with the recombinant baculovirus resulted in the production of a ll5K to ll8K glycoprotein which was cleaved intracellularly into two subunits ofM r 55K and 63K to 65K. The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of M r 120K to 122K. The recombinant proteins produced in E. coli and in insect cells elicited EHV-l-specific antibodies in goats as demonstrated by Western blot analysis. The gpl4 expressed in insect cells induced antibodies with virus-neutralizing activity. In contrast, the truncated gpl4 expressed by E. coli failed to elicit neutralizing antibodies. The results suggest that posttranslational modification of the EHV-1 gpl4 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.

Immunization with glycoprotein C of equine herpesvirus-1 is associated with accelerated virus clearance in a murine model

“…EHV-1 glycoprotein B (gB) expressed by a recombinant baculovirus was also able to induce neutralizing antibody [18]. In contrast EHV-1 glycoprotein H (gH) produced and tested in the same systems [16,26], evoked ELISA antibody and cell mediated immune responses, but did not induce neutralizing antibody or protect against EHV-1 infection.…”

Immunization with Attenuated Equine Herpesvirus 1 Strain KyA Induces Innate Immune Responses That Protect Mice from Lethal Challenge

“…Common features include replication in the respiratory mucosae, the development of pneumonitis, cell-associated viremia, and abortion (7,8). Various EHV-1 glycoproteins, including gB, gC, gD, and gH, were capable of inducing neutralizing antibodies (9)(10)(11)(12)(13)(14). EHV-1-specific CD8 ϩ class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) were identified in the peripheral blood mononuclear cells of infected horses, reaching maximal levels 2 to 3 weeks postinfection (15,16).…”

A single plasmid transfection that offers a significant advantage associated with puromycin selection in Drosophila Schneider S2 cells expressing heterologous proteins

“…It is believed that insect cells possess most of the protein processing capabilities that occur in mammalian cells. Therefore, it is likely that a recombinant protein expressed in insect cells may contain the normal activity (Osterrieder et al 1994). However, recombinant proteins expressed in S2 cells typically contain lower molecular masses than the native proteins (Altmann et al 1999).…”