Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky’s disease in infected pigs

• Published in: Protein expression and purification 2013-07, Vol.90 (1), p.1-8
• Main Authors: Serena, María Soledad, Geisler, Christoph, Metz, Germán Ernesto, Corva, Santiago Gerardo, Mórtola, Eduardo Carlos, Larsen, Alejandra, Jarvis, Donald L., Echeverría, María Gabriela
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Viral - blood; Baculoviridae - genetics; Baculovirus; ELISA; Enzyme-Linked Immunosorbent Assay; GC content; Glycoprotein; Herpesvirus 1, Suid - isolation & purification; Insect cell; Male; Mice; Mice, Inbred BALB C; Pseudorabies - diagnosis; Pseudorabies - virology; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sensitivity and Specificity; Suid herpesvirus; Suid Herpesvirus 1; Swine; Viral Envelope Proteins - genetics; Viral Envelope Proteins - isolation & purification; Viral Envelope Proteins - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2013.04.008

•We produce a full-length recombinant gE protein of SHV-1.•We used betaine as a PCR enhancer to facilitate amplification of the entire gE gene.•The gE protein was expressed successfully at high levels.•The protein reacted strongly with sera from SHV-1 infected pigs in an ELISA assay.•The ELISA test has sensitivity and specificity comparable to currently available commercially tests. Suid Herpesvirus 1 (SHV-1) is the etiological agent of Aujeszky’s disease (AD), which affects swine herds worldwide and causes substantial economic losses due to animal mortality and lost productivity. In order to eradicate SHV-1, vaccination programs using viruses lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable and sensitive tests that can detect SHV-1 infection, yet distinguish between infected and vaccinated pigs. To meet this demand, we used the baculovirus-insect cell system to produce immunologically authentic full-length recombinant gE protein for use in a serum ELISA assay. As previous efforts to clone the gE gene had failed due to its extremely high GC-content (75% average), we used betaine as a PCR enhancer to facilitate amplification of the entire gE gene from the Argentinian CL15 strain of SHV-1. The cloned gE gene was expressed at high levels in recombinant baculovirus-infected insect cells and reacted strongly with sera from SHV-1 infected pigs. We used the recombinant gE protein to develop a local indirect ELISA test with sensitivity and specificity comparable to currently available commercial tests. Thus, recombinant gE produced in baculovirus-infected insect cells is a viable source of antigen for the detection of SHV-1 in ELISA tests. We also provide evidence supporting a potential application of this recombinant form of gE as a SHV-1 subunit vaccine.