Abstract
Wild type (wt) and cell culture adapted (ca) strains of the Coronavirus PEDV differ in their ability to cause diarrhea in neonate piglets: the wt strains are virulent; the ca strains are attenuated. Comparison of the available nucleotide sequences obtained from the different viral isolates revealed almost complete sequence identity with the exception of variations and truncations in open reading frame 3 (ORF3) observed exclusively in ca-PEDV isolates. In order to study the biological function(s) of the putative ORF3 product, the molecule was expressed as a heterodimeric fusion protein in E.coli. ORF3 was fused in frame to the alkaline phosphatase gene. Simultaneously, the construct was designed to form specific heterodimers by inclusion of the well known leucine zipper motiv of Jun and Fos. The heterodimerization partner contained the E.coli heat-labile enterotoxin subunit B (LTB) to allow specific binding to the eukaryotic cell receptor GM1. Our results indicate that heterodimeric fusion protein containing a truncated form of ORF3 was produced in high amounts, carried the expected ORF3 epitope, showed phosphatase activity, and was able to bind to the GM1 receptor. In contrast, a fusion protein containing the entire sequence of the ORF3 product was produced in minute amounts, indicating that it may have biological activity in prokaryotes, which led to the reduction of the amounts of proteins expressed.
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Keywords
- Porcine Epidemic Diarrhoea Virus
- Prokaryotic Expression
- Neonate Piglet
- Periplasmic Extract
- Alkaline Phosphatase Gene
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References
Aitken R., Brock J., Sinclair M.C., 1994, Receptor-binding subunits from cholera toxin and heat-labile enterotoxin as immunological carriers, in: Bacterial protein toxins, Zbl. Bakt. Suppl. 24, (A Freer et al., eds), Gustav Fischer, Stuttgart, pp 467–473.
Crameri R., Suter M., 1993, Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production, Gene 137: 69–75.
Duarte M., Tobler K., Bridgen A., Rasschaert D., Ackermann M. and Laude H., 1994, Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF, Virology 198: 466–476.
Holmgren J., Lönnroth I., Svenerholm, L., 1973, Tissue receptor for cholera exotoxin: postulated struture from studies with GM1 ganglioside and related glycolipids, Infect. Immun. 8: 208–14.
Tobler K. and Ackermann M., 1995, PEDV leader and junction sites. In: Corona- and Related Viruses (P.J. Talbot and G.A. Levy, eds.), Adv. Exp. Med. Biol. 380: 541–542.
Utiger A., Tobler K., Bridgen A., Suter M., Singh M., Ackermann M., 1995, Identification of proteins specified by porcine epidemic diarrhoea virus. In: Corona- and Related Viruses (P.J. Talbot and G.A. Levy, eds.), Adv. Exp. Med. Biol. 380: 287–290.
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© 1998 Springer Science+Business Media New York
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Schmitz, A., Tobler, K., Suter, M., Ackermann, M. (1998). Prokaryotic Expression of Porcine Epidemic Diarrhoea Virus ORF3. In: Enjuanes, L., Siddell, S.G., Spaan, W. (eds) Coronaviruses and Arteriviruses. Advances in Experimental Medicine and Biology, vol 440. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-5331-1_100
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DOI: https://doi.org/10.1007/978-1-4615-5331-1_100
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