Stable Expression and Purification of the Ectodomain of Porcine Epidemic Diarrhea Virus (PEDV) Spike (S) Protein

Since late 2010, new variants of porcine epidemic diarrhea virus (PEDV) have arisen in China, spread to Asia and North America in the end of 2013, and cause severe watery diarrhea in pigs of all ages and extremely high mortality rate in neonatal piglets resulting in significant economic losses. Recent studies indicate that the spike (S) gene of Taiwan new PEDV strains are closely related to global non-S INDEL PEDVs but significantly different from historic and traditional PEDV vaccine strains, that might explain the failure of cross protection of current available vaccines in pigs. The S protein is considered as a promising candidate for development of a subunit vaccine against PEDV. We present the development of a stable expression system using mammalian cells for production of full-length ectodomain of S protein from a non-S INDEL PEDV isolated from Taiwan.

To establish the S protein expressing stable cell line, sequence of S protein of a Taiwan non-S INDEL strain was cloned into pcDNA 3.1/V5-His-TOPO® vector. After transfecting the vector into HEK293 cell line for 48 hrs, the cells were selected by G418 for more than 2 weeks to generate a stable 293 cell line expressing PEDV S protein. The recombinant S protein was further purified by metal-binding affinity of histidine (TALON Superflow, GE healthcare), desalted by PD-10 desalting columns (GE healthcare), and concentrated by VivaspinTM (GE healthcare). The expression and the size of the expressed PEDV S protein were confirmed by the western blot and immunocytochemistry staining (ICC) using anti-V5 (invitrogen) and antiPEDV monoclonal antibodies.

A 293 cell line stably expressing the PEDV S protein has been successfully established. The expressing of the PEDV S protein was detected in more than 60% cells of the stable cell line for more than 20 passages by ICS using anti-V5 monoclonal antibody. By western blot assays, the size of the S protein, about 180kDa, was detected and was consistent with the predicted size of the protein. The expressed S protein was also further characterized and confirmed by anti-PEDV S monoclonal antibodies to prove the recombinant PEDV S protein exhibiting similar biological nature as the PEDV S protein.

The PEDV S protein is majorly responsible for viral entry via interactions with specific host cell receptors and for induction of neutralizing antibodies. The PEDV S protein from the stable 293 cell line may be a promising candidate for development of a subunit vaccine or diagnostic ELISA assays for global PEDVs.

Disclosure of Interest: None Declared.