Evaluation of the Efficacy of a Recombinant Adenovirus Expressing the Spike Protein of Porcine Epidemic Diarrhea Virus in Pigs

Liu, Xinsheng; Zhao, Donghong; Zhou, Peng; Zhang, Yongguang; Wang, Yonglu

e S protein gene of the highly virulent Chinese genotype GIIa PEDV strain CH/HBXT/2018 was amplified and sequenced, and the length of the complete coding sequence was 4158 bp, corresponding to 1386 aa. e synthesized S protein gene containing restriction enzyme BioMed Research International sites and a His-tag was confirmed by sequencing (data not shown). e recombinant transfer bacmid pDC316-S-EGFP was confirmed by restriction enzyme digestion and sequencing (data not shown). e expression levels of the recombinant virus rAd-PEDV-S were assessed in vitro by real-time fluorescence quantitative RT-PCR, and the results showed that the PEDV S gene was well expressed at high levels ( Figure 1(a) ). Expression of PEDV S by the recombinant virus rAd-PEDV-S was further assessed by western blot assay, and the results revealed 153 kDa protein bands, consistent with the predicted size of the S protein, in HEK293A cells transfected with rAd-PEDV-S (Figure 1(b) ). No specific protein bands were observed in HEK293A cells infected with Ad-blank alone (Figure 1(b) ). e results of the fluorescence dilution assay also showed that the recombinant virus rAd-PEDV-S could be well replicated in HEK293A cells and that its titers could reach 10 11 PFU/mL (Figure 1(c) ).