Production of Recombinant S1 Protein of Porcine Epidemic Diarrhea Virus in Recombinant CHO Cells for Application in Indirect ELISA
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The N-terminal S1 subunit of the spike protein (S1 protein) of porcine epidemic diarrhea virus (PEDV) is recognized as a potential diagnostic antigen for detecting anti-PEDV immunoglobulin A (IgA) levels in colostrum, a key indicator for assessing passive immunity against PEDV infection. Given the advantages of producing Fc-fusion proteins in mammalian cells, it is important to investigate how the addition of the Fc region affects the diagnostic performance of the PEDV-S1 protein. In this study, we successfully produced full-length PEDV-S1 protein fused with an Fc region (PEDV-S1-Fc protein) via transient gene expression in recombinant Chinese hamster ovary (rCHO) cells adapted to serum-free suspension culture. The resulting expression showed significantly higher volumetric productivity than previously reported. The aglycosylated form of PEDV-S1-Fc protein was also generated using PNGase F treatment of the purified protein, as well as tunicamycin treatment to inhibit glycosylation during cell culture. Notably, neither the addition of the Fc region nor the removal of N-glycans markedly affected the diagnostic function, as demonstrated by indirect ELISA using PEDV-positive and PEDV-negative colostrum samples. Moreover, the diagnostic performance of the PEDV-S1-Fc protein was further validated using total IgA purified from PEDV-positive colostrum. In summary, the full-length Fc-fused PEDV-S1 protein produced in rCHO cell culture exhibited high productivity and purity, making it a promising antigenic candidate for indirect ELISAbased detection of anti-PEDV IgA in colostrum.
Abstract
The N-terminal S1 subunit of the spike protein (S1 protein) of porcine epidemic diarrhea virus (PEDV) is recognized as a potential diagnostic antigen for detecting anti-PEDV immunoglobulin A (IgA) levels in colostrum, a key indicator for assessing passive immunity against PEDV infection. Given the advantages of producing Fc-fusion proteins in mammalian cells, it is important to investigate how the addition of the Fc region affects the diagnostic performance of the PEDV-S1 protein. In this study, we successfully produced full-length PEDV-S1 protein fused with an Fc region (PEDV-S1-Fc protein) via transient gene expression in recombinant Chinese hamster ovary (rCHO) cells adapted to serum-free suspension culture. The resulting expression showed significantly higher volumetric productivity than previously reported. The aglycosylated form of PEDV-S1-Fc protein was also generated using PNGase F treatment of the purified protein, as well as tunicamycin treatment to inhibit glycosylation during cell culture. Notably, neither the addition of the Fc region nor the removal of N-glycans markedly affected the diagnostic function, as demonstrated by indirect ELISA using PEDV-positive and PEDV-negative colostrum samples. Moreover, the diagnostic performance of the PEDV-S1-Fc protein was further validated using total IgA purified from PEDV-positive colostrum. In summary, the full-length Fc-fused PEDV-S1 protein produced in rCHO cell culture exhibited high productivity and purity, making it a promising antigenic candidate for indirect ELISAbased detection of anti-PEDV IgA in colostrum.