[Expression and immunogenicity of porcine deltacoronavirus S-trimer protein]

This study aims to express the S-trimer protein of porcine deltacoronavirus (PDCoV) and study its immunogenicity. Targeting the extracellular domain (D20-N1 077) of PDCoV S protein, we introduced the signal peptide sequence (secretoglobin family 1D member 1, SCGB1D1) at the N-terminus and the T4 phage Foldon motif at the C-terminus, and cloned the recombinant sequence into pcDNA3.1(+) vector. The S-trimer fusion protein was expressed in the ExpiCHO expression system and purified, and the molecular weight of the S-trimer fusion protein was analyzed by non-denaturing polyacrylamide gel electrophoresis and molecular sieve gel filtration chromatography. The purified S-trimer fusion protein was used to immunize New Zealand white rabbits to prepare hyperimmune rabbit serum. The titer of specific IgG antibodies in rabbit hyperimmune serum against PDCoV S was measured by indirect ELISA, and the neutralizing antibody titer in the hyperimmune rabbit serum was measured by the serum microneutralization test. The application of hyperimmune rabbit serum was verified by Western blotting and immunofluorescence assay (IFA). The results showed that the PDCoV S-trimer fusion protein was successfully expressed and purified, and its molecular weight was about 660 kDa, which was consistent with the molecular weight of the trimer. Compared with previous studies, this study innovatively introduced the T4 phage Foldon motif into the S protein to stabilize the trimer structure. The S-trimer fusion protein ExpiCHO expressed in the eukaryotic expression system had complete glycosylation and a structure closer to that of the natural S protein. The prepared hyperimmune rabbit serum against rabbit PDCoV S-trimer fusion protein showed the specific antibody titer of 1:218 700 and the neutralizing antibody titer of 1:1 215.9, being able to be applied in laboratory detection of PDCoV by Western blotting and IFA. In summary, the PDCoV S-trimer protein expressed in this study has good immunogenicity, providing biomaterials for the subsequent study of PDCoV S protein structure, the screening of specific monoclonal antibodies, and the development of subunit vaccines.