Eukaryotic expression of porcine deltacoronavirus S1 protein and preparation of polyclonal antibody

Abstract:

Background Porcine deltacoronavirus (PDCoV) is a porcine enteric coronavirus first reported in 2012, mainly causing gastrointestinal diseases characterized by diarrhea, vomiting, and dehydration in pigs. PDCoV infection is difficult to be distinguished from porcine epidemic diarrhea and porcine contagious gastroenteritis because of the similarity of their clinical symptoms. PDCoV has four structural proteins, and the spike (S) protein is located on the surface of PDCoV particles. The S protein consists of two subunits, S1 and S2, which are the main targets of virus-neutralizing antibodies.Objective To express the PDCoV S1 protein by the eukaryotic system and prepare polyclonal antibody against the purified PDCoV S1 protein.Methods The S1 gene of PDCoV CHN-JS-2018 strain was amplified by RT-PCR and ligated into the eukaryotic expression vector pCAGGS. Two eukaryotic expression plasmids named pCAGGS-PDCoV-S1-mFc and pCAGGS-PDCoV-S1-hFc were constructed and sequenced. Then the HEK293T cells were transfected with these two plasmids respectively, and the recombinant proteins were expressed, purified, and identified by indirect immunofluorescence assay (IFA) and Western blotting. The purified recombinant protein PDCoV-S1-mFc was used to immunize BALB/c mice, and polyclonal antibodies against it were prepared. The reactivity and titer of the polyclonal antisera were determined by IFA and indirect ELISA, respectively.Results The constructed eukaryotic expression plasmids pCAGGS-PDCoV-S1-mFc and pCAGGS-PDCoV-S1-hFc were expressed in HEK293T cells and the expressed proteins specifically bound to the positive serum of mice immunized with PDCoV. The titer of polyclonal antibodies against the recombinant PDCoV S1 protein reached more than 1:64 000, and the serum neutralization (SN) titer was 1:185, which indicated that the recombinant protein could be used in indirect IFA and Western blotting for detection of PDCoV.Conclusion The recombinant PDCoV S1 protein expressed by the eukaryotic expression system has strong immunogenicity and can induce mice to produce antibodies with a high titer. The prepared polyclonal antibody has good reactivity and neutralization activity. This study lays a foundation for the research on the diagnosis of PDCoV, preparation of monoclonal antibodies against PDCoV S1 protein, and development of subunit vaccines against PDCoV.