Rescue and identification of a recombinant porcine epidemic diarrhea virus carrying a tagged peptide at the N terminus of S protein
[Background]Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus(PEDV)is a severe porcine infectious disease causing serious losses to the pig industry.The spike(S)protein,one of the structural proteins of PEDV,is responsible for viral infection and entry in host cells.The structure and function of S protein are a research hotspot in the molecular biology of PEDV.The S protein of PEDV has two functional domains:the N-terminal S1 domain and the C-terminal S2 domain.Deletion of the S1N domain(position:19-233 aa)did not impair the survival or propagation of the PEDV strain DR13att in vitro.[Objective]To know whether a tagged protein or peptide can substitute the S1N domain,we introduced the HA tag and soybean ascorbate peroxidase(APEX2)gene respectively into the S1N domain of DR13att to establish recombinant viruses by targeted RNA recombination.[Methods]The S1N domain(position:1-234 aa)of the transfer vector p-PEDV-DR13att was respectively substituted with HA gene and APEX2 gene and the transfer vectors were linearized.The RNA of linearized transfer vectors were electrotransferred to the LR7 cells infected with mPEDV,and the recombinant PEDV was rescued on Vero cells.The recombinant virus was validated by observation of cytopathic effect(CPE),RT-PCR,sequencing,indirect immunofluorescence assay(IFA),and Western blotting.Finally,the titer of each recombinant virus was measured and the growth curve of each recombinant virus was plotted to reveal the growth characteristics of the recombinant viruses and their parent virus.[Results]The constructed recombinant viruses were rescued and passaged.The recombinant virus rPEDV-DR13-S1N-HA(rPEDV-DSH)carrying the HA tag instead of the S1N domain showed CPE in P1.The recombinant virus rPEDV-DR13-S1N-APEX2(rPEDV-DSA)carrying APEX2 had not presented CPE until blind passage to P4.The RT-PCR,sequencing,IFA,and Western blotting of rPEDV-DSH and rPEDV-DSA in P4 confirmed that rPEDV-DSH with the HA tag was successfully rescued,while rPEDV-DSA was not rescued.The growth curves indicated that rPEDV-DSH had a similar growth trend but decreased proliferation level compared with the parent strain DR13att.[Conclusion]The recombinant virus rPEDV-DSH carrying a HA tag in the S1N domain(position:1-234 aa)was successfully rescued,which laid a foundation for further research on the interaction mechanism between the S protein and host cells.
万方数据
维普
