Study On E Protein Of Porcine Epidemic Diarrhea Virus Involved In Virus Replication

Porcine epidemic diarrhea(PED)is a highly contagious viral disease of swine.The main features are vomiting,anorexia,watery diarrhea,dehydration and weight loss.Pigs of all ages are susceptible to the disease,and the case fatality rate of newborn piglets can reach 100%,seriously harming the development of China's pig industry.The genome of Porcine epidemic diarrhea virus(PEDV)is about 28 kb,with the essential genes occurring in the order 5' UTR-ORP1a-ORF1b-S-ORF3-E-M-N-3'UTR.ORF1a and ORF1b will generate 16 non-structural proteins(nsp1?nsp16)after self-cutting after translation.At present,there are few studies on E proteins and non-structural proteins of PEDV,and their functions are not yet clear.This study will focus on the necessity of E protein in PEDV replication,and identify the expression time and intracellular location of several non-structural proteins(Nsp2?Nsp3-Ac?Nsp5)in PEDV-infected cells.The specific research is as follows:1.Analyze the expression time and intracellular localization of several non-structural proteins in infected Vero cells by IFA at 3hp.i,6hp.i,9hp.i,12hp.i,24hp.i.The results show that these non-structural proteins are expressed in the early stage of PEDV infection,especially Nsp2 can be detected with antibodies at 3hp.i.The specific fluorescence of the proteins is gathered in the form of fluorescent focus in the nucleus or dispersed in the cytoplasm,showing a discrete small structure similar to the endoplasmic reticulum or small vesicles.2.The E proteins with different tags were expressed by prokaryotic expression system and eukaryotic expression system respectively.The E protein expressed by insect cells could not be eluted from Ni-NTA Resin.Only the E protein expressed by the prokaryotic expression system was successfully purified.At the same time,the E polypeptide was designed and synthesized.New Zealand white rabbits were immunized with the E protein and E polypeptide to obtain corresponding polyclonal antibodies.However,IFA and Western Blot identification showed that antibodies can only recognize their immune antigens,but not the natural E protein of PEDV and the E protein overexpressed by cells.3.Construct a recombinant expression plasmid containing the full-length E gene named pWPI-E.Then co-transfect 293T cells with pWPI-E,lentiviral packaging plasmid(psPAX2)and lentiviral envelope plasmid(pMD2.G)to package the recombinant lentivirus.BHK-E cell line and Vero-E cell line stably expressing E protein were successfully prepared by infecting target cells with recombinant lentivirus and drug screening,and the expression of E protein in cell line was relatively quantified by Real-time PCR.4.In vitro homologous recombination method was used to reconstruct a full-length clone pSB2?-PEDV-?nsp2 of PEDV which deleting all the nsp2 gene,and a infectious clone pSB2?-PEDV-?E of PEDV which deleting most of the E gene sequence and containing point mutations in transcription control sequence and start codon.The pSB2?-PEDV-?E was used to rescue the recombinant virus rPEDV-?E,and the results showed that rPEDV-?E could be rescued and passaged only in BHK-E cells and Vero-E cells which stably expressing E protein,proving that E protein is indispensable in the replication of PEDV.In summary,this study shows that E protein is necessary for the replication of PEDV.At the same time,it was proved that Nsp2,Nsp3-Ac and Nsp5 proteins were expressed in the early stage of PEDV infection and had similar intracellular localization.This study laid the foundation for further exploration of the functions of E protein and Nsp protein.At the same time,recombinant viruses lacking the E protein can be developed into new and safe vaccines.Anti-Nsp2 antibody can be used as early diagnostic tool for PEDV infection,and it is of great significance for the diagnosis and prevention of G2 subtype PEDV.