Study On The Effect Of Porcine Epidemic Diarrhea Virus Nsp15 Protein On Cell Transcriptome And Its Function

Porcine epidemic diarrhea virus(PEDV)is a kind of positive stranded RNA virus with high pathogenic to piglets.It can cause vomiting,acute diarrhea and dehydration in pigs,and death in severe cases,which has caused huge economic loss to pig industry.PEDV is a member of the genus ? coronavirus of the coronavirus family,which only infects vertebrates,such as human,mouse,pig and bird.Its genome length is about 27-32 kb,which is the largest genome among the known RNA viruses.PEDV genome encodes 7 open reading frames(ORFs),among which ORF1 b encodes viral polyprotein pp1 b,which is lysed into non-structural protein nsp12-16.Nsp15 has viral endonuclease activity and is involved in viral replication and pathogenesis.However,it has not been reported to analyze the influence of PEDV nsp15 protein expression on the gene expression profile of host cells at the transcriptome level,and to explore the regulatory effect of nsp15 on cell genes.In this paper,the influence of PEDV nsp15 protein on cell transcriptome was explored,and new functions of nsp15 protein were found,so as to deepen our understanding of the pathogenesis of PEDV.Firstly,PEDV nsp15 gene was constructed into p MAL-c2 X and p CAGGS-HA vectors respectively by molecular cloning technology,and the prokaryotic expression vectors p MAL-c2X-nsp15 and p CAGGS-HA-nsp15 were obtained.Prokaryotic:p MAL-c2X-nsp15 was transformed into BL21(DE3),and the expression temperature,expression time and inducer concentration were explored to optimize the expression conditions of the nsp15 recombinant protein.After the obtained recombinant protein glue was recovered,mice were immunized for several times to prepare polyclonal antibody against nsp15,and the specificity of the antibody was identified by western blot.Eukaryotic aspects: the eukaryotic expression vector p CAGGS-HA-nsp15 was transfected into IPEC-J2 cells and Vero cells,respectively.The expression of nsp15 protein was confirmed by immunofluorescence and western blot experiments.After cell RNA was extracted,the transcriptome sequencing was performed for the eukaryotic associated transcriptome.Through the analysis of transcriptome data,a total of 35538 genes were detected by Vero cell sequencing,and 606 differential genes were screened out,including 372 up-regulated genes and 234 down-regulated genes.GO enrichment showed that the differentially expressed genes were significantly enriched in 33 GO classifications,and 28 genes involved in molecular functions,such as metal ion transmembrane transporter activity and potassium ion channel activity.A total of21654 genes were obtained by IPEC-J2 cell sequencing,among which 415 genes were differentially expressed,including 136 up-regulated genes and 279down-regulated genes.These differentially expressed genes were significantly enriched in 32 GO classifications,of which 25 were involved in biological processes.KEGG enrichment analysis showed that the differentially expressed genes were mainly concentrated in immune-related pathways such as influenza A,NOD-like receptor signaling pathway and TNF signaling pathway.In addition,we statistically analyzed the differential SNP and In Del sites in the transcriptome of IPEC-J2 cells and analyzed the differential sites occurring in the exon region.Chemokines regulate physiological processes such as inflammation and immune response,and transcriptome sequencing showed that nsp15 significantly down-regulated the expression of CCL5,CXCL8,and CXCL10 chemokines.PEDV-infected cells can induce a sharp increase in the expression of chemokines,in order to explore whether chemokines can regulate the replication and proliferation of the virus.In this study,overexpression vectors of CCL5,CXCL8 and CXCL10 chemokines were constructed,which were found to significantly inhibit the proliferation of the virus after overexpression in IPEC-J2 cells.In summary,the prokaryotic expression of nsp15 recombinant protein was obtained by optimizing the expression conditions,and nsp15 antiserum was prepared by immunizing mice,and the transcriptome files of nsp15 transfected Vero cells and IPEC-J2 cells were reported for the first time,transcriptome data showed that nsp15 could significantly down-regulate the expressions of CCL5,CXCL8 and CXCL10 chemokines,thus contributing to the intracellular replication and proliferation of the virus.These results have broadened our understanding of nsp15's promotion of viral replication and pathogenesis,and provided new ideas for the research and development of anti-PEDV specific drugs.