Study On The Role Of ACE2 In PEDV-infected IPEC-J2 Cells

Porcine epidemic diarrhea(PED)is a contact intestinal disease caused by Porcine epidemic diarrhea virus(PEDV),which mainly manifests clinically as watery diarrhea,dehydration and vomiting in pigs.The virus can cause disease in pigs of all ages,and is most harmful to newborn piglets,with mortality rate of affected piglets close to 100%.However,the mechanism of diarrhea caused by PEDV has not been elucidated,and the receptor of PEDV-infected piglets has not been clarified,and it was generally believed that porcine aminopeptidase(p APN)was the receptor of its infected host in past studies,but this conclusion was overturned in later studies.Current studies suggest the existence of multiple receptors for PEDV infection,but the true functional receptor has not been conclusively demonstrated.In this study,we first performed transcriptomic sequencing of the intestine of PEDV-infected piglets,and validated the transcriptome sequencing results,from which we found that PEDV infection can significantly reduce angiotensin-converting enzyme 2(ACE2)expression,followed by bioinformatics prediction analysis of the structure and properties of ACE2,and finally detected the effect on PEDV replication by regulating the expression of ACE2,it was determined that ACE2 can regulate PEDV infection,but there is no reciprocal relationship between ACE2 and PEDV S protein,and ACE2 is not a receptor for PEDV infection.The contents and results of this thesis are as follows:1.Preliminary mining and validation of PEDV infection affecting differentially expressed genes in the piglet intestine After piglets were instilled with PEDV-containing small intestinal tissue grinds and developed clinical signs of PED,jejunal tissue was immediately dissected and collected for transcriptomic sequencing,while small segments of intestine were collected for PCR to identify whether PEDV successfully infected the intestine.The genes that were significantly changed in the transcriptome sequencing results were screened,and four up-regulated genes and four down-regulated genes were selected among those functionally annotated as membrane-related,and the RNA of tissue samples was extracted to verify the changes of mRNA by RT-qPCR;then the PEDV-LJX strain was used to infect IPEC-J2 cells to construct a PEDV-infected cell model,and to identify at the cellular level by PCR We then used the PEDV-LJX strain to infect IPEC-J2 cells,constructed a PEDV-infected cell model,identified whether PEDV successfully infected the cells at the cellular level by PCR,and verified the accuracy of sequencing results by RT-qPCR.The results showed that the PEDVinfected piglet intestinal and cellular models were successfully constructed;the RT-qPCR results showed that six of the eight genes screened were consistent with the trend of transcriptome sequencing results,and two were opposite to the sequencing results,and the sequencing results had a certain degree of confidence,and the six genes with consistent transcriptome sequencing results were also verified at the cellular level.Combining the functions of the validated proteins and the research progress,we believe that the study of ACE2(angiotensin-converting enzyme 2)may have important research value,therefore,the follow-up study was carried out around ACE2.2.Bioinformatics analysis of ACE2The sequence of ACE2 protein was downloaded from the NCBI database and compared with human ACE2to analyze the similarity of ACE2 protein.The physicochemical properties,hydrophobicity,transmembrane structure,signal peptide,phosphorylation site and glycosylation site of ACE2 protein and the tertiary structure of ACE2 protein were analyzed by online software to predict the protein network that may have interactions with ACE2.The prediction analysis speculated that porcine ACE2 has high similarity with human ACE2 in terms of properties and structure,which may predict a role of ACE2 in the infestation process of porcine coronavirus.3.Study on the effect of ACE2 on PEDV replication(1)IPEC-J2 cells were infected with PEDV,and the changes of ACE2 mRNA at different time points were detected by RT-qPCR,and the changes of ACE2 total protein at different time points were detected by Western-blot.The results showed that ACE2 expression was down-regulated after PEDV infection,and the relative mRNA expression was significantly decreased at 24h and 36h(P<0.05,P<0.01),and the total protein expression was significantly decreased at 36h in the PEDV-infected group(P<0.01).(2)The expression of ACE2 was regulated by ACE2 overexpression plasmid pEGFP-ACE2 and interference plasmid pLVX-shRNA,and the changes of PEDV M mRNA at different time points were detected by RTqPCR,the changes of PEDV N total protein by Western-blot and immunofluorescence,and the changes of PEDV virus titer by TCID50.The results showed that the expression level of PEDV M mRNA increased significantly at 12h,24h,36h and 48h after pretreatment with overexpression plasmid pEGFP-ACE2(P<0.05,P<0.01),the expression of PEDV N protein increased significantly at 12h,24h,36h and 48h(P<0.05),and the titer of PEDV virus increased significantly at 12h,48h significantly increased(P<0.05,P<0.01,);PEDV M mRNA expression level significantly decreased at 12h,24h,36h and 48h after pretreatment with interfering plasmid pLVX-shRNA2(P<0.01),PEDV N protein expression significantly increased at 12h,24h,36h and 48h(P<0.05,P<0.01,),and PEDV viral titers significantly decreased at 24h and 48h(P<0.01).The above results indicated that regulation of ACE2 upregulation could promote PEDV replication,and inhibition of ACE2 expression could reduce PEDV replication.the Co-IP results showed that there was no reciprocal relationship between PEDV N protein and ACE2.4.Preliminary investigation of the interaction between ACE2 and PEDV S proteins The eukaryotic expression plasmids pTT5-PEDV S1 and p PCDNA3.1-ACE2 were cotransfected into HEK293T cells,and whether there was an interaction between ACE2 and PEDV S proteins was examined by CoIP.The results showed that there was no direct interaction between ACE2 and PEDV S protein,and ACE2regulated the infection and replication of PEDV probably through other pathways rather than direct interaction.