The Screening Of The Protein Interacting With The Cytoplasmic Tail Of CD163 And The Sequencing Analysis Of Transcriptome Of Cells Infected With PRRSV

Porcine reproductive and respiratory syndrome(PRRS),caused by PRRSV,is an infectious disease causing heavy economic losses to the swine industry worldwide.The virus genome is approximately 15.4 kb in length,containing ten open reading frames(ORFs).PRRSV is an RNA virus,and is a single strand of positive strand RNA,It's its characterized by non-segmental,polyadenylation,with capsule,and there is are very strict cell tropism.PRRSV-infected host cells rely on the binding of their ligands to the receptors on the cells,so the study of cell receptors facilitates elucidating the relationship between the virus and the host cell in order to further inform the mechanism by which the virus enters the cell and the effect of the virus on the host cell.The phenotype of the cells for PRRSV infection is decided by the presence or absence of the cellular receptors.CD163 is one of the important receptors.This receptor plays an important role in the release of the Virus genomeand the release of the virus.In addition,the receptor can also reproduce PRRSV in non-sensitive cell lines.CD163 is an extracellular protein,which comprises a signal peptide followed by nine SRCR domains,a single TM segment and a short cytoplasmic tail.The study found that the CD163 cytoplasmic tail may be joined with the host signaling mechanism to modulate PRRSV replication,but its regulatory mechanism is not clear,which requires us to further analyze the CD163 cytoplasmic tail involved in the mechanism of viral infection,which helps to reveal the mechanism of PRRSV infection,and has important guiding values for anti-PRRSV-infection drug development.In this study,a GST-CD163-tail recombinant vector was constructed and introduced into the prokaryotic expression system.The GST-CD163-tail protein was purified by Sepharose 4B beads.The purified protein was used as a bait protein to interact with Marc-145 at 0h,24 h and 48 h after PRRSV infection.The protein was identified by the interaction of PRRSV and CD163 cytoplasmic tail.The results of Pull down were screened by mass spectrometry to a protein that could respond toviral infection(protein elongation factor-1).In addition,small RNA and m RNA sequencing of PRRSV infection for 12h(M12)and non-infected(M0)Marc-145 were performed by high-throughput sequencing.Three replicates were set in each group.A total of 6 mi RNAs and m RNAs were analyzed in the experimental group M12 and the control group M0,and the differentially expressed mi RNAs and differentially expressed genes(m RNAs)were screened in both samples.The results showed that there were 12up-regulated and4 down-regulated mi RNAs,552down-regulated and 768up-regulated in the m RNA.Subsequently,the target genes were predicted by RNA hybrid,PITA and mi Randa.The predicted target genes and differentially expressed genes were subjected to GO analysis and KEGG pathway enrichment respectively.Finally,the corresponding target gene of mi RNA and the differentially expressed genes were analyzed by mi RNA and m RNA.GO gene analysis and KEGG pathway enrichment and regulatory network analysis were performed on the associated genes.These analyses were designed to screen out the key regulatory factors or candidate genes associated with the viral infection from differential mi RNAs and differentially expressed genes.Through the above analyses,this study speculated that novel mi RNAs of novel-70 and mi R-27a-5P may be closely related to PRRSV infection.The experimental group will further verify the specific functions of these two mi RNAs during PRRSV infection.