Preliminary Study On The Mechanism Of Regulation Of Salmonella Carry Via Activation IQGAP2/Rho GTPase Pathway By MiR-124 And SipA

Salmonella is a Gram-negative bacterium that can cause enteritis,typhoid and other illnesses.The pathogenicity of Salmonella is mainly related to a specific region of the pathogenicity island,which is distributed in clusters on the chromosomes encoding pathogenicity related genes.This paper is divided into two parts to study the virulence functions of different virulence proteins in Salmonella.First,miR-124 is a significantly up-regulated miRNA in peripheral blood of piglets infected with Salmonella typhimurium,suggesting that miR-124 may play an important role in the pathogenesis of Salmonella typhimurium.In this study,transcriptomic analysis of peripheral blood mononuclear cells(PBMCs)of piglets treated with miR-124 sponge and Salmonella typhimurium was performed.In order to investigate the function of miR-124 in Salmonella infection,deep sequencing technology was used to analyze the transcriptome of peripheral blood mononuclear cells(PMBCs)of piglets treated with Salmonella typhimurium and miR-124 inhibition,and through a series of experimental methods to reveal the survival and proliferation mechanism of Salmonella typhimurium in infected cells.Secondly,SipA gene is an important bacterial pathogenic factor of pathogenic island I,which can affect signal transmission and cytoskeletal structure of host cells.This study aims to study and apply the target gene,and construct recombinant suicide plasmid p RE-SipA through genetic engineering technology.In the future,it can realize a variety of applications such as efficient expression of foreign genes,and research on gene function.1.Transcriptome analysis of peripheral blood mononuclear cells(PBMCs)of piglets treated with miR-124Transcriptome analysis of PBMCs isolated from peripheral blood of piglets treated with miR-124 inhibition and Salmonella typhimurium showed that 2,778 genes were differentially expressed in Salmonella typhimurium+miR-124 inhibition treatment and2,271 genes were differentially expressed in Salmonella typhimurium treatment compared with the control group.1,301 genes were differentially expressed(FDR<0.05,FC>2.0)by inhibition of Salmonella typhimurium+miR-124 compared with treatment of Salmonella typhimurium.Pathway analysis showed that MAPK signaling pathway,ribosome signaling pathway and T cell receptor signaling pathway were the most significantly enriched pathways in miR-124 inhibition+differentially expressed genes of Salmonella typhimurium and Salmonella typhimurium before and after treatment(FDR<0.05).Report analysis and electrophoretic mobility transfer analysis showed that miR-124 is a key regulator of the IQ motifs targeting Gtpase-activated protein 2(IQGAP2).In cell culture,miR-124 decreased the up-regulation of CDC42 and RAC1 mediated by Salmonella typhimurium(P<0.05).Compared with the control group,the number of Salmonella typhimurium in PBMCs cells treated with miR-124 and IQGAP2-si RNA increased,especially at 12 h after infection(P<0.05).Therefore,miR-124/IQGAP2/CDC42 may be the target pathway of Salmonella typhimurium infection in PBMCs of piglets.2.Construction of recombinant suicide plasmid p RE-SipARefer to Salmonella typhimurium ATCC 14028 strain(Gen Bank entry number:NZ＿CP043907.1)SipA gene sequence.The Oligo7 software was used to design specific primers for the upper and downstream fragments of SipA gene,and PCR amplified the upper and downstream nucleotide fragments of SipA gene,which were connected with p MD18-T vector respectively and transformed into E.coli DH5α receptor cells.The positive clones were identified by PCR amplification,and the band size was exactly consistent with the expected size.The upper and downstream fragments connected by p MD18-T vector were cloned into p RE112 suicide plasmid and transformed into E.coli SM10 λpir receptive cells to identify the recombinant suicide plasmid p RE-SipA by PCR amplification.In this study,miR-124/IQGAP2/Rho GTPase was found to be the target pathway of Salmonella typhimurium infection in PBMCs of piglets.The recombinant suicide plasmid p RE-SipA was successfully constructed,and the 1042 bp sequence of the upper and downstream fragments of SipA gene was successfully inserted into the p RE112 suicide plasmid by PCR amplification and gene sequencing analysis,and no base mutation occurred.