Preliminary Analysis Of Goats Epithelial Cells Infected By Pasteurella Multocida Infection In Host Cells And The Expression Of RecN Gene

The common disease Pasteurella multocida（Pm）with charactertics of short incubation period,fast propagation speed,high fatality rate,and fulminant epidemic causes considerable amount loss to stockbreeding.Although 135 years have passed since Pm was first known to us,there is still no clear understanding of its DNA repair and recombination function and bacterial adhesion process.In order to further understand how Pm adhesion on the host cell surface leads to host disease,a sheep bronchial epithelial cell model of infection with P.multocida HN01 strain was constructed and high-throughput sequencing technology was used to screen out the differentially expressed genes（DEGs）.This study first determine the multiplicity of infection MOI=100,infection time select 4 h and 6 h method,established the P.multocida HN01 strain model of the epithelial cells of the infected sheep.The DEGs were screened by GO and KEGG analysis,and the pathway of DEGs was investigated and validated from the direction of bacterial adhesion.With respect to m RNA sequencing results,the control group got 43938063 effective reads while the treatment group got 35452136 and 44565947 effective reads at 4 h and 6 h,respectively.The DEGs was selected by comparison:29 genes were up-regulated and 88genes were down-regulated in D＿T 4h vs Ctrl group.In the D＿T 6 h vs control group,238genes were up-regulated and 261 genes were down-regulated.With the help of GO and KEGG enrichment analysis,a total of 5186 genes were enriched into 310 pathways of KEGG metabolism.Through STEM analysis,231 genes were found to show slow and then rapid decline in expression,and 157 genes showed slow and then rapid growth.Three DEGs related to bacterial adhesion,ITGA3,TMSB10 and RHOC,were selected and tested with the same q RT-PCR results.In terms of mi RNA sequencing results,DEGs was selected by comparison:160 genes were up-regulated and 130 genes were down-regulated in D＿T 4h vs Control group by comparison.In D＿T 6h vs Ctrl group,213 genes were up-regulated and 125 genes were down-regulated.The correct DEGs verified by q RT-PCR were selected:ITGA3,TMSB10and RHOC were further analyzed,and the q RT-PCR verification was carried out by taking9 mi RNA intersected in the target gene prediction and STEM analysis.The results showed that in addition to PC-3p-19778＿398 and oan-mi R-1386＿L+1＿1 other chi-mi R-128-3p,PC-3p-4951＿2010,chi-mi R-103-5p＿R-7,PC-3p-9125＿1006,bta-mi R-2285i＿R+1＿1SS6C T,chi-mi R-106b-5p and bta-mi R-138 were the same as expected.In order to explore the function of rec N（genes required for gene recombination and regulation of cell response to DNA damage）genes,this study constructedp ET-28a（+）-rec N recombinant plasmid and carried out corresponding biogenic analysis on rec N by transforming it into E.coli BL21（DE3）competent cells.The results showed that 66.94ku rec N protein was induced in this experiment.After analysis,the molecular formula of rec N protein is C₇₈₆₄H₁₃₀₀₉N₂₄₆₃O₂₉₆₅S₃₇₇,the extinction number is 47410,and the theoretical isoelectric point（PI）is 5.61 and acidic.Its total average hydrophilicity（GRAVY）is 0.571,which is the same as the inclusion body protein expressed in the test.Its instability coefficient is 42.66,which is an unstable protein.The secondary structure prediction was mainly alpha helix（Hh,64.87%）and irregular curl（Cc,21.00%）.By hydrophobicity analysis,the protein was predicted to have 3 regions with high hydrophobicity and 9regions with high hydrophilicity.This experiment provides a theoretical basis for exploring the function of rec N gene of Pm.To sum up,this paper provides a new idea for the study of rec N gene of Pm at the protein level for the preparation of Pm vaccine and interspecific identification of Pm.Studies at transcriptome level have shown that the bacterial adhesion mechanism of Pm is regulated simultaneously by multiple genes and mi RNAs.The genes significantly enriched in the regulation of actin cytoskeleton and the up-regulation and down-regulation of adhesion spots may provide the material basis for bacterial invasion of cells,while the genes significantly enriched in the regulation of actin cytoskeleton may enhance and promote the adhesion process between bacteria and matrix.At the same time,these differential genes will be regulated by the corresponding mi RNAs,and through the complex interaction between the two,eventually lead to the occurrence of diseases.