Transcriptome Study On The EBL Cells Stimulated By Mycoplasma Bovis-Derived LAMPs And The Effects Of PIM2 On IL-1? Secrection From EBL Cells

Mycoplasma bovis(M.bovis)infection can cause diseases including calf pneumonia,arthritis and mastitis,which has been an increasingly important factor that threatens the cattle industry.Study on pathogenicity and pathogenesis of M.bovis has aroused more and more concern since M.bovis infection was first reported in China,2008.However,the pathogenicity and pathogenesis of M.bovis have not been fully elucidated so far.In this study,EBL cells stimulated by M.bovis lipid-associated membrane proteins(LAMPs)were sequenced by RNA-seq,and the differentially expressed genes(DGEs)were analyzed.To systematically study the immune response of EBL cells to M.bovis LAMPs,the biological processes and signal pathways of DEGs were analyzed using bioinformatics technique.Based on IL-1? release regulation,MAPK and NF-?B signaling pathway,the PIM2 gene which significantly up-regulated and was associated with NF-?B signaling pathway was screened.Furthermore,the effect of PIM2 gene on the secretion of IL-1? from EBL cells was also studied.The PIM2 gene was colned,prokaryotic expressed,and the PIM2 polyclonal antibody was preparated.Our main results are as follows:1.RNA sequencing of M.bovis-derived LAMPs induced group and control group706 DEGs,364 up-regulated genes and 342 down-regulated genes,were screened after RNA-seq and data quality control(FC?1.5,p<0.05);Further analysis of biological processes and signaling pathways indicated that the function of DEGs coding proteins involves cell adhesion,apoptosis and cell growth,and 40 of DEGs were associated with NF-?B or MAPK signaling pathways,including 23 up-regulated genes and 17 down-regulated genes;Moreover,the expressions of the DEGs(PIM2,F2RL1 and PTPN22)were further verified by real-time PCR,and all these genes were up-regulated in LAMPs stimulated EBL cells,which was consistent with the RNA-seq identification.2.Cloning,prokaryotic expression and polyclonal antibody preparation of PIM2According to the above analysis,the PIM2 gene which was associated with the NF-?B signaling pathway was selected for the subsequent experiments.The PIM2 gene was successfully cloned and proved to be bovine PIM2 gene by sequencing.In addition,the sequence information was analyzed by DNAstar and the results showed a 936 bp length coding region which could encode a polypeptide of 311 amino acids.The theoretical molecular weight and isoelectric point were 34314.71 Da and 5.563,respectively.Neither transmembrane region nor signal peptide were dectected by the TMHMM and Signal IP4.1 software.The recombinant plasmid pET-28a-PIM2 was successfully constructed,the recombinant protein rp-PIM2 was obtained by optimizing the induction and purification conditions,and the PIM2 polyclonal antibody was successfully prepared.3.Subcellular localization of PIM2 gene and its effect on IL-1? secretion in EBL cellsBased on the full length of cloned PIM2 gene,the recombinant pEGFP-C1-PIM2 green fluorescent reporter plasmid was successfully constructed and its subcellular localization was observed by laser confocal microscopy to confirm transient expression in the cytoplasm.Furthermore,the recombinant eukaryotic expression plasmid pCMV-C-HA-PIM2 was successfully constructed.We preliminary confirmed a decrease of IL-1? expression in EBL cells after PIM2 overexpression.