Cloning And Expression Of Porcine MNSFβ And Analysis Of Its Spatial Expression Patterns

MNSFβ（monoclonal non-specific suppressor factor β）is a natural immunosuppressivefactor which has been reported to be involved in various biological processes includingimmune responses, cell division, stress response, cell apoptosis and nuclear transport. MNSFβplays an important role in the immune system. MNSFβ conjugates to endophilin II with alinkage between the C-terminal Gly74and Lys294in mouse liver and macrophages whichmay be implicated in phagocytosis of macrophages and inflammatory reaction mediated byDectin-1. MNSFβ inhibits the TNFα production in TLR-2mediated signal transduction，but itis not clear which protein interacts with it. MNSFβ covalently conjugates to pro-apoptoticprotein BCL-G and regulates the mitogen-activated protein kinase (MAPK) pathway byinhibiting the activation of extracellular signal-regulated kinase (ERK). However, we stilldon’t know the function of MNSFβ-BCL-G in the apoptotic signal pathway. To confirm thefunction of MNSFβ in swine and to lay the foundation for studying the role of MNSFβ in theapoptotic signal pathway, we performed experiments as following:1. In this study, the full-length sequence of porcine MNSFβ was predicted in silicon andthe cDNA was obtained through RT-PCR from porcine spleen for the first time. Softwareswere used to analyze its nucleic acid and protein sequences. Results showed that the fulllength of porcine MNSFβ was402bp encoding133amino acids with only one exon. PorcineMNSFβ consisted of an ubiquitin-like protein fused to the ribosomal protein S30. MNSFβwas highly conservative in different species and the homology of MNSFβ protein betweenswine and human was93.98%.2. The mRNA expression levels of porcine MNSFβ in different tissues (heart, liver,kidney, spleen, lung, thymus, tonsil, lymphonodi mandibulares, superficial inguinal lymphnodes, hilar adenopathy and nodi lymphatic mesenteric) were detected by qRT-PCR. Tissueexpression profile analysis showed that porcine MNSFβ was widely expressed in most ofimmune tissues, highest in liver but not in lung, suggesting that MNSFβ may play animportant role in immune response. 3. Porcine MNSFβ was subcloned into a eukaryotic expression vector pEGFP-C1toconstruct pEGFP-MNSFβ. The recombinant plasmid containing a green fluorescence reportergene was transfected into SUVECs (Swine Umbilical Vein Endothelial Cells) usinglipofectamine-2000. Fluorescence microscope showed that fusion protein EGFP-MNSFβ wasat the best expression level36hours post transfection. According to the fluorescence positionof the cells, we concluded that porcine MNSFβ was localized in both cytoplasm and nucleusby confocal scanning laser microscope. Western blot analysis was applied to confirm theexpression of porcine MNSFβ in vitro.4. MNSFβ was also subcloned into a prokaryotic expression vector pET-32a to constructrecombinant plasmid pET-MNSFβ. The fusion protein His-MNSFβ was expressed in E.coliBL21. Western blot analysis showed that we got the fusion protein of right size, so we purifiedthe fusion protein His-MNSFβ. It provided the materials for further research of diagnostic reagent ofporcine MNSFβ protein.