The Relationships Of Porcine Immune-related Molecules & Inflammatory Reaction Induced By Staphylococcal Enterotoxins

Staphylococcal Enterotoxins(SEs) are soluble extracellular proteins excreted by staphylococcus bacterial strains, which are highly stable and highly heat-resistant and can cause a multitude of different diseases. SEs function as potent gastrointestinal toxins and superantigens that trigger inflammantion. However, the molecular details of mechanisms underlying such behavior remain unclear. In this study, the characters and functions of several immune surface molecules were revealed, and the impact of SEs on immunocyte were discussed.In this study, six immune-related porcine surface molecules CD28, CD86, CD25, IL17 A, MHC I and MHC II were amplified by PCR using total RNA isolated from porcine T/B/PBMC/PAM cells as the template. Then PCR products were cloned into pUC T simple and sequenced. Sequence comparison results showed that the complete CDs sequence of the six molecules shared more than 95% identity with that logged in GenBank/NCBI. Analysis of the six sequences by secondary structure prediction indicated that except for IL17 A, which was an extracelluar protein, all the other five molecules were with a single transmembrance domain. Additionally, there was an Ig domain structure in CD28, CD86, MHC I and MHC II respectively, and two CCP structural domains in CD25. Furthermore, the glycosylation site, antigenic epitope and phosphorylation site of these six molecules were predicted for further study of cellular signal transduction.The biochemical characteristics of the six immune-related proteins were analyzed by bioinformatics analysis, and the recombinant CD28, CD86, CD25, IL17 A, MHC I and MHC II were obtained by constructing prokaryotic expression vector. Six recombinant plasmids p ET28a-X were expressed in E.coli BL21(DE3) and purified recombinant proteins were obtained through denaturizing of inclusion bodies. The subcelluar localization and advanced structures of the recombinant proteins were predicted, and the interaction between SEs and swine MHC II molecule were analyzed by homology models to evaluate the impacts on the superantigen activities of staphylococcal enterotoxins.To evaluate the inflammation induced by enterotoxins superantigen, the transcription of chemokines and their receptors(CXCL2/CXCL8-CXCR2, MCP-1-CCR2, CCL25-CCR9, CCL28-CCR10 and CXCL10-CXCR3) engaged in cell-mediated immunity on porcine intestinal epithelial cells/PBMCs/PAM cells, and the six immune-related porcine surface molecules were detected by the relative fluorescence quantitative RT-PCR(qRT-PCR). It showed that SEs induced all chemokines and their receptors secretion after 48 h or 72 h of culture, indicating the recruitment of immune cells to the inflammatory sites after SEs stimulation. On the contrary, the effects of SEs on the intestinal tract, peripheral blood and alveolar were variable. The chemokines and their receptors expressed on PBMCs and PAM were significantly suppressed by SEO-HIS inoculation after 72 h of injection, while the transcripation of CD28, CD25, IL17 A and MHC II in intestinal tract were significantly increased after 4 hours of SEO-HIS stimulation, which might be related to body’s immune system in the regulation of diversity, multi-directional, and temporal and spatial variation.