Cloning,Expression,Activity Identification And Preliminary Study On Intestinal Epithelial Function Of Porcine Interleukin-22

Interleukin-22(IL-22),a member of the IL-10 cytokine family,mainly produced by the lymphatic system cells of innate and adaptive immunity,including ?? T cells,y8 T cells,NKT cells and innate lymphoid cells(ILCs).IL-22 primarily targets nonhematopoietic epithelial and stromal cells,where it acts different effects on cell proliferation,tissue regeneration,cellular defense and inflammation.Especially,IL-22 in the gut is to stimulate intestinal epithelial cells to produce antimicrobial peptides,enhance the intestinal epithelial barrier of mucus and promote intestinal repair.Pigs are suffering from intestinal mucosal damage or enteritis due to intestinal pathogens,contaminated environment and diet,but there is no good alternative in addition to antibiotics treatment for enteritis.Therefore,it is very important to explore whether interleukin-22 can relieve enteritis and maintain intestinal mucosal barrier,but its mechanism is not clear.Therefore,our research mainly studies the prokaryotic expression of recombinant porcine interleukin-22 and whether the recombinant porcine interleukin-22 can relieve the apoptosis of intestinal epithelial cells and the infection of intestinal pathogens.1.In this study,the prokaryotic expression was used to obtain the recombinant porcine IL-22 protein.After recombination of porcine interleukin 22 gene into pET-32a(+)vector,the recombinant plasmid pET-32a(+)-pIL-22 was transformed into E.coli BL21(DE3).It was found that the recombinant protein existed in the form of inclusion body,which was denatured in urea,gradient dialysis and purified by a Ni-NTA column,refolded porcine IL-22 was obtained,the protein was approximate 35.6 kDa as expected and purity reached 95.7%through Quantity One software analysis.2.The IPEC-J2 cells of porcine jejunal epithelial cells were treated with recombinant porcine IL-22.The results showed that the recombinant porcine IL-22 had the biological activity of activating STAT3 signal pathway and it could protect the intestinal mucosal barrier,not cause inflammation of the intestinal epithelial cells.Apart from it,it could also alleviate the apoptosis induced by Deoxynivalenol(DON)and stimulate epithelial cells secrete pBD-1 to reduce the adhesion and apoptosis rate of enterotoxigenic E.coli ETEC K88.In conclusion,we successfully obtained recombinant porcine IL-22 by prokaryotic expression and proved that it can activate STAT3 signal pathway and play an important role in relieving intestinal epithelial cell apoptosis and resisting the infection of enterotoxigenic E.coli,which projected a new protective role for porcine IL-22 to repair the intestinal injury and treat the intestinal inflammation of pigs.It can also provide theoretical basis for further study of the biological role of porcine IL-22.