Cloning And Expression Of Porcine Interleukin 22

Porcine interleukin 22 is a member of the interleukin-10 family of cytokines, mainly produced by activated T cells and NK cells, having anti-inflammatory activity and immunoregulatory function. At present, the related research of human and murine IL-22 has been reported, but there is no any report on the study of porcine IL-22. The main biological function of 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. To construct different expression systems of p IL-22 gene is significant for developing p IL-22 related drugs, treating intestinal disease in swine, improving the intestinal immune system and raising the quality of pork.According to homologous sequences of IL-22 gene in human, monkeys, cows, goats and house mouse which had published in the NCBI, primers of p IL-22 gene were designed and full-length sequence of porcine interleukin 22 protein was amplified by RT-PCR with the c DNA from lung. Firstly, RNA was extracted from different pig tissues and then reverse transcription to obtain c DNA. We researched the expression level of p IL-22 in different tissues by RT-q PCR and found that the relative expression quantity of p IL-22 in muscle, liver and lung is higher than other tissues. Secondly, bioinformatics was used to analyze the physicochemical properties and structural characteristics of p IL-22 and three-dimensional model was constructed by homology modeling. Thirdly, p IL-22 gene was inserted into prokaryotic expression vector p ET28a(+) and eukaryotic expression vector pc DNA?3.1 /myc-His(-) A via polymerase chain reaction technique and enzyme connection, respectively. The recombinant vector p ET28a-IL-22 was transformed into E.coil expression stain Rosetta-gami 2(DE3) p Lys S, IPTG was used to induce the expression of recombinant protein p IL-22 / His. SDS-PAGE showed a specific band of molecular mass about 22 KDa which was consistent with the expected size of 21.94 k Da of p IL-22. The inclusion body was dissolved by freezing thawing method and then the recombinant protein was purified by Ni affinity chromatography. What’s more, the endotoxin-free vector pc DNA3.1-IL-22 was transfected into piglet jejunum epithelium IPEC-J2 and the expression level of p IL-22 was detected by RT-q PCR. The results showed that the expression level of p IL-22 was about 12 times in transfected group than non-transfected, so p IL-22 was highly expressed in the IPEC-J2. Finally, the IPEC-J2 cells which had been successfully transfected plasmid pc DNA3.1-IL-22 were infected by Enterotoxigenic Escherichia coli 196 and the expression level of p BD-1 and p IL-22 were detected by RT-q PCR. Results showed upregulation of p IL-22 could lead to the expression levels of p IL-22 rise, that p IL-22 could stimulate epithelial cells secrete antimicrobial peptides with anti-infective biological activity.In summary, in this study we had successfully cloned porcine interleukin 22 gene and used prokaryotic expression system and eukaryotic expression system to produce recombinant protein p IL-22. It is meaningful for providing different means to study the mechanism of p IL-22 and providing theoretical basis for further study of the biological role of p IL-22.