Cloning, Prokaryotic Expression Of Porcine Interleukin15 Gene (pIL-15) And The Bioactivity Study Of PIL-15

Interleukin-15(IL-15) is a kind of new interleukin found in1997 which has the similarbiological fuctions to Interleukin-2 (IL-2) but has no sequence homology. Its distribution isfar wider than that of IL-2 in vivo. IL-15 plays a great important role in mmune regulationand immune response, especially in the immune response to tumor. Porcine interleukin-15has similar biological fuctions with human interleukin-15.A pair of specific primers based on the reported sequence in GenBank (NM214390)were designed to clone the cDNA sequence encoding RongChang porcine interleukin-15from the PBMC stimulated with ConA. The cDNA was amplified, inserted into thePMD 18-T vector, sequenced and analyzed by bioinformatics, then proved to be IL-15 geneby enzyme digestion and Sequencing. The ORF was composed of 489 nucleotidesencoding 162 amino acids. The result showed that the gene and deduced amino acidshomology between the Rong Chang pIL-15 and other pIL-15 sequences published in theGenBank was both 99.4%. The gene sequence homology between Rong Chang pIL-15 andthe IL-15 of human, Macaca, Ovis aries, bovine, canine and feline range from 84.1 to89.8%, but only 69.6% and 27.8% campared with musculus and chicken. The homology ofdeduced amino acids between Rongchang porcine IL-15 and other mammalian IL-15 wasfairly high, but only 34.8% compared with that of chicken. Phylogenetic tree analysisindicated that porcine IL-15 had close evolutionay relationship with other mammalianIL-15, but had distant relationship with chicken IL-15. There was distinct resemblancebetween porcine IL-15 and porcine IL-2 by structural domain prediction analysis.The pIL-15 mature protein gene was inserted into pMD18-T vector then pMD18-T-pIL-15 and pET-32a(+) were both digested by EcoRⅠand XholⅠand the products werelinked. Recombinant expression plasmid pET-32-mpIL-15 was constructed by orientingthe pIL-15 gene into pET-32a(+), then the recombinant plasmid was transformed intoBL21(DE3). The recombinant expression strain was induced by IPTG, and then subjected to SDS-PAGE. The results showed that the pIL-15 fusing with pET-32a was expressed asinclusion body, and the product was about 34kD in size. The expression amount of fusionprotein was about 39.3% of the total thalline protein. The expression conditions were opt-imized and the optimal condition, IPTG 0.4mmol/L and induced 4h at 37℃, was obtained.The expressed inclusion body was purified and annealed, and its biological activitywas detected by MTT. The results indicated that the recombinant porcine IL-15 proteinhad a great capacity of stimulating the proliferation of porcine peripheral blood cells,which showed the recombinant porcine IL-15 protein had a high biological activity. Thisresearch laid a foundation for the futher study of porcine IL-15.