Cloning And Expression In E.coli And Insect Cells Of Chicken Interleukin-15 Gene

Interleukin-15 (IL-15) is a multifunctional cytokine that plays a major role in promoting cell growth and differentiation. IL-15 is produced mainly by natural immunocyte. In mammals, IL-15 shares many in vitro functions with IL-2. however, both cytokines bind to receptors with uniqe a chians enabling IL-15 and IL-2 to mediate very different functions in vivo. Recent results have shown that chicken cytokines share similar biological activity with mammalian cytokines, especially, in immunological enhancement of IL-2 and IFN- . Chicken IL-15(ChIL-15), Which was discovered in 2001, plays a main role in activating NK cells and CD8+ memory T cells, and may therefore have potential as a vaccine adjuvant.According to published ChIL-15 gene sequence, a pair of primers were designed and synthesized to clone ChIL-15 cDNA from ConA-activated chicken splenocytes by RT-PCR. Recombinant plasmid pUC19ChIL-15 carrying the ChIL-15 gene was constructed. The gene encoding mature ChIL-15 (mChIL-15) was amplified from pUC19ChIL-15 by PCR and cloned into pMD 18-T vector. After digested with Kpn I /Pst I , the mChIL-15 gene was subcloned into prokaryotic expression vector pPRoEX橦Ta and transformed into E.coli DH5a .The transformant was induced by IPTG, and the recombinant ChIL-15(rChIL-15) was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by ProBond?Nickel-Chelating Resin. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane to identify rChIL-15 by Western blot. The rChIL-15 was used to immune the guinea pig to obtain rChIL-15 polyclonal antibody. The mChIL-15 gene from recombinant plasmid pPRoChIL-15 was subcloned into transfer vector pMelBacB and a recombinant vector pMelChIL-15 was constructed. Co-transfection of Sf9 insect cells with pMelChIL-15 and baculovirus linear DNA was performed. After three times purification, a recombinant baculovirus designated as rBac-ChIL-15 was obtained. The infected cells were collected at different time post-infection and analyzed by SDS-PAGE and Western blot.The results showed that the open reading frame of ChIL-15 cDNA encompassed 564 base pairs(bp) and encoded a protein of 187 amino acids with three potential N-linked glycosylation sites, four conserved cysteine residues, two out-of-frame ATG initiation codons in the 5' untranslated region, and a signal peptide consisting of 66 amino acids. When it was compared with the published sequence of ChIL-15 cDNA, 7 mutant sites were found, and 5 amino acids were changed in predicted amino acids, which indicated that ChIL-15 may be polymorphic. The rChIL-15 was expressed in E.coli and was approximately 18kDa in size, which accounted for 30% of total protein. The purified rChIL-15 and rChIL-15 polyclonal antibody was obtained. Expression of rChIL-15 in rBac-ChIL-15 infected Sf9 cells was detected by SDS-PAGE and Western blot, the expressed product had a molecular weight of approximately 22kDa and the expression level of the recombinant protein was up to 25% of total cell protein .