Cloning,Expression and Phylogenetic Analysis of Interleukin-18(IL-18) Gene in Xiaoshan Chickens

The ChIL-18 mRNA was isolated from chicken splenocytes 10h after stimulated with LPS.ChIL-18 gene was amplified from chicken primordial spleen cells induced by lipopolysaccharide(LPS) with RT-PCR(Genbank accession,AY628648).The RT-PCR products were inserted into prokaryotic(pGEX-4T-2) expression vectors.One 591 bp cDNA fragment,containing complete open reading frame(ORF) of ChIL-18 gene,was obtained in RT-PCR.The deduced ChIL-18 amino acid(aa) sequence of Xiaoshan chicken is the same as that from HD-11 cell except 2aa difference.Phylogenetic analysis showed that the avian IL-18s together,apart from the mammalian sequences,separated with a long branch between them.Recombinant ChIL-18 protein was obtained in prokaryotic expression system,and its polyclonal antibody was performed in rabbits.We obtained ChIL-18 gene from the activated splenocytes of Xiaoshan chicken.The recombinant ChIL-18 protein expressed in E.coli strain BL21 has immunoreactivity and immunogenicity.These results gave a basis for further research of the bioactivity of ChIL-18.To further understand the evolution of chicken interleukin-18(IL-18),and also with the eventual aim of using cytokine to influence the immune response to pathogens in chicken.