Abstract：The recent studies discovered bovine leukocyte interleukin-32 gene (IL-32) is a new type of inflammatory cytokine and has many biological functions. It can induce the immune cells to product immune factors and chemical factors and play an important role in autoimmune/inflammatory diseases. And IL-32 can promote cell apoptosis, inhibite the growth of mycobacterium tuberculosis and other mycobacterium's infections, and improve the ability of resistance to disease of IL-32γ. In this study, we cloned the bovine IL-32β and IL-32γ genes, which were alternative splicing isoform of IL-32β, from Qinchuan cattle(Bos taurus) spleen. The result showed that the IL-32β gene was located on bovine chromosome 25th. There were five bases difference between the cloned bovine IL-32β and the published sequence in NCBI, but the differences did not affect the protein secondary structures, suggesting that these nucleotide differences were the SNPs of IL32 gene in Qinchuan cattle. The bovine IL-32γ had an additional sequence fragment from the second intron of IL-32 gene comparing with IL-32β, and that sequence encoded additional 47 amino acids. In order to study the function of IL-32γ, we constructed the eukaryotic expression vector of pIRES-IL32γ-GFP, which was stably transfected into mouse(Mus musculus) macrophages RAW264.7. After selection, the IL32γ overexpressing mouse macrophage cells were obtained. At the same time, the macrophages that were stably transfected with human IL-32γ gene were used as a positive control. Real-time PCR analysis showed that the bovine IL-32γ gene had similar effect as that of human IL-32γ to regulate the expression of cytokines, such as IL-1β, IL-6, MIP2 and TNFα. This study provided the crucial information for further study on the biological functions of bovine IL-32γ gene.
