Cloning And Expression Of Bovine And Goat Interleukin-18 Gene

Interleukin-18 (IL-18), a novel discovered cytokine is considered to be an usful adjuvant and therapeutics in control of diseases in intensive herd industries. Such studies of IL-18 and its receptors are useful for the progress of immunology. We isolated and sequenced a 480bp cDNA encoding mature goat interleukin-18 (gIL-18) from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The gIL-18 gene was cloned into pMD18-T vectors and sequenced. Nucleotide sequence of gIL-18 shares high homology with cattle and sheep`s. Conserved amino acid sequence at the enzymed site of caspase-1 and two key amino acid about its bioactivity are also observed. The gIL-18 gene from the recombinant plasmid was sub-cloned into pET32a(+) vector. The recombinant plasmid pET-gIL-18 in E.Coli BL21(DE3) was introduced by 1mmol/L IPTG for 4h or more time for expression. The fusion protein of pET and gIL-18 was expressed. SDS-PAGE analysis suggests that the recombinant protein is about 38ku. The recombinant protein can induce IFN-γproduction in MDBK cells after denature,renature and purified. Western-blotting indicated recombinant gIL-18 showed well reaction to its specific multi-antibodies. IL-18 mRNA from cells and tissues of goat are detected by RT-PCR,in whichβ-actinis is as a inner-control. The IL-18 mRNA was constitutively detected in goat alveolar macrophages with or without LPS, While, enhanced expression was detected in splenocytes and liver cells if treated by LPS, and can be weakly detected in PBMC treated by activator. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues. Some virus genome of vaccinia subgenus in poxvirus encoded IL-18BP gene, which can block IL-18 activity but no sequence similarity to membrane IL-18 receptors. Virus genome in sheep pox subgenus also be induced having such homologous gene. A pair of primers spanned the ORF of goat IL-18BP gene stemming from sheep poxvirus genome were designed. We cloned this gene and expressed it in E.Coli with pET32a(+) plasmid. After being purified,the recombinant fusion protein can inhibit goat IL-18 induce IFN-γproduction in MDBK. We amplified bovine mature IL-18 gene from the spleen cells in a milch cow by RT-PCR...