Production and bioactivity of recombinant tilapia IL-1β
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To study the biological activity of interleukin-1β(IL-1β), a proinflammatory cytokine, in nile tilapia, Oreochromis niliticus, the recombinant tilapia IL-1βwas produced in E. coli cells based on pQE vector. Ni-NTA (nitriloacetic acid) metal affinity chromatography was used to purify recombinant protein. The eluted fractions exhibited a single band of protein with a molecular weight of about 25kDa, which is in close agreement with 25.4 kDa predicted by the cDNA sequence. The biological activity of the purified recombinant tilapia IL-1βwas tested through its effects on IL-1βgene expression, which are known as IL-1β inducible genes in mammals and fishes. IL-1βgene expression induced by poly I:C, a synthetic double stranded RNA, was also assessed in tilapia head kidney cells. IL-1βgene expression was analysed using QPCR (quantitative polymerase chain reaction). The ratio of the indicated gene expression was expressed as the relative mRNA level to β-actin mRNA level, which is constitutively expressed in macrophages. Consequently, head kidney cells incubated for three hours with rIL-1β(10, 2, 1 μg/ml) showed a dose dependent increase in IL-1βmRNA levels and 1 μg/ml of poly I:C was also able to induce IL-1βgene expression in head kidney in tilapia.
Abstract
To study the biological activity of interleukin-1β(IL-1β), a proinflammatory cytokine, in nile tilapia, Oreochromis niliticus, the recombinant tilapia IL-1βwas produced in E. coli cells based on pQE vector. Ni-NTA (nitriloacetic acid) metal affinity chromatography was used to purify recombinant protein. The eluted fractions exhibited a single band of protein with a molecular weight of about 25kDa, which is in close agreement with 25.4 kDa predicted by the cDNA sequence. The biological activity of the purified recombinant tilapia IL-1βwas tested through its effects on IL-1βgene expression, which are known as IL-1β inducible genes in mammals and fishes. IL-1βgene expression induced by poly I:C, a synthetic double stranded RNA, was also assessed in tilapia head kidney cells. IL-1βgene expression was analysed using QPCR (quantitative polymerase chain reaction). The ratio of the indicated gene expression was expressed as the relative mRNA level to β-actin mRNA level, which is constitutively expressed in macrophages. Consequently, head kidney cells incubated for three hours with rIL-1β(10, 2, 1 μg/ml) showed a dose dependent increase in IL-1βmRNA levels and 1 μg/ml of poly I:C was also able to induce IL-1βgene expression in head kidney in tilapia.
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