Yan Y, Wang Q, Niu L L, Deng J B, Yu J Q, Zhang J X Wang Y Z, Yin M M, Tan X M
Key Laboratory for Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China.
Chengdu Zoo, Chengdu, China.
Genet Mol Res. 2014 Nov 19;13(4):9687-700. doi: 10.4238/2014.November19.1.
Interleukin 18 (IL-18), as a member of IL-1 superfamily, is an important pleiotropic cytokine that modulates Th1 immune responses. In this report, we cloned and identified a homolog of IL-18 in giant panda (Ailuropoda melanoleuca) (designated as AmIL-18) from peripheral blood mononuclear cells stimulated with lipopolysaccharide. The open readin g frame of AmIL-18 cDNA is 579 bp encoding a deduced protein of 192 amino acids. AmIL-18 gDNA fragments contained 5 exons and 4 introns. The amino acid sequence of AmIL-18 shared 23.9 to 87.0% identity with other species. To evaluate the effects of AmIL-18 on the immune response, we expressed the recombinant AmIL-18 in Escherichia coli BL21 (DE3). The fusion protein PET-AmIL-18 was purified by nickel affinity column chromatography and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The biological function of purified PET-AmIL-18 was determined on mouse splenocytes by quantitative real-time polymerase chain reaction. INF-γ and other cytokines were increased when stimulated by PET-AmIL-18, particularly when combined with recombinant human interleukin 12, while a Th2-type cytokine, interleukin-4, was strikingly suppressed. These results will provide information for the potential use of recombinant proteins to manipulate the immune response in giant pandas and facilitate the study to protect this treasured species.
白细胞介素18（IL-18）作为IL-1超家族的成员，是一种重要的多效性细胞因子，可调节Th1免疫反应。在本报告中，我们从经脂多糖刺激的外周血单核细胞中克隆并鉴定了大熊猫（Ailuropoda melanoleuca）中IL-18的一个同源物（命名为AmIL-18）。AmIL-18 cDNA的开放阅读框为579 bp，编码一个由192个氨基酸组成的推导蛋白。AmIL-18基因组DNA片段包含5个外显子和4个内含子。AmIL-18的氨基酸序列与其他物种的同一性为23.9%至87.0%。为了评估AmIL-18对免疫反应的影响，我们在大肠杆菌BL21（DE3）中表达了重组AmIL-18。融合蛋白PET-AmIL-18通过镍亲和柱层析纯化，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质免疫印迹分析进行验证。通过定量实时聚合酶链反应在小鼠脾细胞上测定纯化的PET-AmIL-18的生物学功能。当受到PET-AmIL-18刺激时，尤其是与重组人白细胞介素12联合使用时，INF-γ和其他细胞因子增加，而Th2型细胞因子白细胞介素-4则受到显著抑制。这些结果将为重组蛋白在调控大熊猫免疫反应中的潜在应用提供信息，并有助于开展保护这一珍贵物种的研究。
