Xie Jiasong, Belosevic Miodrag
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada; Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
Dev Comp Immunol. 2015 Jan;48(1):76-85. doi: 10.1016/j.dci.2014.09.006. Epub 2014 Sep 19.
We report on the functional characterization of RIP2 of the goldfish. Quantitative expression analysis of goldfish RIP2 revealed the greatest mRNA levels in the spleen, monocytes and splenocytes. We generated a recombinant form of the molecule (rgRIP2) and determined that anti-human RIP2 polyclonal antibody specifically recognized recombinant goldfish RIP2 (rgRIP2). Goldfish RIP2 activity was inhibited by the p38 MAPK pathway inhibitor SB203580. Treatment of goldfish macrophages with LPS, PGN, MDP, Poly I:C, heat-killed and live Mycobacterium marinum, and heat-killed Aeromonas salmonicida differentially changed the expression of RIP2 at both mRNA and protein levels. Co-immunoprecipitation assays indicated that RIP2 interacted with Nod1 and Nod2 receptors in eukaryotic cells. The results of dual luciferase reporter assay revealed that RIP2 over-expression caused the activation of the NF-κB signal pathway. In addition, RIP2 was involved in the regulation of the production of TNFα-2 and IL-1β1 in goldfish macrophages exposed to M. marinum.
我们报道了金鱼RIP2的功能特性。金鱼RIP2的定量表达分析显示，其在脾脏、单核细胞和脾细胞中的mRNA水平最高。我们制备了该分子的重组形式（rgRIP2），并确定抗人RIP2多克隆抗体能特异性识别重组金鱼RIP2（rgRIP2）。金鱼RIP2的活性受到p38丝裂原活化蛋白激酶（MAPK）途径抑制剂SB203580的抑制。用脂多糖（LPS）、肽聚糖（PGN）、胞壁酰二肽（MDP）、聚肌苷酸-聚胞苷酸（Poly I:C）、热灭活和活的海鱼分枝杆菌以及热灭活的杀鲑气单胞菌处理金鱼巨噬细胞，会在mRNA和蛋白质水平上不同程度地改变RIP2的表达。免疫共沉淀分析表明，RIP2在真核细胞中与Nod1和Nod2受体相互作用。双荧光素酶报告基因检测结果显示，RIP2的过表达导致核因子κB（NF-κB）信号通路的激活。此外，RIP2参与了暴露于海鱼分枝杆菌的金鱼巨噬细胞中肿瘤坏死因子α-2（TNFα-2）和白细胞介素-1β1（IL-1β1）产生的调节。
