Lü Aijun, Hu Xiucai, Li Li, Pei Chao, Zhang Chao, Cao Xianglin, Kong Xianghui, Nie Guoxing, Li Xuejun, Sun Jingfeng
College of Fisheries, Henan Normal University, Xinxiang, 453007, China,
Fish Physiol Biochem. 2015 Aug;41(4):899-907. doi: 10.1007/s10695-015-0056-7. Epub 2015 Apr 17.
Voltage-dependent anion channel (VDAC) is located in the mitochondrial outer membrane, which plays a crucial role in regulating cell life and death. In this study, the tissue distribution of olive flounder Paralichthys olivaceus VDAC2 (PoVDAC2) was detected by quantitative real-time PCR and Western blot analysis. The qRT-PCR results showed that the expression level of PoVDAC2 was abundant in heart, muscle and gill tissues. Western blot analysis revealed a protein of 32 kDa detected in all six tissues. Furthermore, a recombinant eukaryotic expression plasmid pEGFP-N3-PoVDAC2 was successfully constructed and transiently expressed the fusion protein in fish cell lines. Subcellular localization indicated that PoVDAC2-GFP was distributed in a punctate mitochondria-like pattern throughout the cytoplasm in flounder embryonic cells (FEC). The distribution of native VDAC2 in untransfected fish cells was also investigated by immunofluorescence microscopy. The punctate VDAC2 fluorescence signals of both FEC and EPC cells were identically observed in the cytoplasm but not in the nucleus. These results laid a foundation for investigating the functional relevance of VDAC response to pathogens in flounder.
电压依赖性阴离子通道（VDAC）位于线粒体外膜，在调节细胞生死中起关键作用。在本研究中，通过定量实时PCR和蛋白质免疫印迹分析检测了牙鲆（Paralichthys olivaceus）VDAC2（PoVDAC2）的组织分布。定量实时PCR结果显示，PoVDAC2在心脏、肌肉和鳃组织中表达水平丰富。蛋白质免疫印迹分析显示在所有六种组织中均检测到一条32 kDa的蛋白条带。此外，成功构建了重组真核表达质粒pEGFP-N3-PoVDAC2，并在鱼类细胞系中瞬时表达了融合蛋白。亚细胞定位表明，PoVDAC2-GFP在牙鲆胚胎细胞（FEC）的整个细胞质中呈点状线粒体样分布模式。还通过免疫荧光显微镜研究了未转染鱼类细胞中天然VDAC2的分布。在FEC和EPC细胞的细胞质中均观察到点状的VDAC2荧光信号，但在细胞核中未观察到。这些结果为研究牙鲆中VDAC对病原体反应的功能相关性奠定了基础。
