鰤鱼诺卡氏菌DmpA基因的克隆及亚细胞定位研究

摘要

鰤鱼诺卡氏菌(Nocardia seriolae)是鱼类诺卡氏菌病的主要病原,为兼性胞内菌.生物信息学分析显示鰤鱼诺卡氏菌DmpA基因的表达产物为分泌蛋白,且可能定位于宿主细胞的线粒体上.通过构建重组质粒pEGFP-DmpA和pcDNA-DmpA、鰤鱼诺卡氏菌胞外产物鉴定、亚细胞定位、过表达等方法,对鰤鱼诺卡氏菌DmpA基因进行了克隆、亚细胞定位及初步功能研究.结果表明在鰤鱼诺卡氏菌胞外产物中鉴定到了DmpA蛋白,证实其为分泌蛋白.亚细胞定位研究发现DmpA-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明DmpA蛋白并不定位在线粒体上.DmpA-GFP融合蛋白的表达会改变FHM细胞线粒体的分布为团块状.DmpA在细胞中的过量表达对细胞核无明显影响,表明该基因无诱导细胞凋亡的功能.通过对鰤鱼诺卡氏菌DmpA的克隆、亚细胞定位和过表达研究,为进一步研究该基因的功能和深入了解鰤鱼诺卡氏菌的致病机理奠定了基础.%Nocardia seriolae,a facultative intracellular bacterium,is the main pathogen of fish nocardiosis. Bioinformatics analysis showed that the DmpA gene of N. seriolae encoded a secreted protein,and may be co-located with mitochondria of the host cell. In this study, the recombinant plasmids pEGFP-DmpA and pcDNA-DmpA were constructed,extracellular product of N. seriolae was identified,and the subcellular localization and over-expression of DmpA were carried out. The results showed that the protein DmpA was identified in the extracellular products of N. seriolae,and confirmed as a secreted protein. Subcellular localization of DmpA-GFP fusion proteins were evenly distributed in the whole cell of FHM cells,and were not coincided with the distribution of mitochondria,indicating that the protein DmpA was not co-localized with the mitochondria. The expression of DmpA changed the distribution of mitochondria into lumps in the FHM cell. The over-expression of DmpA in FHM cells had no significant effect on the nucleus,revealing that the gene had no function on inducing cell apoptosis. The cloning,subcellular localization and over-expression of DmpA from N. seriolae laid the foundation for further studying the function of the gene and promoting the understanding of the pathogenic mechanism of N. seriolae.