一种(鱼师)鱼诺卡氏菌酪氨酸蛋白磷酸酶基因的克隆及功能初步研究

這鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,可导致鱼类慢性系统性肉芽肿疾病.這鱼诺卡氏菌全基因组序列分析发现了一个酪氨酸蛋白磷酸酶(protein tyrosine phosphtase,PTP)基因,生物信息学分析显示该基因很可能编码一个靶向定位于宿主细胞线粒体的分泌蛋白.本实验对這鱼诺卡氏菌PTP进行了基因克隆、分泌蛋白鉴定、亚细胞定位、过表达和线粒体膜电位检测,结果显示,在這鱼诺卡氏菌胞外产物中质谱鉴定到了PTP肽段,证实其为分泌蛋白.亚细胞定位研究观察到PTP-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明這鱼诺卡氏菌PTP蛋白并未靶向定位于线粒体.亚细胞定位和过表达研究都显示PTP蛋白在FHM细胞中表达后,细胞核出现固缩浓染、凋亡小体等明显的细胞凋亡特征.通过线粒体膜电位检测表明,在pcDNA-PTP转染后48 h,线粒体跨膜电位被明显破坏,说明這鱼诺卡氏菌PTP很可能是一种可诱导细胞凋亡的细菌蛋白.通过对這鱼诺卡氏菌PTP开展基因克隆和功能初步研究,为进一步揭示该基因的功能和深入了解這鱼诺卡氏菌的分子致病机理奠定了基础.

Gene cloning and preliminary function study of protein tyrosine phosphatase from Nocardia seriolae

Nocardia seriolae is the main pathogen of fish nocardiosis, which is a chronic systemic granuloma disease of fish. A gene of protein tyrosine phosphatase (PTP) was found by analyzing the whole genome sequence of N. seriolae, and bioinformatics analysis showed that the PTP gene mayencode a secreted protein which could possibly target host cell mitochondria. In this study, the gene cloning, subcellular localization, over-expression and mitochondrial membrane potential detection were carried out. The results showed that the protein PTP was identified in the extracellular products of N. seriolae, which confirmed that PTP was a secreted protein. Subcellular localization of PTP-GFP fusion proteins were evenly distributed in the whole cell of FHM cells, and did not coincide with the distribution of mitochondria, which indicated that the protein PTP was not targeted at mitochondria. Typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were found when PTP protein was expressed in FHM cells by both subcellular localization and over-expression studies. The mitochondrial membrane potential was significantly damaged in FHM at 48h after the transfection of pcDNA-PTP, which confirmed that PTP was a bacterial protein which can likely induce cell apoptosis. The gene cloning and preliminary function study of PTP from N. seriolae have laid the foundation for further research on the gene and for promoting the understanding of the pathogenic mechanism of N. seriolae.