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鱼诺卡氏菌动力蛋白调节蛋白robl/LC7的亚细胞定位和功能初步研究
引用本文:夏立群,陈锐敏,廖保山,苏泽杰,徐亮,童邦卓,黄嘉慧,鲁义善.鱼诺卡氏菌动力蛋白调节蛋白robl/LC7的亚细胞定位和功能初步研究[J].水产学报,2018,42(3):419-430.
作者姓名:夏立群  陈锐敏  廖保山  苏泽杰  徐亮  童邦卓  黄嘉慧  鲁义善
作者单位:广东海洋大学水产学院,广东海洋大学水产学院;广东海洋大学深圳研究院;广东省水产经济动物病原生物学及流行病学重点实验室,广东海洋大学水产学院,广东海洋大学水产学院,广东海洋大学水产学院,广东海洋大学水产学院,广东海洋大学水产学院
基金项目:国家自然科学基金(41406172),广东省科技发展专项资金项目(2016A050502061),广东省自然科学基金(2014A030313602),深圳大鹏新区产业发展专项资金项目(KY20160207),广东省攀登计划项目(pdjh2016b0234)
摘    要:鱼类诺卡氏菌病是一种慢性系统性肉芽肿疾病,鱼诺卡氏菌是其主要病原。鱼诺卡氏菌全基因组生物信息学分析,发现了一个可能靶向定位于宿主细胞线粒体的分泌蛋白——动力蛋白调节蛋白robl/LC7。为了对鱼诺卡氏菌robl/LC7的亚细胞定位和功能进行初步研究,实验对鱼诺卡氏菌robl/LC7进行了基因克隆、真核表达重组质粒构建、分泌蛋白鉴定、亚细胞定位、过表达和凋亡检测。结果显示,成功克隆了鱼诺卡氏菌robl/LC7基因并构建了其真核表达质粒p EGFP-robl/LC7和pc DNA-robl/LC7;鱼诺卡氏菌分泌蛋白质谱鉴定证实robl/LC7为分泌蛋白;亚细胞定位研究显示robl/LC7-GFP融合蛋白呈全细胞分布,不与线粒体共定位;凋亡检测发现robl/LC7过表达能诱导FHM细胞凋亡。研究表明,鱼诺卡氏菌robl/LC7是一个不与线粒体共定位的分泌蛋白,其可能通过参与细胞凋亡调控,协助鱼诺卡氏菌在宿主体内生存和免疫逃避,并在鱼诺卡氏菌的致病过程中具有重要作用。

关 键 词:鰤鱼诺卡氏菌  动力蛋白调节蛋白robl/LC7  分泌蛋白  亚细胞定位  细胞凋亡
收稿时间:2017/1/19 0:00:00
修稿时间:2017/4/20 0:00:00

Subcellular localization and preliminary function study of dynein regulation protein robl/LC7 from Nocardia seriolae
XIA Liqun,CHEN Ruimin,LIAO Baoshan,SU Zejie,XU Liang,TONG Bangzhuo,HUANG Jiahui and LU Yishan.Subcellular localization and preliminary function study of dynein regulation protein robl/LC7 from Nocardia seriolae[J].Journal of Fisheries of China,2018,42(3):419-430.
Authors:XIA Liqun  CHEN Ruimin  LIAO Baoshan  SU Zejie  XU Liang  TONG Bangzhuo  HUANG Jiahui and LU Yishan
Institution:Fisheries College of Guangdong Ocean University,,,,,,Fisheries College of Guangdong Ocean University
Abstract:Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. A dynein regulation protein robl/LC7, which likely to be secreted and to target host cell mitochondria, was found by the bioinformatics analysis on the whole genome sequence of N. seriolae. In order to determine the subcellular localization and study the preliminary function of robl/LC7 from N. seriolae, the gene cloning, eukaryotic expression plasmid construction, secreted protein identification, subcellular localization and apoptosis detection of robl/LC7 were carried out in this study. The results showed that robl/LC7 was successfully cloned from N. seriolae and the eukaryotic expression plasmid of pEGFP- robl/LC7 and pcDNA- robl/LC7 were constructed. Mass spectrometry analysis of extracellular products from N. seriolae confirmed that robl/LC7 was a secreted protein. Subcellular localization of robl/LC7-GFP fusion protein discovered that the green fluorescence distributed in the whole cell and did not co-localize with mitochondria. In addition, apoptosis assay revealed that apoptosis was induced by the over-expression of robl/LC7 in FHM cell. Thus, robl/LC7 from N. seriolae is a secreted protein which does not co-localize with mitochondria. It may help the host cell survival and immune escape of N. seriolae by participating the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.
Keywords:Nocardia seriolae  dynein regulation protein robl/LC7  secreted protein  subcellular localization  cell apoptosis
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