鰤鱼诺卡氏菌DmpA基因的克隆及亚细胞定位研究
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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p EGFP-DmpA和pc DNA-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 analysisshowed 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 p EGFP-DmpA and pc DNA-DmpA were constructed,extracellular product of N. seriolae was identified,andthe subcellular localization and over-expression of DmpA were carried out. The results showed that the protein DmpA was identified in theextracellular products of N. seriolae,and confirmed as a secreted protein. Subcellular localization of DmpA-GFP fusion proteins were evenlydistributed in the whole cell of FHM cells,and were not coincided with the distribution of mitochondria,indicating that the protein DmpA wasnot co-localized with the mitochondria. The expression of DmpA changed the distribution of mitochondria into lumps in the FHM cell. The overexpression 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 thegene and promoting the understanding of the pathogenic mechanism of N. seriolae.
Nocardia seriolae,a facultative intracellular bacterium,is the main pathogen of fish nocardiosis. Bioinformatics analysisshowed 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 p EGFP-DmpA and pc DNA-DmpA were constructed,extracellular product of N. seriolae was identified,andthe subcellular localization and over-expression of DmpA were carried out. The results showed that the protein DmpA was identified in theextracellular products of N. seriolae,and confirmed as a secreted protein. Subcellular localization of DmpA-GFP fusion proteins were evenlydistributed in the whole cell of FHM cells,and were not coincided with the distribution of mitochondria,indicating that the protein DmpA wasnot co-localized with the mitochondria. The expression of DmpA changed the distribution of mitochondria into lumps in the FHM cell. The overexpression 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 thegene and promoting the understanding of the pathogenic mechanism of N. seriolae.
引文
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[19]Beaman BL,Beaman L.Nocardia species:host-parasiterelationships[J].Clinical Microbiology Reviews,1994,7(2):213-64.
[2]黄郁葱,简纪常,吴灶和,等.卵形鲳鲹结节病病原的分离与鉴定[J].广东海洋大学学报,2008,28(4):49-53.
[3]Labrie L,Ng J,Tan Z,Komar C,Ho E,Grisez L.2008.Nocardialinfections in fish:an emerging problem in both freshwater andmarine aquaculture systems in Asia[G]//Bondad-ReansatoM,Mohan CV,Crumlish M,Subasinghe R.Diseases in Asianaquaculture VI.Fish Health Section.Philippines:Asian FisheriesSociety,2008:297-312.
[4]Elkesh A,Kantham KPL,Shinn AP,et al.Systemic nocardiosis in aMediterranean population of cultured meagre,Argyrosomus regius,Asso(Perciformes:Sciaenidae)[J].Journal of Fish Diseases,2013,36(2):141-149.
[5]张建丽,刘志恒.诺卡氏菌型放线菌的分类[J].微生物学报,2001,41(4):513-517.
[6]Beaman B L,Beaman L.Nocardia species:host-parasiterelationships[J].Clinical Microbiology Reviews,1994,7(2):213-264.
[7]罗云蔓,郭晓奎,姜叙诚.病原菌逃避单核-巨噬细胞杀灭策略的研究进展[J].微生物与感染,2010,5(2):117-120.
[8]张瑶,徐平,李文均,等.放线菌蛋白质组学研究进展[J].生物工程学报,2014,30(7):1044-1058.
[9]Xia L,Cai J,Wang B,et al.Draft genome sequence of Nocardia seriolae ZJ0503,a fish pathogen isolated from Trachinotus ovatus inChina[J].Genome Announcements,2015,3(1):e01223-14.
[10]夏立群,王蓓,夏洪丽,等.鰤鱼诺卡氏菌培养条件及培养基的优化[J].南方水产科学,2013,9(3):51-56.
[11]Laurence F,Colette G,Abdellatif C,et al.The Dmp A aminopetidasefrom ochrobactrum antropi LMG7991 is the prototype of a newterminal nucleophile hydrolase family[J].Biochemical Journal,1999,341(Pt 1):147-155.
[12]Komeda H,Asano Y.A Dmp A-homologous protein from Pseudomonas sp.is a dipeptidase specific for beta-alanyl dipeptides[J].FEBS Journal,2005,272(12):3075-3084.
[13]高新星.枯草芽孢杆菌氨肽酶分泌表达、分子改造及生理功能研究[D].无锡:江南大学,2014.
[14]党光辉.结核分枝杆菌酯酶的表达、纯化、结晶及理化特性研究[D].福州:福建农林大学,2013.
[15]Holland C,Mak TN,Zimnyarndt U,et al.Proteomic identificationof secreted proteins of Propionibacterium acnes[J].BmcMicrobiology,2010,10(1):1-11.
[16]Negoro S,Kakudo S,Urabe I,et al.A new nylon oligomerdegradation gene(nyl C)on plasmid p OAD2 from aFlavobacterium sp.[J].Journal of Bacteriology,1992,174(24):7948-7953.
[17]Seyffert N,Pacheco LG,Silva WM,et al.Preliminary serologicalsecretome analysis of Corynebacterium pseudotuberculosis[J].Journal of Integrated OMICS,2011,1(2):193-197.
[18]Barry DP,Beaman BL.Nocardia asteroides,strain GUH-2 inducesproteasome inhibition and apoptotic death of cultured cells[J].Research in Microbiology,2007,158(1):86-96.
[19]Beaman BL,Beaman L.Nocardia species:host-parasiterelationships[J].Clinical Microbiology Reviews,1994,7(2):213-64.