溶藻弧菌全菌可溶性蛋白二维图谱的建立及部分蛋白分子的鉴定  

Proteome reference map for total soluble proteins of Vibrio alginolyticus

作　　者：庞欢瑛[1,2,3,4,5] 李岩[3,4,5] 鲁义善[3,4,5] 简纪常[3,4,5] 吴灶和[3,4,5] 

机构地区：[1]中国科学院南海海洋研究所,广东广州510301 [2]中国科学院研究生院,北京100049 [3]广东海洋大学水产学院,广东湛江524025 [4]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524025 [5]广东省教育厅水产经济动物病害控制重点实验室,广东湛江524025

出　　处：《中国水产科学》2010年第3期404-413,共10页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(30972271);国家科技支撑计划(2007BAD29B05)

摘　　要：本研究采用蛋白质组学技术,建立了溶藻弧菌(Vibrio alginolyticus)ZJ03培养至稳定期的蛋白质组双向电泳图谱,并对部分蛋白分子进行了肽质量指纹图谱分析鉴定。首先将溶藻弧菌接种于TSB培养基28℃培养24 h,利用裂解液裂解细菌,提取全菌可溶性蛋白,荧光染料标记,24 cm pH4 ～ 7的胶条进行等电聚焦,再用12.5%的胶进行SDS-PAGE。2-D胶经过分析,得到902±8个蛋白斑点,重复胶的匹配点数为866±28,匹配率为96%。从双向电泳图谱中选取68个高丰度蛋白质点进行肽质量指纹图谱鉴定,其中60个在NMPDR数据库中得到鉴定,另外8个在NCBI数据库中得到鉴定。鉴定的蛋白中发现Serine protein kinase、purine nucleoside phosphorylase 和Alkyl hydroperoxide reductase subunit C-like protein存在修饰现象,它们在胶上均有2种表现形式。对68个蛋白进行了细胞功能的分类,发现能量代谢蛋白最多,占43%;其次是转运和结合蛋白,占9%;第三是外膜蛋白,占8%。经CMR操纵子预测软件分析预测到2个操纵子,它们均参与了能量代谢过程。研究结果为溶藻弧菌在不同生长条件下的比较蛋白质组学以及该菌强毒株和无毒株的比较研究提供了基础资料。Vibrio alginolyticus is the main Vibrio species isolated from diseased mariculture animals with clinical symptoms of bacterial septicemia and skin ulcer in the South China Sea. This bacterium belongs to the family of Vibrionaceae and is normal inhabitant in estuarine and marine environments. Proteome analysis by two dimensional electrophoresis（ 2-DE） together with mass finger print is a powerful approach for protein resolution and identification of complex biological samples. In this study,a proteome reference map has been constructed for V. alginolyticus in the pI range from 4.0 to 7.0. The techniques for proteome analysis were used to establish reference map of V. alginolyticus in a stationary phase. V. alginolyticus strain ZJ03 was cultured in TSB medium for 24 h at 28℃. Total soluble proteins were extracted with lysis buffer and purified with a 2-D clean-up kit. Then the purified proteins were labeled with Cy3 or Cy5 and separated by 2-DE. 2-DE gels were scanned with Typhoon 9410 and analyzed by DeCyder 6.0. 2-DE image analysis revealed 902±8 protein spots. The matching spots were 866±28 in two repeat electrophoregrams,with matching rate of 96%. The results were obtained from three gels run with 24 cm immobilized pH gradient strips and 12.5% SDS-PAGE gels. Sixty-eight high-abundant spots were chosen for mass spectrometry identification. Among the 68 spots from the 2-DE map,60 spots could match with the proteins in NMPDR Database,and the other 8 spots matched with the proteins in NCBInr Database. Among the 68 proteins,energy metabolism components are the most,accounting for 43%;The second is the transport and binding proteins,accounting for 9%;The third is the outer membrane proteins,accounting for 8%. Two isoforms of serine protein kinase,purine nucleoside phosphorylase and alkyl hydroperoxide reductase subunit C-like protein were proposed. Two operons are proposed which involved in energy metabolism. Ten pathogenicity associated proteins were found. They are OMP TolC,OMP W,OMP A,OMP N,peptide ABC tra

关 键 词：蛋白质组学 溶藻弧菌 全菌可溶性蛋白 肽质量指纹图谱 双向电泳 

分 类 号：S917[农业科学—水产科学]