创伤弧菌外膜蛋白的分离及其抗原性分析  

Antigenicity and identification of Vibrio vulnificus outer membrane proteins

作　　者：田丁[1,2] 林天龙[2] 许斌福[2] 龚晖[2] 

机构地区：[1]福建师范大学生命科学院,福建福州350007 [2]福建农业科学院生物技术研究所,福建福州350003

出　　处：《福建农业学报》2008年第4期337-341,共5页Fujian Journal of Agricultural Sciences

基　　金：国家863资助项目(2006AA100308);福建省科技重点项目(2008N0023);福建省自然科学基金(2008J0057)

摘　　要：分别采用Sarkosyl和PMSF法分离、提取鳗源创伤弧菌FJ03-X2的外膜蛋白,并应用SDS-PAGE和Western-blotting分析比较两种方法提取的外膜蛋白的组分和抗原性。SDS-PAGE分析结果表明,Sarkosyl法分离的主要外膜蛋白分子量集中在14～66 kDa之间;而PMSF法分离的主要外膜蛋白分子量分布在14～92 kDa之间。在两种方法中,分子量38 kDa、36 kDa的外膜蛋白均为高丰度蛋白。两种方法提取的创伤弧菌外膜蛋白经SDS-PAGE后,用兔抗创伤弧菌FJ03-X2血清进行Western-blotting,结果显示,兔抗FJ03-X2高免血清能识别绝大多数外膜蛋白组分,说明FJ03-X2菌株的外膜蛋白具有良好的抗原性。其中,分子量分别为66kDa、47 kDa、44 kDa、38 kDa、36 kDa、34 kDa的外膜蛋白呈强阳性反应,是主要的免疫原组分。同时,经比较分析认为PMSF法提取创伤弧菌外膜蛋白的效果更好。The outer membrane proteins （OMPs） are the major components in outer membrane, which is unique to the gram-negative bacteria and plays an important role in bacterial life functions. This study focuses on the antigenicity and identification of OMPs of Vibrio vulnificus FJ03 - X2, a pathogenic stain isolated from European eel （Anguilla anguilla）. By applying Sarkosyl and PMSF methods, the major OMPs （MOMPs） of V. vulnificus were obtained. The constituents and antigenicities of MOMPs were analyzed by means of SDS - PAGE and Western-blotting. The results showed that the molecular weights of the MOMPs separated by Sarkosyl method were between 14 -66 kDa, and those separated by PMSF method were between 14-92 kDa. The main proteins of the OMPs separated by either method had molecular weights of 38 kDa and 36 kDa. After SDS - PAGE separation, the V. vulnificus OMPs were subjected to the Western-blotting using rabbit antiserum against the intact V. vulnificus FJ03 - X2 cells. The rabbit antiserum could identify most of the MOMPs. The result suggested that the FJ03 - X2 MOMPs had a good antigenicity. Six OMPs of 66 kDa, 47kDa, 44kDa, 38kDa, 36kDa and 34kDa showed significant positive reaction in the test. These proteins, therefore, were believed to be the major components associated with the immunity. It was also found that PMSF was more effective than Sarkosyt method for extracting OMPs from V. vulnificus.

关 键 词：创伤弧菌 外膜蛋白 抗原性 

分 类 号：S941.42[农业科学—水产养殖]