黄海希瓦氏菌多克隆抗体部分特性的分析

Partial characterization of polyclonal antibody against bacterium Shewanella marisflavi

采用免疫学方法,以仿刺参Apostichopus japonicus"化皮病"病原菌——黄海希瓦氏菌Shewanella marisflavi AP629福尔马林灭活疫苗作为免疫原,对新西兰大白兔进行免疫,制备了高效价的多克隆抗体。通过间接酶联免疫、间接免疫荧光和免疫印迹等技术对多克隆抗体的部分特性进行了分析。结果表明:获得的多克隆抗体的效价为1∶32768;多抗与黄海希瓦氏菌标准菌株存在很强的阳性反应,与弧菌和气单胞菌、链球菌和大肠杆菌交叉反应很弱或不存在交叉反应;多抗不仅与菌体本身反应,与菌株表面的分泌物也发生特异性结合。免疫印迹结果显示,多克隆抗体与黄海希瓦氏菌AP629结合的抗原决定簇主要蛋白带有6条,相对分子质量分别为131 000、128 000、113 000、62 000、38 000和33 000,其中38 000和33 000蛋白条带的抗原性最强;与胞外产物结合的抗原决定簇清楚的蛋白条带主要有3条,相对分子质量分别为62 000、38 000和33 000,其中62 000蛋白条带的信号最强。

High titer polyclonal antibody （pAb） against bacterium ShewanelIa marisflavi isolated AP629, a novel pathogen of sea cucumber Apostichopus japonicus, was produced from New Zealand white rabbit with the inactivated vaccine, and partial characterization of the pAb was analyzed with indirect enzyme-lined immunosorbent assay, immunofluoreseence assay and western blot. The results showed that the titer of the pAb was 1 ： 32 768. There was strong reaction of pAb against strain AP629 with S. marisflavi （KCCM 41822）, weak or none that with vibrio,Aeromonas salmonicida, Streptococcus iniae, and Escherichia coll. The binding antigens of pAb was located not only in the membrane of the ceils but also secretion on the surface of strain AP629. Under reducing conditions in western blotting, the major antigenic determinants of the strain AP629 were 131 000,128 000, 113 000,62 000,38 000 and 33 000 ,respectively, in which the 38 000 and 33 000 had stronger signals than the others did. In addition, three major protein antigens of ECP from strain AP629 in molecular weights of 62000,38000 and 33 000 were recognized in western-blot using above pAb, especially protein of 62 000.