鮰爱德华菌黄颡鱼分离株外膜蛋白的抗原性分析  

Antigenic Characterization of the Outer Membrane Proteins of Edwardsiella ictaluri, Isolated from Pelteobagrus fulvidraco

作　　者：李强[1] 刘海燕[1] 黄华[1] 李华[1] 

机构地区：[1]大连海洋大学辽宁省海洋生物资源与生物修复重点实验室,辽宁大连116023

出　　处：《广东海洋大学学报》2011年第3期85-89,共5页Journal of Guangdong Ocean University

基　　金：国家"十二五"科技支撑项目(2011BAD13B03);海洋公益性行业科研专项(200905020);辽宁省海洋与渔业厅项目(201005);辽宁省教育厅高等学校科研计划(2008143)

摘　　要：以黄颡鱼"红头病"致病菌——鮰爱德华菌(Edwardsiella ictaluri)A86作为实验菌株,通过十二烷基肌氨酸钠(Sarkosyl)抽提并结合超速离心的方法提取主要外膜蛋白(OMPs),SDS-PAGE图谱分析结果显示,OMPs主要有6条条带,相对分子质量分别为45 000、42 000、41 000、36 000、30 000和22 000。用A86 OMPs免疫新西兰大白兔,获得多克隆抗体22 mL,抗体效价为1∶20 480,抗体按照1∶2 560稀释时具有较高的特异性,仅与迟缓爱德华菌(E.tarda)、海豚链球菌(Streptococcus iniae)和创伤弧菌(Vibrio vulnificus)存在微弱的交叉反应,与鮰爱德华菌参考株及不同地区的分离株呈现特异性结合。多克隆抗体与OMPs的免疫印迹结果显示,主要有2条明显的免疫反应发色带,相对分子质量分别为36 000和30 000,且36 000蛋白染色最为明显。多克隆抗体与包括菌株A86在内的分离自南北方不同地区的10株鮰爱德华菌全菌免疫印迹试验发现,所有受检菌株反应结果相同,主要有4条明显的反应条带,相对分子质量分别为106 000、54 000、36 000和30 000,其中36 000和30 000两条蛋白带染色最深。因此,认为从黄颡鱼体内分离的鮰爱德华菌OMPs中36 000和30 000蛋白具有很好的免疫原性,36 000蛋白尤甚。Using sodium lauryl sarcosine（Sarkosyl） method combined with super centrifugal purification,the outer membrane proteins（OMPs） were extracted from Edwardsiella ictaluri isolated A86,pathogen of red-headed disease in yellow catfish Pelteobagrus fulvidraco.Subsequently,polyclonal antibody（pAb） against OMPs of strain A86 was produced from New Zealand white rabbit.The result of SDS-PAGE showed the OMPs contained 6 major protein bands with relative molecular mass of 45 000,42 000,41 000,36 000,30 000,and 22 000,respectively.The titer of pAb was 1∶20 480.Cross-reactivity test proved that pAb had weak reaction with E.tarda,Streptococcus iniae and Vibrio vulnificus,and strong reaction with E.ictaluri standard or isolated strains.Two major protein antigens of OMPs in relative molecular mass of 36 000 and 30 000 were recognized in western-blot using above-mentioned pAb.Results of pAb against OMPs reacted with 10 strains of E.ictaluri isolated from P.fulvidraco in western-blot showed the major antigenic determinants of all strains were identical in relative molecular mass of 106 000,54 000,36 000,and 30 000,respectively.Among them the signals of 36 000 and 30 000 were stronger than the others.In conclusion,these results indicated that proteins in relative molecular mass of 36 000 and 30 000 were the major antigens of OMPs of E.ictaluri,especially protein of 36 000.

关 键 词：鮰爱德华菌 黄颡鱼 外膜蛋白 抗原性 

分 类 号：S942.5[农业科学—水产养殖]