摘要
利用蔗糖密度梯度离心法分离纯化洪湖碘泡虫,经反复冻融、液氮研磨提取可溶性蛋白,免疫小鼠制备得到多克隆抗体,并采用间接酶联免疫吸附法(ELISA)、间接免疫荧光试验(IFAT)、免疫印迹分析(Western blotting)对多克隆抗体的免疫特性进行分析。间接ELISA法测定多抗血清的效价为1∶25 600;IFAT荧光定位显示洪湖碘泡虫的抗原主要位于极丝、孢子壳瓣的四周及底部一特定位置;Western blotting印迹分析显示该多抗血清与洪湖碘泡虫可溶性蛋白中约10条带发生反应,该可溶性蛋白中具有免疫原性的蛋白分子质量大小分别约为180、130、78、75、62、52、42、36、34、28ku。该多抗血清与碘泡虫属及单极虫属的一些粘孢子虫种类存在一定程度的交叉反应。
In order to set up a rapid detection method and facilitate immunological tools to screen target antigens of Myxosporidiosis,the polyclonal antibody(PAb)against Myxobolus honghuensis were prepared from mice immunized with soluble protein,which was aquired through freeze-thaw and grinding purified spores after sucrose density gradient centrifugation.Validity and specificity of the PAb was then estimated by ELISA,IFAT and Western blotting.The ELISA titer of the PAb is 1∶25 600.The PAb mainly bound to polar filaments,posterior and the periphery of spores through immunofluorescence analysis.The Western blotting assay showed that the PAb reacted with nearly 10 soluble proteins(180ku,130 ku,78ku,75 ku,62ku,52 ku,42ku,36 ku,34ku,28ku).Cross-reaction between the PAb and several Myxobolus and Thelohanellus species was found.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2015年第6期108-113,共6页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(31172052)
湖北省杰出青年人才基金项目(2011CDA091)
教育部新世纪人才计划基金(NCET-12-0866)
关键词
洪湖碘泡虫
异育银鲫
多克隆抗体
免疫特性
Myxobolus honghuensis
Carassius auratus gibelio
polyclonal antibody
immune characterization
