摘要
目的制备抗尼罗罗非鱼血清免疫球蛋白的单克隆抗体,并对其免疫学特性进行鉴定,运用它们监测嗜水气单胞菌免疫尼罗罗非鱼血清抗体水平。方法采用重组蛋白A亲和层析法纯化尼罗罗非鱼血清免疫球蛋白,纯化蛋白经SDS-PAGE检测后免疫Balb/c小鼠,取免疫鼠脾细胞与Sp2/0细胞融合;用ELISA法对杂交瘤进行筛选以及测定单抗的特性和效价,Western blot和叠加ELISA分析单抗的抗原结合表位;灭活嗜水气单胞菌免疫尼罗罗非鱼,用单抗建立ELISA检测体系监测血清特异性抗体水平。结果 SDS-PAGE电泳条件下血清免疫球蛋白重链和轻链的相对分子质量分别约为77 000和27 000;共获得4株抗罗非鱼血清Ig的单抗,分别命名为3D9、9D4、8G3和7B7,它们的细胞上清ELISA效价为1∶3 200～1∶6 400,抗体Ig亚类均为IgG1;4株单抗都能特异识别罗非鱼免疫球蛋白,而与鲫、草鱼、南方鲇、斑点叉尾鮰、鲈鱼血清无任何交叉反应。3D9识别变性条件下的尼罗罗非鱼Ig重链,9D4、8G3和7B7为轻链特异性。初次免疫后1～15 d尼罗罗非鱼血清中嗜水气单胞菌特异性抗体水平显著上升,第3次免疫后3周抗体水平达到峰值。结论成功制备抗尼罗罗非鱼血清免疫球蛋白单克隆抗体,为尼罗罗非鱼免疫球蛋白的结构分析、免疫应答水平监测和病原诊断等研究提供有力工具。
To prepare monoclonal antibody （mAb） against serum immunoglobulin （Ig） of tilapia and characterize its immunological feature, female Balb/c mice were immunized with tilapia Ig purified by Protein A affinity chromatography. Indirect ELISA and Western blot were employed to screen the hybridoma cells by determining the specificities and titer of the produced mAbs. SDS-PAGE analysis showed that molecular weight of tilapia Ig heavy chain and light chain was 77 kD and 27 kD respectively. Four hybridoma cell strains that could stably secret mAb against the tilapia Ig were obtained, and named as 3D9, 9D4, 8G3 and 7B7, which all belongs to IgGl. Titer of the four mAbs was ranged from 1：3 200 to 1：6 400 tested by ELISA. 3D9 was able to recognize the heavy chain of tilapia Ig, while the other three mAbs could bind to light chain of Ig. Further experiments proved that all of them only bound to Ig of tilapia Oreochromis niloticu specifically, and have not any cross reaction with the Ig of Cienoyharyngodoni dellus, Carrassius auratus, Ietalurus Punetaus, Silurus meridiona!is Chen or Lateolabrax japomcus. Using mAbs, tilapia serum antibodies against A eromonas hydrophila could be detected by ELISA at day 8 after the first immunization, and the peak of titer was appeared at the third week after the third immunization. All of these results demonstrated that the prepared mAbs were highly specific to the Ig of tilapia, and had great potential to be used for analyzing the structure of tilapia Ig, monitoring the immunity level and pathogen diagnosis.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2013年第8期663-667,共5页
Immunological Journal
基金
西南大学基本科研业务费专项基金项目(XDJK2012C082)
