摘要
为获得刺激隐核虫幼虫膜蛋白单克隆抗体并初步分析单抗特性,提取刺激隐核虫幼虫膜蛋白,免疫Balb/c小鼠,取脾细胞与SP2/O细胞融合,以间接ELISA筛选阳性杂交瘤细胞。通过ELISA方法检测单克隆抗体的亚类及效价;通过SDS-PAGE和western blot分析单抗特异性结合的蛋白,并以串联质谱方法对所识别条带进行鉴定;采用免疫荧光法分析单抗的抗原结合位点和特异性。结果得到3株能稳定分泌刺激隐核虫幼虫膜蛋白抗体的细胞株(5D11AG5、5H9BG3、6E11CE7)。这3株细胞株产生的抗体亚型均为IgM,ELISA效价分别为1∶3200、1∶25600、1∶51200。单抗5D11AG5和单抗5H9BG3株能够识别~35kDa的刺激隐核虫幼虫膜蛋白线性抗原表位,单抗5D11AG5所识别的蛋白与数据库中刺激隐核虫表面抗原蛋白、嗜热四膜虫微管蛋白(tubulin)的肽段具有较高覆盖率。免疫荧光结果显示单抗5D11AG5和单抗5H9DG3识别部位主要在虫体表面近胞口的前端,而单抗6E11CE7识别虫体表面外膜。刺激隐核虫幼虫膜蛋白单克隆抗体的制备,为刺激隐核虫功能性蛋白的筛选、纯化和功能分析提供了条件。
This study aimed to prepare and characterize the monoclonal antibodies （McAbs） against the membrane proteins in theronts of Cryptocaryon irritans. For the study, Balb/c mice were immunized with the proteins extracted from the theronts. Spleen cells from the mice were fused with SP2/O cells to confirm the presence of hybridoma cells using indirect ELISA. Then, the ascites containing MeAbs were collected from the mice containing hybridoma cells for antigen identification in Western-blot and immunofluorescence assays. Three hybridoma antibodies, i. e. , 5D11AGS, 5H9BG3 and 6E11CET, were found to consistently induce the target antigens. These McAbs in the mouse ascitic fluids were all IgM type. The titers of the 3 McAbs ascites were 1 ： 3200, 1 ： 25600 and 1： 51200, respectively. Western-blot assay indicated that McAly5D11AG5 and McAb-SH9BG3, in particular, recognized the proteins with a molecular weight approximating 35 kDa. The peptides recognized by McAb-SDllAG5 had a high homology with the surface antigens of C. irritans and the tubulin of Tetrahymena thermophila. According to the immunofluorescence assay, MeAb-SD11AG5 and McAb-5 H9BG3 recognized the antigens mostly on the front, while McAb-6EllCE7 on the external surface, of the membrane. The results obtained would aid further study to separate and screen the functional proteins of C. irritans.
出处
《福建农业学报》
CAS
北大核心
2017年第5期469-475,共7页
Fujian Journal of Agricultural Sciences
基金
福建省科技重大专项专题(2013NZ0002-5)
福建省平台建设项目(2009N2003)
"948"计划项目(2011-Z11)
福建省科技计划项目--省属公益类科研院所基本科研专项(2016R1018-4)
关键词
刺激隐核虫
幼虫
膜蛋白
单克隆抗体
白点病
Cryptocaryon irritans
theront
membrane protein
monoclonal antibody
white spot disease
