刺激隐核虫幼虫膜蛋白单克隆抗体的制备与特性分析

池洪树; 刘晓东; 郑在予; 龚晖; 柯巧珍; 林能锋

doi:10.19303/j.issn.1008-0384.2017.05.001

刺激隐核虫幼虫膜蛋白单克隆抗体的制备与特性分析

doi: 10.19303/j.issn.1008-0384.2017.05.001

1.
福建省农业科学院生物技术研究所/福建省水产病害防治工程技术研究中心, 福建福州 350003
2.
大黄鱼育种国家重点实验室, 福建宁德 352100

基金项目:

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

详细信息

作者简介:
池洪树(1987-), 男, 研究实习员, 主要从事水产动物病原学研究(E-mail:chs321@qq.com)

通讯作者:
龚晖(1971-), 男, 博士, 研究员, 主要从事水产动物病原学与免疫学研究(E-mail:ghxfjm@163.com)

中图分类号: S941.5
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出版历程
- 收稿日期: 2017-01-16
- 修回日期: 2017-03-16
- 刊出日期: 2017-05-01

Preparation and Characterization of Monoclonal Antibodies Against Membrane Proteins in Theronts of Cryptocaryon irritans

1.
Institute of Biotechnology, Fujian Academy of Agricultural Sciences/Fujian Aquaculture Disease Control and Prevention Center, Fuzhou, Fujian 350003, China
2.
State Key Laboratory of Large Yellow Croaker Breeding, Ningde, Fujian 352103, China

摘要

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~35 kDa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。
- 刺激隐核虫 /
- 幼虫 /
- 膜蛋白 /
- 单克隆抗体 /
- 白点病
Abstract: 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 McAbs were collected from the mice containing hybridoma cells for antigen identification in Western-blot and immunofluorescence assays. Three hybridoma antibodies, i.e., 5D11AG5, 5H9BG3 and 6E11CE7, 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 McAb-5D11AG5 and McAb-5H9BG3, in particular, recognized the proteins with a molecular weight approximating 35 kDa. The peptides recognized by McAb-5D11AG5 had a high homology with the surface antigens of C. irritans and the tubulin of Tetrahymena thermophila. According to the immunofluorescence assay, McAb-5D11AG5 and McAb-5H9BG3 recognized the antigens mostly on the front, while McAb-6E11CE7 on the external surface, of the membrane. The results obtained would aid further study to separate and screen the functional proteins of C. irritans.
- Cryptocaryon irritans /
- theront /
- membrane protein /
- monoclonal antibody /
- white spot disease

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图 1 幼虫膜蛋白SDS-PAGE电泳

注：M为标准分子质量蛋白；1为刺激隐核虫幼虫膜蛋白。

Figure 1. SDS-PAGE analysis on membrane proteins of theront

下载: 全尺寸图片幻灯片

图 2 单抗腹水的效价

Figure 2. Antibody titer of McAbs ascites

下载: 全尺寸图片幻灯片

图 3 免疫印迹分析单抗结合的抗原

注：M为蛋白质分子量标准(低)；1为一抗为McAb-5D11AG5；2为一抗为McAb-5H9BG3；3为一抗为McAb-6E11CE7；4为一抗为阳性免疫鼠血清；5为阴性对照；6为空白对照。

Figure 3. Western blot analysis on McAbs for binding antigens

下载: 全尺寸图片幻灯片

图 4 免疫荧光分析单抗识别的刺激隐核虫抗原位点

注：A为一抗为McAb-5D11AG5；B为一抗为McAb-5H9BG3；C为一抗为McAb-6E11CE7；D为阴性对照。

Figure 4. Immunofluorescence analysis of McAbs for binding antigens

下载: 全尺寸图片幻灯片

表 1 单抗亚类鉴定

Table 1. McAbs isotyping

表 2 质谱分析结果

Table 2. Analytical of MS/MS

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