| 摘要: |
| 以前期已制备的抗WSSV 囊膜蛋白单克隆抗体4G9(Ab1)杂交瘤细胞生产小鼠腹水, 腹水经辛酸-硫酸铵法、Protein G 亲和层析法纯化后, 用以制备兔抗血清, 所得血清经纯化得到粗提的兔抗WSSV 独特型抗体(Ab2)。采用竞争酶联免疫吸附实验、间接免疫荧光法、斑点免疫印迹和蛋白免疫印迹等实验方法分析了Ab2 特性, 结果表明：Ab2 能识别Ab1 并与WSSV 竞争Ab1 的抗原结合位点, 是具有模拟WSSV 特性的抗独特型抗体; Ab2 能与中国对虾血细胞结合, 并能部分阻断WSSV 与血细胞膜的结合; 其与中国对虾血细胞膜上结合的蛋白分子量分别为94.5、51.5 和27.0kDa,由此推断, 这三个蛋白为WSSV 在中国对虾血细胞膜上的结合蛋白。本研究结果可为进一步研究WSSV 侵染机理提供资料。
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| 关键词: 独特型抗体, 对虾白斑症病毒, 竞争酶联免疫吸附实验, Dot-blot, 间接免疫荧光法, Western-blot |
| DOI：10.11693/hyhz201002019019 |
| 分类号: |
| 基金项目:国家重点基础研究发展计划“973”, 2006CB101806 号; 国家高技术研究发展计划“863”, 2006AA100312 号 |
附件 |
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| PRODUCTION AND CHARACTERIZATION OF RABBIT ANTI-IDIOTYPIC ANTIBODY OF WHITE SPOT SYNDROME VIRUS (WSSV) |
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WEI Xiu-Mei, SHENG Xiu-Zhen, TANG Xiao-Qian, XING Jing, ZHAN Wen-Bin
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Laboratory of Pathology and Immunology of Aquatic Animals, Laboratory of Mariculture Ministry of Education of China (LMMEC), Ocean University of China
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| Abstract: |
| During the past more than ten years since a white spot syndrome virus (WSSV) disease outbreak in 1993, many research papers have been published on the pathogen, pathology, detection and diagnostic methods of the disease. Recent works focused mainly on the infection mechanism and searching the strategies of prophylaxis and control of WSSV infection. The purpose of this work was to produce rabbit anti-idiotypic antibody of WSSV (Ab2), analyze its characterization, and find a new way to study WSSV infection mechanism. Anti-WSSV envelope protein monoclonal antibody (Ab1), which was produced previously, was purified from ascitic fluid using caprylic acid-ammonium sulfate (CA-AS) and Protein G agrose, then used to immunize rabbit, and finally Ab2 was purified from rabbit antiserum using CA-AS. The Ab2 was characterized by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIFA), Dot-blot and Western-blot. Results of competitive ELISA indicate that Ab2 could recognize Ab1 specifically and inhibit competitively the binding of WSSV with Ab1, implying that Ab2 could compete against the same paratope on Ab1 with WSSV, and represent an internal image of WSSV. In the meantime, IIFA proved that Ab2 could bind to the haemocyte membrane of Chinese shrimp (Fenneropenaeus chinensis). The results of both Dot-blot and ELISA blocking experiments show that Ab2 could block the binding of WSSV to haemocyte membrane. By Western-blot, three protein bands of 94.5, 51.5 and 27.0kDa were recognized specifically by Ab2, and they were considered to be WSSV binding protein on Chinese shrimp haemocyte membrane.
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| Key words: Idiotypic antibody, WSSV, ELISA, Dot-blot, IIFA, Western-blot |
