DeSheng Kong, HuaiRan Liu, JiaSen Liu, Zuo Yu, Qian Jiang, DongChun Guo, XiaoLiang Hu, FengJie Wang, QianQian Huang, LianDong Qu
Zoonosis of Natural Foci, State Key Laboratory of Veterinary Biotechnology, Harbin Vet Res Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang District, Harbin, 150001, People's Republic of China,
Appl Microbiol Biotechnol. 2015 Jul;99(14):5951-66. doi: 10.1007/s00253-015-6571-8. Epub 2015 Apr 18.
The VP60 protein of rabbit hemorrhagic disease virus (RHDV) is a structural protein with important roles in viral replication and assembly. In this study, we immunized BALB/c mice with the RHDV-TP strain. Six monoclonal antibodies (mAbs) were selected and characterized by enzyme-linked immunosorbent assay, Western blotting, and indirectly immunofluorescence analysis (IFA). All six mAbs (AD4, AG10, BC9, BE8, BH3, and DE2) had positive reactions with recombinant VP60 as analyzed by IFA, but only two (AG10 and DE2) reacted with denatured RHDV by Western blotting. Fifty-four partially overlapping fragments of the VP60 gene were expressed with His or Glutathione S-transferase (GST) tags to identify the epitopes recognized by AG10 and DE2. These two epitopes were located at the C-terminal of VP60 and were longer (64 and 53 amino acids, respectively) than normal B cell epitopes. However, both AG10 and DE2 also interacted with RHDV2 VP60 expressed in insect cells. Amino acid alignments of the AG10 and DE2 epitope regions between RHDV and RHDV2 VP60 indicated several mutations, suggesting that the epitopes recognized by the mAbs AG10 and DE2 were discontinuous. Epitope immunogenicity was evaluated by inoculating specific pathogen-free rabbits with saline, purified DE2 epitope, or RHDV inactive vaccine. Rabbits immunized with the DE2 epitope developed high levels of RHDV-specific antibodies but no cellular immune response and died after challenge with RHDV-HYD isolate. Despite their lack of neutralizing activity, these mAb reagents and epitopes may have useful clinical applications and will be valuable tools in further studies of the structure and function of the RHDV VP60 protein.
兔出血症病毒（RHDV）的VP60蛋白是一种结构蛋白，在病毒复制和组装过程中发挥着重要作用。在本研究中，我们用RHDV-TP株免疫BALB/c小鼠。筛选出6种单克隆抗体（mAb），并通过酶联免疫吸附测定、蛋白质印迹法和间接免疫荧光分析（IFA）对其进行表征。通过IFA分析，所有6种mAb（AD4、AG10、BC9、BE8、BH3和DE2）与重组VP60均呈阳性反应，但通过蛋白质印迹法检测，只有2种（AG10和DE2）与变性的RHDV发生反应。表达了54个VP60基因的部分重叠片段，并带有His或谷胱甘肽S-转移酶（GST）标签，以鉴定AG10和DE2识别的表位。这两个表位位于VP60的C末端，比正常B细胞表位更长（分别为64和53个氨基酸）。然而，AG10和DE2也与在昆虫细胞中表达的RHDV2 VP60相互作用。RHDV和RHDV2 VP60之间AG10和DE2表位区域的氨基酸比对显示存在多个突变，这表明mAb AG10和DE2识别的表位是不连续的。通过用生理盐水、纯化的DE2表位或RHDV灭活疫苗接种无特定病原体的兔子来评估表位的免疫原性。用DE2表位免疫的兔子产生了高水平的RHDV特异性抗体，但没有细胞免疫反应，在用RHDV-HYD分离株攻击后死亡。尽管它们缺乏中和活性，但这些mAb试剂和表位可能具有有用的临床应用价值，并且将成为进一步研究RHDV VP60蛋白结构和功能的有价值工具。
