Wang Xiaoyan, Gao Honglei, Gao Yulong, Fu Chaoyang, Wang Zhi, Lu Guili, Cheng Yu, Wang Xiaomei
Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, China.
J Virol Methods. 2007 Aug;143(2):194-9. doi: 10.1016/j.jviromet.2007.03.016. Epub 2007 May 3.
To map the epitopes of VP2 protein of chicken anemia virus (CAV), VP2 was expressed as a fusion protein in Escherichia coli BL21 (DE3). The Western blot demonstrated that recombinant VP2 protein could be recognized by sera of chickens infected with CAV. Female BALB/c mice were immunized with purified recombinant VP2 produced in E. coli BL21 (DE3) and seven VP2-specific monoclonal antibodies (MAbs) were developed. The results of Western blot showed that all the seven MAbs recognized the recombinant VP2 protein expressed in the baculovirus and reacted with MDCC-MSB1 cells infected with CAV by indirect immunofluorescence assay. The VP2 protein was dissected into 21 overlapping fragments, expressed as fusion peptides in E. coli and used for epitope mapping by pepscan analysis. ELISA and Western blot assays indicated that most of MAbs reacted with the 12th and 13th fragments (amino acids 111-136) and one of them reacted with the 3rd fragment (amino acids 21-36). The linear immunodominant epitope of VP2 was located mainly in amino acid residues 111-126 and 121-136.
为了绘制鸡贫血病毒（CAV）VP2蛋白的表位图谱，VP2在大肠杆菌BL21（DE3）中作为融合蛋白表达。蛋白质免疫印迹法表明，重组VP2蛋白能被感染CAV的鸡血清识别。用在大肠杆菌BL21（DE3）中产生的纯化重组VP2免疫雌性BALB/c小鼠，获得了7种VP2特异性单克隆抗体（MAb）。蛋白质免疫印迹法结果显示，所有7种MAb均能识别杆状病毒中表达的重组VP2蛋白，并通过间接免疫荧光试验与感染CAV的MDCC-MSB1细胞发生反应。将VP2蛋白切割成21个重叠片段，在大肠杆菌中作为融合肽表达，并通过肽扫描分析用于表位图谱绘制。酶联免疫吸附测定和蛋白质免疫印迹试验表明，大多数MAb与第12和13个片段（氨基酸111 - 136）反应，其中一种与第3个片段（氨基酸21 - 36）反应。VP2的线性免疫显性表位主要位于氨基酸残基111 - 126和121 - 136处。
