| 抗鸡贫血病毒结构蛋白单克隆抗体制备及其对应抗原表位分析 |
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| 引用本文： | 王晓艳,高宏雷,高玉龙,程宇,王笑梅.抗鸡贫血病毒结构蛋白单克隆抗体制备及其对应抗原表位分析[J].农业生物技术学报,2007,15(4):557-561. |
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| 作者姓名： | 王晓艳 高宏雷 高玉龙 程宇 王笑梅 |
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| 作者单位： | 1. 中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,禽传染病实验室,哈尔滨,150001
2. 新疆农业大学,乌鲁木齐,830052 |
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| 摘 要： | 以纯化的原核表达的鸡贫血病毒结构蛋白为抗原，免疫6-8w的雌性Balb/c鼠, 经过三次免疫后，取其脾细胞与SP2/0骨髓瘤细胞进行融合，以纯化的蛋白为抗原进行ELISA检测，将阳性细胞孔经过三次有限稀释，共获得6株能稳定分泌特异性抗体的阳性细胞株。间接免疫荧光试验证实6株单抗可以与鸡贫血病毒感染的MDCC-MSB1细胞发生特异性反应，Western blot结果显示单抗与杆状病毒表达的VP1重组蛋白可以发生特异性反应。利用单克隆抗体亚型鉴定试剂盒进行亚型鉴定，结果表明6株单抗均为IgG1亚型，且所有单抗的轻链均为κ链。用单克隆抗体对分段表达的VP1蛋白进行免疫印迹分析，初步鉴定了筛选出的单抗所对应的抗原表位，其中有四株单抗识别的表位位于VP1 218-274位氨基酸之间，另外两株单抗识别的表位分别位于275-301位、324-369位氨基酸之间。
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| 关 键 词： | 鸡贫血病毒（CAV） 结构蛋白（VP1） 单克隆抗体 抗原表位 |
| 文章编号： | 1006-1304（2007）04-0557-05 |
| 收稿时间： | 2006-09-27 |
| 修稿时间： | 2006-11-24 |
Development of Monoclonal Antibodies Against VP1 Protein of Chicken anemia virus and Its Identification of Epitopes |
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| WANG Xiao-yan,GAO Hong-lei,GAO Yu-long,CHENG Yu,WANG Xiao-mei.Development of Monoclonal Antibodies Against VP1 Protein of Chicken anemia virus and Its Identification of Epitopes[J].Journal of Agricultural Biotechnology,2007,15(4):557-561. |
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| Authors: | WANG Xiao-yan GAO Hong-lei GAO Yu-long CHENG Yu WANG Xiao-mei |
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| Institution: | 1.Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China; 2.Xinjiang Agricultural University, Urumq 830052, China |
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| Abstract: | Female, 6-8w Balb/c mice were immunized with the purified recombinant fusion protein. After three immunizations, the spleen cells from Balb/c immunized were fused with mouse myeloma cells SP2/0. An indirect ELISA with purified VP1 protein as coated antigen was used to screen antibody-producing hybridomas. By 3 cycles of limited dilutions, Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. All MAbs reacted with MDCC-MSB1 cell infected with CAV by indirect immunofluorescence assay (IFA) and they recognized the VP1 protein expressed in Baculovirus by Western blot. This demonstrated that all MAbs have good specificity. The subtype of all MAbs is IgG1 and light chains of all MAbs are kappa. Taking advantage of VP1 fragments expressed, the epitopes corresponding to the MAbs are analyzed. The results showed that the monoclonal antibodies 1C5、2F3、4D2 and 4E8 recognized the same epitope, which was located in 218-274aa. Moreover, the two other epitopes were identified and they were located in 274-301, 324-369aa, respectively. |
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| Keywords: | Chicken anemia virus(CAV) structural protein(VP1 protein) monoclonal antibody VP1 epitope |
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