鸭坦布苏病毒E蛋白的原核表达及多克隆抗体的制备 |
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引用本文: | 王勇,殷冬冬,宫晓华,王瑞,许漩,唐井玉,兰梦蝶,王桂军,马晨,张和平.鸭坦布苏病毒E蛋白的原核表达及多克隆抗体的制备[J].中国微生态学杂志,2016(2). |
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作者姓名: | 王勇 殷冬冬 宫晓华 王瑞 许漩 唐井玉 兰梦蝶 王桂军 马晨 张和平 |
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作者单位: | 安徽农业大学动物科技学院,安徽农业大学动物科技学院,安徽农业大学动物科技学院,安徽农业大学动物科技学院,安徽农业大学动物科技学院,安徽农业大学动物科技学院,安徽农业大学动物科技学院,安徽农业大学动物科技学院,内蒙古农业大学 乳品生物技术与工程教育部重点实验室,内蒙古农业大学 乳品生物技术与工程教育部重点实验室 |
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摘 要: | 目的用RT-PCR技术扩增鸭坦布苏病毒(DTMUV)AH-F10株E基因,并克隆至pET32a(+)载体,构建重组表达质粒pET32a-E,表达E蛋白。方法重组表达质粒转化感受态细胞BL21(DE3),经IPTG诱导后,获得含6个His标签的融合蛋白,大小约54kDa。表达的蛋白以包涵体形式存在。对目的蛋白进行纯化,用纯化的E蛋白免疫Balb/c小鼠,制备多克隆抗体血清。结果SDS-PAGE和Western blot试验结果表明E基因在大肠埃希菌中成功表达,并能与抗DTMUV多克隆抗体产生特异性反应,具有良好的反应原性。间接免疫荧光试验表明免疫小鼠后获得的多克隆抗体能与DTMUV反应。结论本研究为DTMUV新型疫苗和诊断试剂盒的进一步研究奠定了基础。
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关 键 词: | 鸭坦布苏病毒 E基因 原核表达 多克隆抗体 |
Prokaryotic expression of E protein of Duck Tembusu virus and
preparation of polyclonal antibodies |
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Abstract: | Abstract: Objective To construct, express and purify the envelope protein of Duck Tembusu virus (DTMUV). Methods The E gene of Duck Tembusu virus AH-F10 strain was amplified by RT-PCR and inserted into the multiple cloning sites of pET32a(+) vector. The recombinant plasmid was transformed into BL21 (DE3). The expression of E protein was induced with IPTG. The 6His-E protein was expressed in a form of inclusion body with a molecular weight of 54 kDa. Six Balb/c mice were injected with the purified protein for preparation of E protein specific polyclonal antibodies. Results SDS-PAGE and Western blot analysis showed that the E protein was successfully expressed in BL21. Indirect immunofluorescence assay (IFA) showed that the polyclonal antibody from the inoculated mice was able to react with DTMUV. Conclusion The study laid a foundation for further study on new vaccines and diagnosis kits of DTMUV. |
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