抗鸭坦布苏病毒非结构蛋白1(NS1)多克隆抗体的制备和应用  

Preparation and application of polyclonal antibody against non-structural protein 1(NS1) of duck Tembusu virus

作　　者：周祺[1] 苏观志 邢雪 周晓雅[1] 石亦鹏 唐中文 宋祥军 刘红梅 王桂军[1,2] ZHOU Qi;su Guanzhi;XING Xue;ZHOU Xiaoya;SHI Yipeng;TANG Zhongwen;SONG Xiangjun;LIU Hongmei;WANG Guijun(College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036;Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Hefei 230036, China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]兽医病理生物学与疫病防控安徽省重点实验室,安徽合肥230036

出　　处：《细胞与分子免疫学杂志》2017年第10期1422-1427,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点研发计划科研专项(2016YFD0500805);兽医病理生物学与疫病防控安徽省重点实验室专项(SYZD2017002)

摘　　要：目的原核表达鸭坦布苏病毒(DTMUV)非结构蛋白1(NS1)蛋白,并制备小鼠抗NS1多克隆抗体。方法利用PCR从DTMUV AH-F10株c DNA中获得NS1基因,亚克隆至p ET-32a中进行原核表达,对表达产物进行鉴定。使用含尿素的Tris缓冲液洗去杂蛋白再梯度复性,纯化重组蛋白,并免疫小鼠制备小鼠抗NS1多克隆抗体,琼脂扩散试验测定抗体效价,Western blot法鉴定该抗体的特异性与反应原性,将抗体应用于病毒的间接免疫荧光法(IFA)检测。结果获得鼠抗NS1蛋白多克隆抗体,效价达到1∶8;Western blot结果表明该多抗血清具有良好的特异性和反应性,且可以应用于病毒的IFA检测。结论成功制备了鼠抗NS1多克隆抗体。Objective To express prokaryotically the non-structural protein 1（NS1） of duck Tembusu virus（DTMUV）and prepare NS1-specific polyclonal antibodies. Methods The NS1 gene of DTMUV strain AH-F10 was amplified by PCR,followed by subcloning and expression in the prokaryotic vector p ET-32 a. The recombinant NS1 protein was successfully expressed in Escherichia coli BL21（ DE3）,and purified with hydroxymethyl urea and renatured by gradient centrifugation.The BALB/c mice were immunized with the purified recombinant NS1 protein to prepare polyclonal antibodies against the NS1 protein. Furthermore,the titer of the polyclonal antibodies was determined by agar diffusion test（ AGP）,and the specificity of the polyclonal antibodies was verified by Western blotting and indirect immunofluorescence assay（IFA）. Results Polyclonal antibodies against NS1 protein in serum was successfully obtained with an AGP titer of 1 ∶ 8. Western blotting and IFA demonstrated that the serum with polyclonal antibodies had a high-level specificity and reactivity to the NS1 protein of DTMUV. Conclusion Polyclonal antibodies against DTMUV NS1 protein were successfully prepared and validated in this study.

关 键 词：鸭坦布苏病毒 非结构蛋白1(NS1) 多克隆抗体 原核表达 

分 类 号：S852.43[农业科学—基础兽医学] S834[农业科学—兽医学]