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, China.
Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Hefei 230036, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Oct;33(10):1422-1427.
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 pET-32a. 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.
目的 原核表达鸭坦布苏病毒（DTMUV）非结构蛋白1（NS1）并制备NS1特异性多克隆抗体。方法 通过PCR扩增DTMUV株AH-F10的NS1基因，随后亚克隆并在原核载体pET-32a中表达。重组NS1蛋白在大肠杆菌BL21（DE3）中成功表达，用羟甲基脲纯化并用梯度离心复性。用纯化的重组NS1蛋白免疫BALB/c小鼠以制备抗NS1蛋白的多克隆抗体。此外，通过琼脂扩散试验（AGP）测定多克隆抗体的效价，并通过Western印迹和间接免疫荧光试验（IFA）验证多克隆抗体的特异性。结果 成功获得血清中抗NS1蛋白的多克隆抗体，AGP效价为1:8。Western印迹和IFA表明，含有多克隆抗体的血清对DTMUV的NS1蛋白具有高度特异性和反应性。结论 本研究成功制备并验证了抗DTMUV NS1蛋白的多克隆抗体。
Zotero 插件