Graduate Institute of Animal Vaccine Technology, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuehfu Road, Neipu, Pingtung, 91201, Taiwan, Republic of China.
BMC Vet Res. 2019 Sep 2;15(1):313. doi: 10.1186/s12917-019-2059-6.
Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID assay require days of waiting time. We sought to develop a quick dot blot assay for BEFV titering.
Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli (E. coli)-expressed BEFV G1 region, and 3) E. coli-expressed BEFV N protein. Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays.
E.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering.
牛暂时热病毒（BEFV）可引起牛发热和肌肉僵硬，给牛和奶牛场带来经济负面影响。在病毒控制的灭活疫苗制造过程中，确定病毒滴度很重要，但传统方法，如蚀斑测定和 TCID 测定，需要数天的等待时间。我们试图开发一种用于 BEFV 滴度测定的快速斑点印迹分析。
为了在斑点印迹分析中检测 BEFV，制备了三种不同的 BEFV 抗原来制备针对 BEFV 的初级抗体：1）纯化的 BEFV 颗粒，2）大肠杆菌（E. coli）表达的 BEFV G1 区，和 3）E. coli 表达的 BEFV N 蛋白。结果表明，针对纯化的 BEFV 颗粒产生的抗体可以检测 BEFV 颗粒，但也显示出来自 BHK-21 细胞的蛋白质的高背景水平。针对 E. coli 表达的 BEFV G1 区产生的抗体不能在斑点印迹分析中检测到 BEFV 颗粒。最后，针对 E. coli 表达的 BEFV N 蛋白产生的抗体在斑点印迹分析中以高信噪比检测到 BEFV 颗粒。
E. coli 表达的 N 蛋白是产生抗血清的合适抗原，该抗血清可以以高信噪比检测 BEFV 颗粒。可以使用该抗血清开发一种快速且经济的 BEFV 滴度测定的斑点印迹分析试剂盒。
