Yazdani Fereshteh, Bakhshesh Mehran, Esmaelizad Majid, Sadigh Zohre Azita
Department of Animal Virology, Research and Diagnosis, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Organization Extension (AREEO), Karaj, Iran.
Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Organization Extension (AREEO), Karaj, Iran.
Vet Res Forum. 2017 Summer;8(3):209-213. Epub 2017 Jun 15.
Bovine ephemeral fever is an acute and arthropod-borne viral disease of cattle and water buffalo which occurs seasonally in most of the world tropical and subtropical regions. The epizootic feature of the disease has been reported in Iran with serious economic consequences. The surface glycoprotein G of bovine ephemeral fever virus (BEFV) is composed of 4 antigenic sites (G1-G4) and plays the main role for eliciting neutralizing antibodies and protective immunity. The G1 - epitope is a linear antigenic site and conserved among BEFV strains. In order to develop an ELISA test based on G1-epitope as coating antigen, this study was carried out to express the recombinant G1-epitope of BEFV in prokaryotic system. Using PCR and specific primers, a length of 88 amino acid of the G glycoprotein of BEFV including G1- epitope was amplified and cloned into the expression vector pGEX-4T-1, with the GST moiety. The recombinant plasmid (pGEX-4T-1-G1) was then transformed into BL and expression of fusion protein was induced by 0.10 mM IPTG. The maximum expression of the fusion protein was obtained at 16 hr post induction as verified by SDS-PAGE electrophoresis, and it was also confirmed that this protein bearing G1- epitope is sufficiently biologically active to bind to anti-BEFV serum in western blot experiment.
牛流行热是一种由节肢动物传播的牛和水牛急性病毒性疾病，在世界上大多数热带和亚热带地区呈季节性发生。伊朗已报告了该病的流行特征，并造成了严重的经济后果。牛流行热病毒（BEFV）的表面糖蛋白G由4个抗原位点（G1-G4）组成，在引发中和抗体和保护性免疫方面起主要作用。G1表位是一个线性抗原位点，在BEFV毒株中保守。为了开发一种以G1表位作为包被抗原的ELISA检测方法，本研究开展了在原核系统中表达BEFV重组G1表位的工作。使用PCR和特异性引物，扩增出BEFV G糖蛋白包括G1表位的88个氨基酸长度，并将其克隆到带有GST部分的表达载体pGEX-4T-1中。然后将重组质粒（pGEX-4T-1-G1）转化到BL中，用0.10 mM IPTG诱导融合蛋白表达。SDS-PAGE电泳验证在诱导后16小时获得融合蛋白的最大表达，并且在蛋白质印迹实验中也证实该带有G1表位的蛋白具有足够的生物活性以与抗BEFV血清结合。
