Dashprakash M, Venkatesan G, Kumar Amit, Sankar M, Arya Sargam, Ramakrishnan M A, Pandey A B, Mondal B
Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar, Nainital, Uttarakhand, 263 138, India.
Arch Virol. 2019 Apr;164(4):1049-1058. doi: 10.1007/s00705-019-04170-8. Epub 2019 Feb 18.
Goatpox is an economically significant transboundary viral disease of goats that is caused by goatpox virus (GTPV). This study describes the prokaryotic expression of the GTPV ORF117 protein, a homologue of vaccinia virus A27L, and evaluation of its diagnostic potential in ELISA. The GTPV ORF117 gene was cloned into the pET32a vector to express recombinant ORF117 protein (rA27L) in E. coli BL21-CodonPlus (DE3)-RIPL. The bacterial expression of the protein was confirmed by western blot analysis using anti-GTPV polyclonal antibodies that detected rA27L, which is ~ 35 kDa in size. rA27L was affinity purified under native conditions and used to assess the antibody response in an optimized indirect ELISA. The purified antigen specifically reacted with anti-GTPV and anti-SPPV serum in ELISA. A preliminary screening of random and purposive serum samples (n = 520) from sheep and goats using this optimized ELISA gave a positivity rate of 19.4 % with a diagnostic specificity of 88.7% and diagnostic sensitivity of 98.5% when compared to the gold standard serum neutralization test. Our results suggest that the indirect ELISA based on the rA27L protein has potential for serosurveillance and seromonitoring of GTPV in goats.
山羊痘是由山羊痘病毒（GTPV）引起的一种对山羊具有重要经济意义的跨界病毒性疾病。本研究描述了痘苗病毒A27L同源物GTPV ORF117蛋白的原核表达，并评估了其在酶联免疫吸附测定（ELISA）中的诊断潜力。将GTPV ORF117基因克隆到pET32a载体中，以在大肠杆菌BL21-CodonPlus（DE3）-RIPL中表达重组ORF117蛋白（rA27L）。使用检测到大小约为35 kDa的rA27L的抗GTPV多克隆抗体，通过蛋白质免疫印迹分析证实了该蛋白的细菌表达。rA27L在天然条件下进行亲和纯化，并用于在优化的间接ELISA中评估抗体反应。纯化的抗原在ELISA中与抗GTPV和抗绵羊痘病毒（SPPV）血清特异性反应。与金标准血清中和试验相比，使用这种优化的ELISA对绵羊和山羊随机和有目的采集的血清样本（n = 520）进行初步筛查，阳性率为19.4%，诊断特异性为88.7%，诊断敏感性为98.5%。我们的结果表明，基于rA27L蛋白的间接ELISA在山羊GTPV血清监测和血清学监测方面具有潜力。
