Development and Preliminary Application of an Indirect ELISA to Detect Infectious Bovine Rhinotracheitis Virus Using Recombinant Glycoprotein D of IBRV Strain SD
Development and Preliminary Application of an Indirect ELISA to Detect Infectious Bovine Rhinotracheitis Virus Using Recombinant Glycoprotein D of IBRV Strain SD
复制标题
IBRV SD株重组糖蛋白D间接ELISA检测传染性牛鼻气管炎病毒的建立及初步应用
DOI：
10.9775/kvfd.2015.14890
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发表时间：
2016-07-01
影响因子：
0.7
通讯作者：
He, Hongbin
中科院分区：
文献类型：
--
作者：
Song, Lingling;Zhang, Hui;He, Hongbin
糖蛋白D（gD）是牛传染性鼻气管炎病毒（IBRV）的主要结构蛋白。它能诱导体液免疫和细胞免疫，是制备IBR诊断试剂的理想蛋白。在DNAstar分析中，以IBRV基因组DNA为模板，通过PCR扩增gD片段的主要抗原区，并将其构建到重组质粒pET 32a-gD中。经IPTG诱导表达融合蛋白。经SDS-PAGE和Western-blotting鉴定后，用Ni-NTA试剂盒进行镍离子亲和层析纯化，并将其作为包被抗原，建立了牛传染性鼻气管炎病毒（IBRV）抗体的间接ELISA检测方法。交叉反应性检测表明，重组抗原与其他常见病毒性疾病（牛流行热、牛病毒性腹泻、牛肠道病毒感染、牛冠状病毒病）阳性血清无交叉反应，特异性强。对1315份临床血清进行检测，抗体阳性率为23.7%（311/1315），与商品化的IBRV全病毒ELISA法比较，符合率为96.8%。该方法稳定、灵敏，为国内IBR流行病学和疾病鉴定提供了一种快速、简便的血清学诊断方法。
Glycoprotein D (gD) is the major structural protein of infectious bovine rhinotracheitis virus (IBRV). It can induce both humoral and cellular immunity thus it is a preferable protein for IBR diagnostic reagent. Regarding to DNAstar analysis, the major antigenic region of the gD fragment was amplified by PCR using the IBRV genomic DNA as a template, and subsequenly constructed into the recombinant plasmid pET32a-gD. The fusion protein was expressed upon IPTG induction. The fusion protein was purified by immobilized Ni ion affinity chromatography with a Ni-NTA Kit after verification by SDS-PAGE and Western-blotting analysis, then was utilized as a coating antigen to detect antibodies to infectious bovine rhinotracheitis virus (IBRV) in an indirect ELISA method. Cross-reactivity examinations have showed that the recombinant antigen had no cross reaction with positive sera of other common viral diseases (bovine ephemeral fever, bovine viral diarrhea-mucosal disease, calf diarrhea, bovine intestinal virus infection, bovine coronavirus disease) which indicating a strong specificity. Application of this diagnostic method in 1315 clinical serum samples displayed an antibody positive rate of 23.7% (311/1315), with respect to a coincidence rate of 96.8% as compared to a commercialized IBRV whole virus ELISA. Our method is stable and sensitive hence provides a quick and convenient serological diagnosis favoring epidemiology and disease identification of domestic IBR.