Development and characterization of a stable Vero cell line constitutively expressing Peste des Petits Ruminants Virus (PPRV) hemagglutinin protein and its potential use as antigen in enzyme-linked Immunosorbent assay for serosurveillance of PPRV
Development and characterization of a stable Vero cell line constitutively expressing Peste des Petits Ruminants Virus (PPRV) hemagglutinin protein and its potential use as antigen in enzyme-linked Immunosorbent assay for serosurveillance of PPRV
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DOI：
10.1128/cvi.00273-06
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发表时间：
2006-12-01
影响因子：
--
通讯作者：
Singh, R. K.
中科院分区：
文献类型：
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作者：
Balamurugan, V.;Sen, A.;Singh, R. K.
我们开发并表征了组成型表达小反刍兽疫病毒 (PPRV) 血凝素 (H) 蛋白的稳定 Vero 细胞系，并评估了其在酶联免疫吸附测定 (ELISA) 中作为诊断抗原的潜在用途。通过逆转录(RT)-PCR扩增疫苗株(Sungri-96)的PPRV H基因，克隆到真核表达载体(pTarget)中，随后转染并在Vero细胞中表达。使用G418抗生素选择压力（400至600μg/ml）重复传代20次后形成稳定的Vero细胞系。分别通过PCR和RT-PCR测定证实了PPRV H基因在Vero细胞基因组中的整合及其基因组转录，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western blotting对70-kDa PPRV H蛋白进行了表征。重组蛋白分别在竞争性、夹心和间接 ELISA 中与 PPRV 抗 H 中和单克隆和多克隆抗体发生特异性反应，表明该蛋白的天然形式得到表达。分别使用 306 份和 146 份山羊田血清样品，在竞争性 ELISA 和间接 ELISA 中对全病毒进行蛋白质评估；获得了具有高度相对诊断特异性（分别为 93.53% 和 100%）和敏感性（分别为 99.04% 和 79.16%）的可比较结果。这项研究表明，PPRV H 蛋白可以成为非流行国家的安全抗原的可持续来源，而无需处理感染性病毒进行血清学诊断。
We developed and characterized a stable Vero cell line constitutively expressing Peste des petits ruminants virus (PPRV) hemagglutinin (H) protein and assessed its potential use as diagnostic antigen in enzyme-linked immunosorbent assay (ELISA). PPRV H gene of the vaccine strain (Sungri-96) was amplified by reverse transcription (RT)-PCR, cloned into a eukaryotic expression vector (pTarget), and subsequently transfected and expressed in Vero cells. A stable Vero cell line was developed after 20 repeated passages by using G418 antibiotic selection pressure (400 to 600 mu g/ml). The integration of PPRV H gene in the Vero cell genome and its genomic transcription were confirmed by PCR and RT-PCR assays, respectively, and the 70-kDa PPRV H protein was characterized by sodium dodecyl sulfate-pollyacrylamide gel electrophoresis and Western blotting. The recombinant protein reacted specifically with PPRV anti-H neutralizing monoclonal and polyclonal antibody in competitive, sandwich, and indirect ELISA, respectively, indicating that the native form of the protein was expressed. Evaluation of the protein in competitive ELISA and indirect ELISA vis a vis whole virus was done using 306 and 146 goat field serum samples, respectively; comparable results were obtained with high degrees of relative diagnostic specificity (93.53% and 100%, respectively) and sensitivity (99.04% and 79.16%, respectively). This study shows that the PPRV H protein could be a sustainable source of safe antigen in countries of nonendemicity without the need to handle infectious virus for serodiagnosis.