College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai St., Xiangfang District, 150030 Harbin, China.
J Clin Microbiol. 2010 May;48(5):1875-81. doi: 10.1128/JCM.01707-09. Epub 2010 Mar 17.
The aim of the current study was to develop a novel diagnostic test for detecting porcine reproductive and respiratory syndrome virus (PRRSV) using phage display technology. The N gene of PRRSV isolate HH08 was cloned following reverse transcription-PCR. Sequence comparison indicated that the N gene shared 96.4% homology to that of North American PRRSV (isolate VR2332) and 35.5% with that of European PRRSV (isolate LV), indicating that the PRRSV isolate was related to the North American PRRSV genotype. The bacterially expressed N protein was used as a target in a biopanning process using a phage display random peptide library. Seven phages expressing different peptides had a specific binding activity with the N protein. The putative binding motifs were identified by DNA sequencing. More importantly, the selected phages harboring specific peptides that recognize the N protein of PRRSV were able to efficiently distinguish PRRSV from other viruses in enzyme-linked immunosorbent assays.
本研究旨在利用噬菌体展示技术开发一种新型的猪繁殖与呼吸综合征病毒(PRRSV)检测诊断方法。通过反转录-聚合酶链反应(RT-PCR)克隆了 PRRSV 分离株 HH08 的 N 基因。序列比较表明,N 基因与北美的 PRRSV(分离株 VR2332)同源性为 96.4%,与欧洲的 PRRSV(分离株 LV)同源性为 35.5%,表明该 PRRSV 分离株与北美的 PRRSV 基因型有关。利用噬菌体展示随机肽库,将细菌表达的 N 蛋白作为生物淘选过程中的靶标。7 个表达不同肽段的噬菌体与 N 蛋白具有特异性结合活性。通过 DNA 测序鉴定了假定的结合基序。更重要的是,筛选到的噬菌体含有可识别 PRRSV N 蛋白的特异性肽段,在酶联免疫吸附试验中能够有效地将 PRRSV 与其他病毒区分开来。