基于原核表达鸭瘟病毒gC蛋白建立的表面等离子共振检测技术的研究

Identification of duck plague based on surface plasmon resonance with DPV gC prokaryotic expression protein

本研究旨在应用表面等离子共振技术(surface plasmon resonance,SPR)建立鸭瘟血清的SPR检测方法。PCR扩增鸭瘟g C基因全长,亚克隆于原核表达载体pET32a中,诱导、表达、纯化鸭瘟g C蛋白,将其作为捕获蛋白偶联芯片,建立了鸭瘟的SPR检测方法。敏感性、特异性和实用性分析结果显示,该SPR方法仅与鸭瘟阳性血清有明显反应,而对鸭流感病毒、番鸭细小病毒、传染性喉气管炎病毒、小鹅瘟病毒、马立克病毒、鸭源沙门菌和鸭源大肠杆菌等血清为阴性反应;倍比稀释鸭瘟阳性血清,SPR的灵敏度可达0.1?g;对留存的19份疑似鸭瘟血清的SPR检测与商业化ELISA的检测结果完全一致。上述结果表明,本研究建立的表面等离子共振技术具有灵敏度高、特异性强、稳定性好、样品免标记等特点,适用于鸭瘟血清的快速检测。

Based on surface plasmon resonance(SPR),a method to detect duck plague serum was established.PCR amplification of duck plague virus g C gene was subcloned into prokaryotic expression vector p ET32 a,then the expressed 65 ku fusion proteins were purified and coupled to SPR Chip.The sensitivity and specificity of the SPR were analyzed.The results showed that the assay was able to accurately detect duck plague virus serum and no cross reactivity with duck influenza virus,Muscovy duck parvovrius,infectious laryngotracheitis,goose parvovirus,Marek’s disease virus,Salmonella and Escherichia coli serum.Detection limit of the SPR was 0.1?g.19 suspected duck plague serum samples were tested and in result the developed SPR is 100%consistent with the ELISA method.The above mentioned results showed that the SPR combined with DPV g C expressed protein has high sensitivity,reliability and stability and that it can be used to detect duck plague serum.