禽呼肠孤病毒P17蛋白基因在大肠杆菌中的表达及其ELISA检测方法的建立

用设计合成的1对跨越禽呼肠孤病毒(ARV)P17非结构蛋白基因完整开放阅读框(ORF)的特异性引物,对ARV S1133株进行了RT-PCR扩增。扩增产物与pGEX-4T-1原核表达载体连接后,转化大肠杆菌BL21感受态细胞。经0.2 mmol/LIPTG诱导,表达的融合蛋白分子质量为42.4 ku,占菌体总蛋白的34%。表达产物P17蛋白经不同浓度尿素纯化后,纯度达到85%。Western-blotting显示,纯化的P17蛋白能与感染ARV的阳性血清反应,说明其具有抗原性。以此重组蛋白为包被抗原初步建立了用于鉴别检测ARV活病毒感染与灭活疫苗免疫的SPF鸡血清的ELISA。

Expression of P17 protein gene of avian reovirus in Escherichia coli and establishment of an ELISA assay for detection of the virus

A pair of primers was designed and synthesized to amplify the whole open reading frame(ORF) of nonstructural protein P17 gene from avian reovirus(ARV) S1133 strain.The amplified product was cloned into pGEX-4T-1 prokaryotic expression vector,and transformed into Escherichia coli BL21 competent cell.The recombination was induced with 0.2 mmol/L IPTG.SDS-PAGE analysis revealed that a 42.4 ku P17 protein was expressed.The purity of urea-purified fusion protein was up to 85%.Western-blot analysis showed that the purified P17 protein could be specifically recognized by the positive sera of avian infected with ARV.An ELISA using the recombinant protein as coating antigen was established which could differentiate the sera of SPF chicken infected with wild ARV from the sera of SPF chicken immunized with live vaccine against ARV.