传染性支气管炎病毒非结构蛋白nsp5在真核细胞中的重组表达及鉴定

摘要

本文以嗜肾型鸡传染性支气管炎病毒（Infectious bronchitis virus,IBV）毒株的RNA为模板,通过RT-PCR扩增获得IBV非结构蛋白5（non-structural protein,nsp5）基因片段后,构建了杆状病毒重组质粒IBVnsp5-Bacmid。IBVnsp5-Bacmid转染Sf9细胞,获得nsp5重组杆状病毒,经（immunofluorescence assay,IFA）和Western blot检测到转染细胞表达的nsp5蛋白。进一步从感染的细胞中纯化重组蛋白,并用纯化的蛋白免疫小鼠制备了抗nsp5的多抗血清,该多抗血清可检测到IBV四川株SC021202感染的DF-1细胞中特异性的nsp5蛋白。结果表明IBV nsp5在Bac-to-Bac真核表达系统中获得了成功表达,而且具有良好的免疫原性和反应原性。%Little is known about the function of non-structural proteins 5（nsp5） of avian infectious bronchitis virus（IBV）.In this study,IBV nsp5 was amplified in RT-PCR using RNA of nephropathogenic IBV strain as template.The recombinant plasmid IBVnsp5-Bacmid was constructed based on Bac-to-Bac baculovirus expression system and transfected into Sf9 cells.The expression of recombinant nsp5 in Sf9 cells was confirmed by IFAT and Western blot analysis.The polyclonal antiserum against nsp5 was prepared using recombinant nsp5 purified from transfected Sf9 cells and demonstrated to react with IBV SC021202 infected DF-1 cells in IFAT.The results indicated that IBV nsp5 was successfully expressed in eukaryotic expression system and had good angenicity.