【摘要】 对肾型鸡传染性支气管炎病毒分离株Ck/Jiangsu/DS10/2008株(DS10株)S基因进行扩增、克隆和序列测定,并与GenBank中的其他25株参考株构建进化树进行比对分析,研究其遗传进化关系。另将S基因克隆至杆状病毒表达系统pFastBac 1载体并转染Sf9昆虫细胞进行表达,经间接免疫荧光鉴定其表达,用琼扩试验验证表达产物的反应性。S基因序列分析表明,S基因高变区域主要集中在157～230位、349～433位、787～886位之间,裂解位点序列为SHRRRRμSI。遗传进化分析表明,DS10与Vic株处于同一分支,而与H120等常用疫苗株相距较远。经间接免疫荧光检测表明,S蛋白在Sf9细胞中得到表达,琼扩试验验证表达产物具有良好的反应性,表明所表达的S蛋白具有良好的生物学活性。本研究表达的S蛋白可为传染性支气管炎诊断抗原的研制、新型疫苗的开发等提供基础材料。更多还原

【Abstract】 The spike(S) gene of nephropathogenic infectious bronchitis virus strain Ck/Jiangsu/DS10/2008(DS10) was amplified by reverse transcriptase(RT)-PCR,cloned into pMD-19T easy vector and sequenced.Phylogenic tree was constructed with other 25 reference viral strains published in GenBank.Subsequently,S gene was cloned and inserted into the pFastBac 1 vector of Bac-to-Bac baculovirus expression system and transfected into Sf9 cell line.The expression of S gene and reactivity of S protein were detected by indirect immunofluorescentassay(IFA) and agar immunodiffusion,respectively.Analysis of the S gene showed that the hypervariable regions were located at 157 bp to 230 bp,349 bp to 433 bp and 787 bp to 886 bp,and the motif of cleavage site was SHRRRR μSI.The results of phylogenesis analysis based on the S1 gene showed that DS10 was in the same branch with Vic strain,but far apart from commonly used vaccine strain such as H120 strain.The results of IFA and agar immunodiffusion showed that S protein was expressed in Sf9 cell line and had a strong reactivity to positive serum.The expressed S protein in this study provided the materials for development of diagnostic antigen and subunit vaccine.更多还原