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)膜蛋白(M)基因进行扩增、克隆和序列测定,并与GenBank中的其它25株参考株的M基因进行比对分析并构建进化树,研究其遗传进化关系。另将M基因克隆至杆状病毒表达系统pFastBac1载体并转染Sf9昆虫细胞进行表达,经间接免疫荧光鉴定,用琼扩试验验证表达产物的反应性,以研究M基因表达产物的生物学活性。测序结果表明DS10的M基因全长为678bp,编码225个氨基酸。遗传进化分析表明,DS10病毒与其它不同致病型的传染性支气管炎病毒共属同一大分支。间接免疫荧光检测表明,M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,以上结果表明所表达的M蛋白具有良好的生物学活性。本研究表达的M蛋白可为其生物学活性研究及其诊断抗原、新型疫苗开发提供基础材料。
To study the phylogenesis of M gene of nephropathogenic infectious bronchitis virus strain Ck/Jiangsu/DS10/2008（DS10）,the M gene was amplified by reverse transcriptase-PCR,cloned into pGEM-T easy vector and sequenced. Phylogenic tree was constructed with other 25 reference viruses which accessed from GenBank. Subsequently,M gene was cloned and inserted into the pFastBac1 vector of Bac-to-Bac baculovirus expression system and transfected into Sf9 cell line. The expression and biological activity were detected by indirect immunofluorescent assay （IFA） and agar immunodiffusion,respectively. The results showed that ORF of M gene was 678 bp and coded 225 amino acids,and DS10 belonged to a subgroup which composed of various pathogenic type viruses. The results of IFA and agar immunodiffusion showed that M protein was expressed in Sf9 cell line and had powerful biological activity. The expressed M protein in this study provided material to study its biological activity and development of diagnosis antigen and subunit vaccine.
出处
《中国家禽》
北大核心
2010年第24期20-23,共4页
China Poultry
基金
江苏省农业科技自主创新项目(cx10(450))
国家公益性行业(农业)科研专项(20080320)
关键词
传染性支气管炎病毒
M基因
遗传进化
杆状病毒表达系统
表达
infectious bronchitis virus
M gene
phylogenesis
Bac-to-Bac baculovirus expression system
expression
