摘要
采用RT-PCR方法扩增了番鸭呼肠孤病毒S14株σC蛋白编码基因,将其克隆到pFastBacHTA载体上,经酶切鉴定及测序,筛选出阳性重组载体pFastBacHTA-σC;将pFastBacHTA-σC转化到DH10Bac感受态细胞中,通过蓝白斑和PCR筛选,获得重组转座子rBacmid-σC。在脂质体介导下将rBacmid-σC转染sf-9昆虫细胞,获得重组杆状病毒rBV-σC;SDS-PAGE和Western-blot分析表明,在sf-9细胞中表达了分子质量约37 ku的σC蛋白,该蛋白能与原核表达的σC蛋白免疫小鼠血清发生特异性免疫反应,这为以表达的σC蛋白为抗原的亚单位疫苗的研制奠定了基础。
The σC-encoding gene of Muscovy duck reovirus strain S14 was amplified by RT-PCR and subcloned into pFastBacHTA donor plasmid. The recombinant pFastBacHTA was identified by digestion with endonuclease enzyme and sequencing. Then the purified plasmid was transformed into DH10Bac Escherichia coli for transposition into the bacmid and the clonies were verified by blue/white selection and PCR amplification. The recombinant baculovirus was transfected into sf-9 cells by CellFectin reagent. SDS-PAGE and Western-blot analysis showed that the expressed protein was 37 ku in molecular mass and exhibited specific immunological activity. These results provide foundation for development of a subunit vaccine with the expressed σC protein against Muscovy duck reovirus infection..
出处
《中国兽医科学》
CAS
CSCD
北大核心
2007年第5期374-377,共4页
Chinese Veterinary Science
基金
国家重点基础研究发展规划(973)项目(2005CB522905)
