摘要
根据Ⅰ型鸭肝炎病毒(DHV-I)基因组序列设计并合成一对特异性引物,通过RT-PCR方法扩增DHV-I(A66株)VP1基因。用限制性内切酶EcoRⅠ/HindⅢ消化VP1基因和转移载体pFastbacⅠ,在T4DNA连接酶作用下将二者连接构建转移质粒pFastbac-VP1。经PCR、双酶切及测序鉴定后将其转化至含穿梭载体Bacmid的感受态细胞DH10bac中,蓝白斑筛选获得重组杆状病毒穿梭质粒rBacmid-VP1,在脂质体的介导下转染昆虫细胞Sf 9,获得重组杆状病毒rvBac-VP1。SDS-PAGE分析结果表明,VP1基因在昆虫细胞中获得表达,表达产物分子量约为2.7×104。Western-blot检测结果表明,表达产物能与抗DHV-I阳性血清发生反应,具有良好的反应原性。
A pair of primers was designed and synthesized according to duck hepatitis virus（DHV） type I genome sequence logined in the GenBank.VP1 gene of A66 strain of DHV-Ⅰ was amplified by RT-PCR.The purified VP1 gene and the vector of pFastbacⅠ were digested with the restriction endonuclease of EcoRⅠ/Hind Ⅲ,then were ligated in the proper condition using the T4 DNA ligase.The donor plasmid pFastbac-VP1 was confirmed by the methods of PCR,restriction endonuclease digestion and sequencing analysis.The purified pFastbac1-VP1 was transformed into competent cells of DH10Bac contaning the shuttle vector Bacmid.The white colonies selected are the positive colonies containing the recombinant shuttle plasmid rBacmid-VP1 through the blue-white screening.rBacmid-VP1 was transfected into the Sf 9 insect cells by lipofectamine 2000.Once the cytopathic effect was found,the rvBac-VP1 can be aquired.SDS-PAGE analysis showed the VP1 gene was expressed in Sf 9 and the expressed protein＇s molecular weight is about 2.7×104.The protein can be recognized by antisera of DHV-Ⅰ,indicating that the protein had good reactiongenicity.
出处
《江苏农业学报》
CSCD
北大核心
2010年第2期364-368,共5页
Jiangsu Journal of Agricultural Sciences
