表达鸡传染性支气管病毒S1蛋白的重组假型杆状病毒的构建

Construction of a Pseudotype Baculovirus Expressing S1 Protein of Infectious Bronchitis Virus

根据鸡传染性支气管炎病毒M41株的基因序列(GenBank:AY851295.1),设计特异性引物,利用RT-PCR方法扩增其S1基因,并将扩增产物克隆到杆状病毒转移质粒pFast-VSV-G-CMV中,获得重组质粒pFast-VSV-G-CMV-S1,进一步将该重组质粒转化到DH10Bac感受态细胞中,得到重组穿梭载体Bacm id-CMV-S1,再利用脂质体转染sf9昆虫细胞,获得重组杆状病毒Ac-V-S1.间接免疫荧光试验表明,该重组杆状病毒可以转导哺乳动物细胞并表达S1蛋白.

According to the sequence of infectious bronchitis M41 （GenBank：AY851295.1） ,the S1 gene was amplified with specific primers and cloned the amplicon into the baculovirus transfer vector pFast- VSV-G-CMV to construct the recombinant plasmid pFast-VSV-G-CMV-S1. Then, the plasmid was transformed into Escherichia coli DH10Bac competent cells to obtain the recombinant shuttle vector Bacmid- CMV-SI after identifying the plasmid by digestion with restriction endonuclease and sequencing. Eventually, the recombinant Bacmid was transfected into sf9 ceils to produce the recombinant baculovirus Ac-V- S1 using the LipofectamineTM2000. Indirect immunofluoresent assay demonstrated the Ac-V-S1 could efficiently transduct the mammalian cells in vitro and the S1 gene was expressed successfully.