摘要
以鸡传染性支气管炎病毒M41毒株基因序列为模板,利用PCR方法扩增膜定位信号缺失的S1基因(dS1),将其亚克隆入杆状病毒表面展示质粒pBACsurf-1中,再次将S1及gp64的基因片段克隆到杆状病毒转移质粒pFastBacTMDual,得到重组质粒pFastBac-gp64-dS1.将该重组质粒转化到DH10Bac感受态细胞中,获得重组穿梭载体Bacmid-gp64-dS1,提取重组Bacmid质粒,以PCR验证其正确性.将阳性重组Bacmid质粒利用脂质体转染Sf9昆虫细胞,获得重组杆状病毒BV-dS1.间接免疫荧光试验表明,该重组杆状病毒可以Sf9昆虫细胞膜表达鸡传染性支气管炎病毒S1蛋白.
The S1 （Spike） protein gene without membrane localization signal of infectious bronchitis virus （dS1） was amplified from infectious bronchitis virus （M41） by PCR and sub-cloned into pBACsurf-1. The fusion gene containing S1 and gp64 was then inserted into baculovirus transfer vector pFastBacTM Dual to construct the recombinant plasmid pFastBac-gp64-dS1. Then, the plasmid was transformed into Esch- erichia coli DH10Bac competent cells to obtain the recombinant shuttle vector Baemid-gp64-dS1. Extracted Baemid-gp64-dS1 was transfeeted into Sf9 cells to produce the recombinant baculovirus BV-dS1 by using LipofectamineTM 2000. Indirect immunofluorescent assay indicated that the recombinant baculovirus BV-dS1 could express S1 protein on infected Sf9 cell membrane.
出处
《华南农业大学学报》
CAS
CSCD
北大核心
2012年第3期398-402,共5页
Journal of South China Agricultural University
基金
国家自然科学青年基金(30800826)
广东省博士启动基金(8451064201001131)
农业微生物学重点实验室开发课题(20090010)
华南农业大学校长基金(5500-K08240)
