表面展示猪圆环病毒2型衣壳蛋白的重组杆状病毒的构建  

Construction of Recombinant Baculovirus Surface-Displayed the Capsid Protein of Porcine Circovirus Type 2

作　　者：程晓亮[1] 林文耀[1] 叶煜[1] 陈筱薇[1] 严常燕[1] 廖明[1] 樊惠英[1] 

机构地区：[1]华南农业大学兽医学院,农业部动物疫病防控重点实验室,广东广州510642

出　　处：《华南农业大学学报》2011年第4期96-100,共5页Journal of South China Agricultural University

基　　金：863计划(2006AA10A205);教育部新世纪优秀人才支持计划(NCET-06-0752);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)

摘　　要：利用PCR方法扩增猪圆环病毒2型核定位信号区缺失的Cap蛋白基因,将其亚克隆入杆状病毒表面展示质粒pBACsurf-1的gp64信号肽和gp64成熟蛋白之间.将此融合片段亚克隆到质粒pcDNA3.1(+),获得重组质粒pcDNA3.1-gp64-dCap.然后将含有CMV启动子,gp64及dCap的基因片段克隆到杆状病毒转移质粒pFastBac-V,得到重组质粒pFastBac-dCap-V.然后将此转化DH10Bac感受态细胞,获得的重组穿梭质粒Bacmid-dCap-V,经脂质体转染Sf9细胞,获得重组杆状病毒Ac-dCap-V.该重组病毒感染Sf9细胞后可以产生典型的细胞病变,转导哺乳动物细胞后经间接免疫荧光试验证明,该重组杆状病毒成功转导哺乳动物细胞并表达dCap蛋白.The open capsid （Cap）protein gene without nuclear localization signal （NLS） of porcine circovirus 2 （PCV2）was amplified by PCR and subcloned into pBACsurf-1 between the upstream gp64 signal sequence and downstresm gp64 mature domain. The fusion gene containing dCap and gp64 was inserted into pcDNA3.1 （ ＋ ） to construct the recombinant plasmid peDNA3.1-gp64-dCap. A fragment of the CMV promoter-gp64-dCap fusion gene cassette was excised from pcDNA3. 1-gp64-dCap and inserted into the baculovirus transfer vector pFastBac-V to construct the recombinant plasmid pFastBac-dCap-V. It was followed by the transformation of plasmid into Escherichia coli DH10Bae competent cells to obtain the recom- binant shuttle vector Bacmid-dCap-V. Eventually, the recombinant Baemid was transfected into Sf9 cells to produce the recombinant baeulovirus Ac-dCap-V using the LipofectamineTM 2000. The Sf9 cells produced cytopathic after infection of recombinant baculoviruses Ac-dCap-V. Indirect immunofluoresent assay demonstrated that the Ac-dCap-V efficiently transducted the mammalian cells in vitro and the dCap protein was expressed successfully.

关 键 词：杆状病毒表面展示系统 猪圆环病毒2型 dCap基因 

分 类 号：S852.65[农业科学—基础兽医学]