脊髓灰质炎病毒Sabin2株结构蛋白VP1在昆虫杆状病毒表达系统中的表达  

Expression of Structural Protein VP1 of Poliovirus Sabin2 Strain in Insect Baculovirus Expression System

作　　者：刘金花[1] 董关木[1] 安祺[1] 曹守春[1] 孔艳[1] 

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2011年第6期625-628,共4页Chinese Journal of Biologicals

基　　金：国家科技支撑计划(2008BAI54B03)

摘　　要：目的在昆虫杆状病毒表达系统中表达脊髓灰质炎病毒(Poliovirus,PV)Sabin2株结构蛋白VP1。方法从Sabin2疫苗原液中RT-PCR扩增PV结构蛋白VP1基因,克隆入pFastBac1质粒,转化含杆状病毒穿梭载体Bacmid的E.coliDH10Bac,获得重组杆状病毒表达质粒Bacmid-VP1,在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒rBac-VP1。将第2代rBac-VP1感染sf9细胞,间接免疫荧光法检测VP1蛋白的表达,并优化感染条件。结果重组杆状病毒表达质粒Bacmid-VP1转化子可扩增出3 203 bp的目的片段;第2代rBac-VP1的滴度为6×107 pfu/ml,其感染的sf9细胞在荧光显微镜下可见绿色荧光,以不同MOI的rBac-VP1感染sf9细胞,VP1蛋白表达量差别不大,而在96 h内,随着感染时间的延长,VP1蛋白的表达量逐渐升高。结论在昆虫杆状病毒表达系统中成功表达了PV Sabin2株结构蛋白VP1,为脊髓灰质炎亚单位疫苗的研制奠定了基础。Objective To express the structural protein VP1 of poliovirus（PV） Sabin2 strain in insect baculovirus expression system.Methods VP1 gene was amplified by RT-PCR from the bulk of PV vaccine prepared with Sabin2 strain,and cloned into vector pFastBac1.The constructed recombinant plasmid pFast-VP1 was transformed to E.coli DH10Bac carrying baculovirus shuttle plasmid Bacmid,and the obtained recombinant plasmid Bacmid-VP1 was transfected to sf9 insect cells in mediation of liposome to prepare recombinant baculovirus rBac-VP1.The sf9 cells were infected with rBac-VP1 of passage 2 and determined for expression of VP1 by IFA,based on which the condition for infection was optimized.Results The target gene fragment at a length of 3 203 bp was amplified from the transformants of Bacmid-VP1.The rBac-VP1 of passage 2 reached a titer of 6 × 107 pfu/ml,with which the infected sf9 cells showed green fluorescence under fluorescent microscope.The expression levels of VP1 in sf9 cells infected with rBac-VP1 at various MOIs showed no significant difference,which increased gradually with the increasing time for infection within 96 h.Conclusion The structural protein VP1 of PV Sabin2 strain was successfully expressed in insect baculovirus expression system,which laid a foundation of development of poliovirus subunit vaccine.

关 键 词：脊髓灰质炎病毒 Sabin2株 VP1蛋白 杆状病毒 

分 类 号：R373.22[医药卫生—病原生物学] Q786[医药卫生—基础医学]