肠道病毒71型类病毒颗粒在昆虫细胞Sf9中的表达  

Expression of Virus-like Particles of Enterovirus 71 in Insect Sf9 Cells

作　　者：徐婧雯[1] 李鸿钧[1] 谢天宏[1] 张光明[1] 尹娜[1] 孙茂盛[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,昆明650118

出　　处：《中国生物制品学杂志》2011年第5期513-516,521,共5页Chinese Journal of Biologicals

摘　　要：目的构建含有肠道病毒71型(Enterovirus 71,EV71)P1和3CD基因的嵌合型重组杆状病毒质粒,探讨3CD蛋白酶对P1蛋白的切割作用及形成类病毒颗粒(Virus-like particles,VLPs)的可能性。方法将EV71的P1和3CD基因克隆至同一pFastBac Dual质粒上,转化感受态大肠杆菌DH10,构建重组杆状病毒质粒Bac-P1-3CD,转染昆虫细胞Sf9,制备重组杆状病毒,经SDS-PAGE、Western blot及电镜对目的基因表达产物进行分析。结果重组杆状病毒质粒Bac-P1-3CD经PCR鉴定证明构建正确。重组杆状病毒感染的Sf9细胞经SDS-PAGE分析,可见相对分子质量约39 000、32 000和26 000的特异条带,大小与EV71的VP1、VP0、VP3蛋白相符;Western blot分析可见与EV71 VP1蛋白大小相近的特异条带;电镜观察可见EV71类病毒颗粒。结论 EV71的P1和3CD嵌合基因可以通过重组杆状病毒在昆虫细胞中表达,3CD蛋白酶可以切割P1蛋白得到VP1抗原,并能形成类病毒颗粒。Objective To construct a chimeric baculovirus plasmid carrying P1 and 3CD genes of enterovirus 71（EV71） and investigate the cleavage of P1 protein with 3CD protease as well as the possibility of formation of virus-like particles（VLPs）.Methods The P1 and 3CD genes of EV71 were cloned into plasmid pFAStBac Dual,and the constructed shuttle plasmid pFastBac Dual-P1-3CD was transformed to E.coli DH10.The obtained recombinant baculovirus plasmid Bac-P1-3CD was transfected to Sf9 cells to prepare recombinant baculovirus,and the expressed products of target genes were identified by SDS-PAGE,Western blot and electron microscopy.Results PCR proved that recombinant baculovirus plasmid Bac-P1-3CD was constructed correctly.Specific protein bands with relative molecular masses of 39 000,32 000 and 26 000 were observed on SDS-PAGE profile of Sf9 cells infected with recombinant baculovirus,which were consistent with those of VP1,VP0 and VP3 of EV71.Western blot showed a specific protein band with similar relative molecular mass to that of VP1 of EV71.The VLPs of EV71 were observed by electron microscopy.Conclusion The P1 and 3CD genes of EV71 may be expressed in insect cells using recombinant baculovirus.3CD protease may digest P1 into VP1 antigen and form VLPs.

关 键 词：肠道病毒A型 人 P1基因 3CD基因 杆状病毒科 类病毒 病毒粒子 

分 类 号：R373.2[医药卫生—病原生物学] Q786[医药卫生—基础医学]