O型FMDVP1-2A基因与猪IFN-γ基因共表达质粒的构建及其在BHK-21细胞中的表达  

Construction of Co-expressing Plasmid of FMDV P1-2A Gene with Porcine IFN-γ Gene and Expression in BHK-21 Cells

作　　者：吴国华[1] 张强[1] 颜新敏[1] 侯俊玲[1] 赵志荀[1] 崔力凡[1] 李健[1] 朱海霞[1] 朱彩珠[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,甘肃兰州730046

出　　处：《华北农学报》2010年第5期11-15,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家科技支撑项目(2006BAD06A11);甘肃省重大科技项目(092NKDA032)

摘　　要：为了构建O型FMDVP1-2A基因与猪IFN-γ基因共表达质粒并在BHK-21细胞中进行表达,本研究利用聚合酶链式反应(PCR)从阳性质粒pGEM-P12A克隆到P1-2A基因,将扩增产物纯化、双酶切后连接到pBudCE-IFN载体,构建重组质粒pBudCE-IFN-P1-2A。重组表达质粒鉴定后,在脂质体介导下转染BHK-21细胞,用RT-PCR和直接免疫荧光试验分别检测P1-2A和IFN-γ在BHK-21细胞中的表达。RT-PCR检测显示,重组表达质粒转染细胞后P1-2A和IFN-γ基因成功转录出mRNA;免疫荧光试验表明目的基因在BHK-21细胞中获得了表达。结果证明,本研究成功构建了重组质粒pBudCE-IFN-P1-2A并在体外进行了表达,这为研制抗FMDVDNA疫苗和IFN-γ的佐剂作用奠定了坚实基础。To construct the co-expressing plasmid of FMDV P1-2A gene with porcine IFN-γ gene and express in BHK-21 ceils. A P1-2A gene of foot-and-mouth disease virus（FMDV）was amplified by PCR from the plasmid pGEM- P12A and subcloned into pBudCE-IFN vector for sequencing after purification and digestion. A recombinant plasmid pBudCE-IFN-P1-2A was constructed and transformed into BHK-21 cells with liposome for in vitro expression. 48 hours after transfection,the mRNAs and expressed proteins of P1-2A and IFN-γ were detected by RT-PCR and direct immunofluorescence assay, respectively. The results showed that P1-2A and IFN-T were successfully transcripted and expressed in BHK-21 cells. It was proved that the recombinant plasmid pBudCE-IFN-PI-2A was successfully constructed and expressed in vitro,providing a solid basis for development of DNA vaccine against FMDV.

关 键 词：FMDV P1—2A基因 IFN-Γ基因 共表达 

分 类 号：Q78[生物学—分子生物学]