用于构建双价禽流感DNA疫苗的双目的基因表达质粒的构建及鉴定  

Construction and evalution of the plasmids expressing two target gene for bivalent avian influenza DNA vaccine developoment

作　　者：黄冬[1] 陈普成[1] 刘兵[1] 唐猛[1] 柳金雄[1] 姜永萍[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2016年第4期257-261,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金："十二五"农村领域国家科技计划(2015BAD12B03/001)

摘　　要：为探索构建禽流感双价或多价DNA疫苗的可行性,本研究以本实验室前期研制的禽流感DNA疫苗p CAGGopti HA5为基础[其中含有禽流感病毒(AIV)A/Goose/Guang Dong/1/96(H5N1)的HA基因],将增强型绿色荧光蛋白(eGFP)报告基因作为第二个目的基因,利用双启动子启动、内部核糖体进入位点(IRES)介导以及通过蛋白Linker编码序列连接融合表达两个目的基因的形式,构建了双启动子重组表达质粒pH5-eGFP、IRES介导的重组表达质粒pHIE以及融合表达重组质粒pH6E。并采用脂质体介导将这3种重组质粒分别转染293T细胞,通过间接免疫荧光试验和western blot方法检测HA和eGFP这两种目的蛋白的瞬时表达情况。结果显示,这3种重组质粒均能够正确表达AIV的HA蛋白和eGFP。其中pH5-eGFP表达出两个独立的目的蛋白,而且互相不影响各自的表达和细胞定位;pH6E则以融合形式表达两种目的蛋白,由于HA蛋白具有膜定位信号,所以融合蛋白主要定位于细胞膜上。本研究结果表明,双启动子表达质粒和双目的蛋白融合表达质粒适合用于构建AIV以及其他相关病原的双价DNA疫苗。To explore the feasibility to construct avian influenza bivalent or multivalent DNA vaccines, three recombinant plasmids expressing two target genes were constructed by inserting the gfp gene into the avian influenza DNA vaccine of p CAGGopti HA5 developed in our previous studies, which containing the HA gene of avian influenza virus（AIV） A/Goose/Guang Dong/1/96（H5N1）. Strategies for construction of the 3 recombinant plasmids expressing the two target genes included pH5-eGFP by introducing secondary promoter for gfp gene expressing, p HIE by ligating the internal ribosome entry site（IRES）between the two genes, and pH6 E by fusing the two genes with protein linker coding sequence to express the HA-eGFP fusion protein. Then the recombinant plasmids were transfected into 293 T cells by cationic liposome method, respectively, and the expressions of HA and eGFP were detected by immunofluorescence assay and western blot. As the results, the HA and eGFP were independently expressed from pH5-eGFP transfected cells without interference each other for expression and cellular sub-locationsin the cells. While the pH5-eGFP fusion protein was expressed from the pH6 E transfected cells and sub-located on the cell membrane due to the membrane localization signal sequence in HA protein. The results implied that the strategies by introducing additional promoter and protein linker encoding sequence for two or more target gene expression were potential to be applied in development bivalent or multivalent DNA vaccines against avian influenza or other diseases.

关 键 词：DNA疫苗 双价表达质粒 EGFP 

分 类 号：S852.65[农业科学—基础兽医学]