转运真核表达盒的重组T7噬菌体构建  

Construction of recombinant bacteriophage T7 delivering eukaryotic expression box

作　　者：徐海[1] 鲍熹[1] 王义伟[1] 卢宇[1] 许梦薇 侯继波[1] 

机构地区：[1]江苏省农业科学院/国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《江苏农业学报》2015年第1期117-121,共5页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(11)4073]

摘　　要：为了构建转运真核表达盒的重组T7噬菌体,并分别在体外、体内条件下检测其标签蛋白质EGFP的表达。以T7噬菌体基因组左侧578 bp处为插入位点,取上游400 bp和下游200 bp片段作为左右同源臂,并在其中插入表达EGFP基因的真核表达盒(EEB),构建同源重组质粒p UC-L-EEB-R。将该质粒载体转化T7噬菌体宿主细菌BL21,在噬菌体繁殖过程中完成同源重组,PCR筛选重组T7-EEB噬菌体。提取该重组噬菌体基因组转染Vero细胞后体外检测EGFP蛋白质表达,纯化的噬菌体免疫小鼠后体内检测EGFP蛋白质表达。结果显示,通过同源重组方法成功构建了携带真核表达盒的重组T7噬菌体,PCR检测和酶切鉴定均证明表达盒已正确插入。T7-EEB基因组转染真核细胞可见明显的EGFP蛋白质表达,免疫小鼠后活体荧光检测到EGFP蛋白质信号,在小鼠肝脏、脾脏组织中RT-PCR检测到EGFP基因的mRNA转录。表明同源重组方法可以用于构建重组T7噬菌体,噬菌体能够转运真核表达盒并实现蛋白质表达。To construct a recombinant T7 phage delivering eukaryotic expression box and to detect the EGFP（ enhanced green fluorecent protein） tag protein expression in vivo and in vitro,respectively,a 400-bp fragment（ L） located at the upstream of 578 bp of T7 genome and a 200-bp fragment（ R） located at the downstream were used as homologous arms.Eukaryotic expression box encoding EGFP protein（ EEB） was inserted into left and right arm to construct homologous recombinant plasmid vector p UC-L-EEB-R. Plasmid vector was transformed into bacterium BL21 where homologous recombinant took place during the T7 phage propagation. EGFP protein expression was identified by phage genome transfection and phage particle immunization. PCR and restriction enzyme digestion analysis demonstrated that eukaryotic expression box was inserted into T7 phage genome correctly. Visible EGFP protein expression was detected both in vivo and in vitro. In mice liver and spleen,mRNA transcription of EGFP gene was detected by RT-PCR as well. These results indicate that homologous recombinanation is capable to construct recombinant T7 phage which could deliver eukaryotic expression box and achieve protein expression.

关 键 词：同源重组 DNA疫苗 真核表达 T7噬菌体 

分 类 号：Q939.48[生物学—微生物学]