狂犬病病毒糖蛋白单、双拷贝基因重组腺病毒穿梭载体的构建及表达  

Construction of recombinant adenoviral shuttle vectors carrying the rabies virus single G and double G genes

作　　者：谭业平[1] 祝艳蕾[1] 孙招金[1] 郭霄峰[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医学报》2010年第6期774-778,共5页Chinese Journal of Veterinary Science

基　　金：农业部公益性行业科研专项(200803014);广东省动物防疫检疫研究专项(粤农[2006]264号)

摘　　要：提取HEP-Flury株狂犬病病毒RNA,RT-PCR方法扩增糖蛋白G基因,并克隆入pMD18-T载体,进行测序鉴定和序列分析。之后双酶切下G基因,将其亚克隆入腺病毒穿梭载体pShuttle-IRES-hrGFP-1,经卡那霉素抗性、PCR、双酶切及测序鉴定,获得重组单G腺病毒穿梭载体pShuttle-G。应用PCR方法在G基因末端引入口蹄疫病毒2A自剪切肽序列,并克隆入T载体,同时构建含G基因的T载体,分别双酶切回收以上2片段并将他们定向亚克隆入pShuttle-IRES-hrGFP-1,经系列鉴定,成功构建由2A自剪切肽序列连接双G基因的重组腺病毒穿梭载体pShuttle-dG。转染以上2个表达质粒入Ad-293细胞,荧光显微镜观察到GFP的表达,斑点印迹试验结果显示G蛋白成功表达。The viral RNA was isolated from the rabies virus （RV） HEP-Flury strain,and then the glycoprotein （G） gene were amplified by RT-PCR and cloned into pMD18-T plasmid.The G gene was then double digested and subcloned into adenoviral shuttle vector pShuttle-IRES-hrGFP-1.The recombinant shuttle vector pShuttle-G carrying G gene was identified by kanamycin resistance,PCR,restriction endonuclease and sequencing.Moreover,the 2A self-shearing peptide gene of foot-and-mouth disease virus （FMDV） was added to the end of G gene by PCR strategy and cloned into T vector.The G＋2A sequence and G gene were respectively cleaved from the pMD18-T plasmids by double restriction endonuclease,and then subcloned directedly into the shuttle vector pShuttle-IRES-hrGFP-1.The recombinant shuttle vector pShuttle-dG carrying double G genes was obtained after being selected by kanamycin resistance,and identitied by restriction endonuclease and sequencing.After transfecting into Ad-293 cells with the above two expression plasmid respectively,GFP expression was observed using fluorescence microscopy,Dot-blot results showed the success of glycoprotein expression.

关 键 词：狂犬病病毒 糖蛋白基因 腺病毒穿梭载体 

分 类 号：S852.65[农业科学—基础兽医学]