狂犬病毒CVS株G基因和IFN-a基因共表达重组腺病毒载体的构建  

Construction of recombinant adenoviral vector co-expressing G gene of RV and IFN-a gene

作　　者：李江涛[1,2] 殷相平[1] 柳纪省[1] 胡永浩[3] 

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 [2]湖州市农业科学研究院,浙江湖州313000 [3]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《甘肃农业大学学报》2008年第6期1-5,共5页Journal of Gansu Agricultural University

基　　金：甘肃省自然基金项目(3Z S051-A25-076);甘肃省科技攻关(2GS0S2-A41-006-02).

摘　　要：通过RT-PCR方法扩增得到狂犬病毒G基因,PCR方法得到IFN-Αa基因并亚克隆入pIRES获得pIRES-a质粒.双酶切G基因和pIRES-a质粒并回收目的基因与腺病毒穿梭载体pAdTrack-CMV连接,再与pAdEasy-1质粒在BJ5183菌中同源重组产生腺病毒载体质粒.线性化后的重组腺病毒质粒转染人胚肾293细胞,通过观察报告基因绿色荧光蛋白的表达鉴定重组的腺病毒.结果表明:该重组病毒质粒经酶切鉴定与预期结果一致;转染293细胞后观察到绿色荧光蛋白的表达,说明成功构建了G和IFN-a双基因共表达重组腺病毒载体.To construct adenoviral vector co-expressing G gene of RV and IFN-a gene by utilizing an internal ribosome entry site （IRES） sequences to link the two genes,the G gene of RV was obtained by reverse transcriptase-polymerase chain reaction （RT-PCR）,and the IFN-a gene was amplified by PCR from a T-vector containing IFN-a cDNAs. The DNA fragments were inserted into T vector and sequenced, then subcloned IFN-a gene into pIRES. The amplification product of G gene and the plasmid of pIRES-a were digested with double restriction endonuclease,and then simultaneously cloned into shuttle vector pAdTrack-CMV. Then the recombinant adenoviral plasmid was obtained by homologous recombinant of the linearized shuttle plasmid and pAdEasy-1 backbone vector in E. coll. Finally the recombinant adenoviral plasmid was digested with Pac I and transfected into cell 293 to pack the adenovirus. Strong green fluorescence were observed by fluorescence microscope. The results showed that the recombinant adenovirus containing G gene and IFN-a gene were successfully constructed, thus provided a basis for the research of recombinant replication-defective adenovirus based RV vaccine.

关 键 词：狂犬病病毒G基因 IFN—a基因 内部核糖体进入位点序列 重组腺病毒 

分 类 号：S852.655[农业科学—基础兽医学]