同源重组法制备口蹄疫病毒多基因重组腺病毒

Construction of Recombinant Adenovirus Vector Containing the Capsid,3C protease and 3D Polymerase Coding Regions of FMDV by Homologous Recombination

通过细菌内同源重组的方法成功构建了含有O型口蹄疫病毒P1-2A和3C蛋白酶基因和3D基因的重组腺病毒表达载体.首先将P1-2A、3C和3D基因亚克隆连接到穿梭质粒pShuttle-CMV上,再将重组穿梭质粒用PmeI线性化后电转化携带有腺病毒骨架载体pAdeasy-1的大肠杆菌BJ5183感受态菌,经细菌内同源重组产生pAdcmv-p12x3c和pAdcmv-p12x3cd重组腺病毒质粒,经序列测定证实目的基因已正确的插入到腺病毒骨架载体中.重组腺病毒质粒经PacI线性化后转染HEK293细胞,转染1w内细胞出现典型病变.取转染细胞裂解液上清连续传代至第4代时,细胞于24~48h内即病变完全,收取接毒后24h细胞进行PCR和RT-PCR检测,表明目的基因已整合到腺病毒基因组内,且在mRNA水平上有表达.取第4、6、8和10代病毒,用蛋白酶K处理后可扩增出目的基因,证明此重组病毒可稳定存在.本研究为FMDV腺病毒活载体疫苗的研究奠定了基础.

Two recombinant replication-defective human adenovirus serotype 5 vector containing foot-and-mouth disease virus (FMDV) serotype O capsid P1-2A, viral 3C protease and 3D polymerase coding region were constructed using the method of homologous recombination in bacteria. Capsid P1-2A, 3C protease and 3D polymerase coding region were subcloned into pshuttle-CMV vector according its original open-reading frame(orf).The positive recombinant pshuttle-CMV vector was linearized and electroporated into Ad-BJ5183 competent cells which had already been transformed with adenovirus skeletal vector pAdeasy-1.Two recombinant adenovirus vector pAdcmv-p12x3c and pAdcmv-p12x3cd were confirmed by Pac I digestion and sequencing, and then transfected HEK293 cell for packaging of recombinant adenovirus. The cell CPE could be observed within one week after transfected, and complete CPE appeared within 24~48h after four round passages. RT-PCR demon- strated that there were expression of the cloned genes. A PCR method has demonstrated that these two recombinant adenovirus could be stably passaged. These results indicated that these two recombinant adenovirus could be used on animals for FMDV recombinant adenovirus vaccine .