O型口蹄疫多基因重组腺病毒的构建及其免疫原性

根据GenBank公布的序列设计1对引物扩增O型口蹄疫病毒P12A3C基因并亚克隆至腺病毒穿梭质粒pShuttle-CMV内.含有目的基因的穿梭质粒(pShuttle-PAC)线性化后和腺病毒骨架载体pAdeasy-1共同电转化入大肠杆菌BJ5183感受态细菌.利用细菌内同源重组法得到重组腺病毒质粒(pAd-PAC).在Lipofectamine2000介导下重组腺病毒质粒转染HEK293细胞得到重组腺病毒(Ad-PAC).半数组织培养感染剂量(TCID_(50))测定、免疫荧光试验(IFA)和连续传代后目的基因的PCR扩增,证实重组病毒滴度为10~(5.5) TCID_(50)/0.1 mL,重组病毒HEK293细胞中得以表达且遗传稳定性良好;动物试验表明,该重组病毒能够诱导小鼠产生较高水平的针对口蹄疫病毒的特异性抗体;本试验为口蹄疫重组腺病毒活载体疫苗的进一步研究和应用奠定了基础.

Construction and immunological characterizations of the recombinant adenovirus expressing multi-genes of the FMDV serotype O

The primers was designed according to the sequences published in GenBank and genomic fragment containing P12A3C genes was amplied by PCR,then cloned into shuttle plasmid pShuttle-CMV.Subsequently the recombinant shuttle plamid(named pShuttle-PAC) was linearized and co-electroporated with the circular adenoviral genome plasmid pAdeasy-1 into competent BJ-5183 bacterial cells.The recombinant adenovirus plasmid(named pAd-PAC) was screened out via homologous recombination.After transfection of pAd-PAC into HEK293 cells,recombinant virus Ad-PAC was obtained and continuously cultured by ten cycles,bearing the titer of 10~(5.5) TCID_(50)/0.1 mL finally.The expression of the protein P12A3C was confirmed by indirect immunofluorescence.The genetic stability of Ad-PAC was examined by serial passages.Mice experiments showed Ad-PAC can induced high specific antiFMDV antibody,but the level of antibody was lower compared with inactivated vaccines.The results indicated the recombinant adenovirus Ad-PAC may provide a foundation of future research for the control strategy of FMD.