口蹄疫病毒P1-2A和3C蛋白重组腺病毒的构建及其免疫原性研究

为研制口蹄疫病毒(FMDV)的新型重组腺病毒疫苗,本研究通过RT-PCR扩增FMDV GD株3C、P1、P1-2A、L-P1和L-2A基因,分别构建重组腺病毒穿梭质粒,并在含有腺病毒骨架质粒pAdEasy-1的BJ5183E.coli中同源重组获得腺病毒重组质粒pAd-3C、pAd-P1、pAd-P1-2A、pAd-L-P1、pAd-L-2A,经PacⅠ线性化后转染AD-293细胞,获得含有目的基因的重组腺病毒。将具有感染能力的复制缺陷型重组腺病毒rAd-3C分别与rAd-P1、rAd-P1-2A、rAd-L-P1、rAd-L-2A共感染Vero细胞,裂解细胞收集细胞上清液并进行小鼠免疫试验。经ELISA检测表明,重组腺病毒rAd-3C分别与rAd-P1-2A、rAd-L-2A共感染Vero细胞上清液能够诱导小鼠产生特异性体液免疫应答。

Recombinant adenovirus expression of FMDV P1-2A and 3C protein and its immune response in mice

To develop a recombinant virus vaccine against foot-and-mouth disease(FMD),the 3C,P1,P1-2A,L-P1 and L-2A genes of FMDV were amplified by RT-PCR,and inserted into the pShuttle-CMV vector to construct the recombinant plasmids which were linearized by Pme Ⅰ and transformed into BJ5183 cells containing the pAdEasy-1 bone plasmid.The infective replication-defective adenovirus rAd-3C was collected after the appearance of CPE,and it was used to infect Vero cell with rAd-P1,rAd-P1-2A,rAd-L-P1 and rAd-L-2A,respectively.It was showed that P1 protein and L-P1 protein were failed to be cleaved by 3C proteinase,while P1 protein was cleaved by 3C proteinase and 2A proteinase.The results of indirect ELISA showed that a certain level of antibodies were induced in mice immunized by the recombinant virus.These results would facilitate to further studies on the recombinant adenovirus live vector vaccine against FMD.