表达A型口蹄疫病毒衣壳蛋白重组腺病毒的构建

Construction of recombinant adenovirus expressing capsid protein of serotype A foot-and-mouth disease virus

为构建表达A型口蹄疫病毒(FMDV)衣壳蛋白的重组腺病毒,本研究通过人工合成A型FMDVP1-2A、2B和3C融合基因,将其克隆到腺病毒穿梭载体pShuttle-CMV中,利用E.coli BJ5183内同源重组将目的基因插入腺病毒骨架质粒pAdEasy-1中,获得携带A型FMDV P1-2A-2B-3C基因的重组AdEasy-1。该重组质粒经PacⅠ线性化后转染AD-293细胞,获得重组腺病毒rAd-A09。经PCR检测,该重组腺病毒在传代过程中目的基因稳定存在,病毒滴度在第8代时可达到108.5TCID50/mL。间接免疫荧光检测和western blot分析表明,rAd-A09在AD-293细胞中产生FMDV的结构蛋白VP0、VP1和VP3。该重组腺病毒的构建为口蹄疫新型疫苗的研究奠定了基础。

To construct the recombinant adenovirus expressing capsid protein of serotype a food-and-mouth disease virus（FMDV）,a fusion gene of P1-2A,2B and 3C was chemically synthesized and cloned into adenovirus shuttle vector pShuttle-CMV.The P1-2A-2B-3C gene was integrated into adenovirus backbone vector pAdEasy-1 by homologous recombination in E.coli BJ5183.Subsequently,the recombinant pAdEasy-1 carrying P1-2A-2B-3C gene was linearized with PacⅠand transfected into AD-293 cells to generate recombinant adenovirus,designated rAd-A09.The titer of rAd-A09 reached 108.5 TCID50/mL at 8th passage in AD-293 cells.The stability of the target gene in rAd-A09 was detected by PCR.Furthermore,the rAd-A09 stably expressing VP0,VP1 and VP3 was confirmed by indirect immunofluorescence assay and western blot analyses of the cell lysates.These results would facilitate the development of FMDV novel vaccine.