猪O型口蹄疫病毒P1／3C基因共表达重组腺病毒的构建与鉴定

Construction and identification of recombinant adenovirus containing P1/3C genes of Swine Foot-and-Mouth Disease virus

通过PCR和RT-PCR扩增出目的基因,将FMDV P1和3C基因通过IRES串联,构建出重组腺病毒穿梭质粒,在BJ5173细菌中同源重组获得同源重组腺病毒质粒,转化XL1-Gold超级感受态细胞以扩大培养,PacⅠ酶切后转染AD-293细胞,纯化获得了重组腺病毒。该重组腺病毒于AD-293细胞连续传代培养,初步浓缩后TCID50为1010.71/mL。应用O型口蹄疫病毒阳性血清进行间接荧光抗体试验,病毒感染的AD-293细胞可见清晰荧光,证明该重组腺病毒对目的基因进行了成功的表达,为FMDVP1/3C基因共表达腺病毒活载体疫苗的研究奠定了基础。

The recombinant shuttle plasmid pShuttle-Pl-IRES-3C was constructed successfully after the FMDV P1 and 3C genes were linked by IRES gene. Then the recombinant shuttle plasmid was linearized by Pme I and then transformed to BJ5183 cells containing the pAdEasy-1 bone plasmid, The homologous recombinant plasmid was transformed to XL-10 GOLD ultracompetent cells in order to obtain this plasmid in a large scale. A sufficient amount of purified recombinant adenovirus plasmid was digest with Pac Ⅰ , and then transfected AD-293 cells using lipsome. After viral stock was passaged, the titer TCID50 was 10^10.71/ml. Obvious fluorescence was observed in the AD-293 cell stained with standard serum of FMDV serotype O by IFA, which showed that the objective genes were expressed successfully. The results lay a foundation for the study of FMDV vaccine.