共表达O型FMDV衣壳蛋白和绿色荧光蛋白重组腺病毒的构建

Construction of recombinant adenovirus co-expressing FMDV serotype O capsid and GFP proteins

为构建表达O型口蹄疫病毒(FMDV)的P12A3C基因及GFP基因的重组腺病毒rAdV-P12aEGFP2a3C,本研究以FMDV的2A基因序列为Linker,将报告基因EGFP插入FMDV的P12A与3C之间。重组腺病毒感染HEK-293细胞后可以观察到绿色荧光,表明EGFP蛋白获得表达。应用FMDV的VP2单克隆抗体4B2对重组病毒感染细胞进行western blot检测,反应条带与FMDV衣壳蛋白VP0和VP3的分子量大小相符,表明FMDV的完整衣壳蛋白和3C蛋白酶也均获得表达,而且EGFP的插入并未影响P1蛋白的表达和3C蛋白酶对P1的正确切割。重组腺病毒的生长特性分析表明,EGFP的插入也未影响该重组腺病毒的增殖特性。上述研究结果显示,表达FMDV衣壳蛋白P12A3C的重组腺病毒可以作为载体,以2A蛋白作为Linker表达一个小分子蛋白,为改进以腺病毒为载体的口蹄疫基因工程疫苗提供了新思路。

To generate the recombinant adenovirus expressing GFP and P12A3C gene of serotype O foot and mouth disease virus （FMDV）, the foreign fusion gene was constructed by insertion of EGFP gene between P1 and 3C genes with a linker sequence encoding selfprocessing peptide 2A derived from the FMDV. The recombinant adenovirus expressing the P12A3C of FMDV and GFP proteins was generated and identified by PCR, fluorescence assay, western blot. The one-step growth curve indicated that the recombinant adenovirus had no difference with the parental adenovirus. To the best of our knowledge, this report was the first description of the recombinant human adenovirus serotype 5 co-expressing P12A3C of FMDV and GFP proteins, which might be used as an attractive way to improve the FMDV vaccine delivered by adenovirus vector.