产肠毒素大肠埃希菌STa-K99融合蛋白重组腺病毒载体的构建及表达

Construction and Expression of Recombinant Adenoviral Vector with STa-K99 Fusion Protein of Enterotoxigenic Escherichia coli

为构建产肠毒素大肠埃希菌(ETEC)STa-K99融合蛋白重组腺病毒载体Ad STa-K99,通过PCR技术分别克隆了STa及K99基因,并与腺病毒穿梭质粒连接构建了穿梭质粒pAd5 STa-K99,将pAd5STa-K99和骨架质粒(含GFP基因)分别用PacⅠ酶切线性化,利用LipofectamineTM LTX&PLUS共转染293细胞进行同源重组包装重组腺病毒,通过PCR及Western blot对重组腺病毒进行鉴定。结果显示,重组腺病毒Ad5STa-K99构建正确并表达融合蛋白,且表达的融合蛋白能够被抗体所识别,为研制由ETEC引起的新生动物腹泻重组腺病毒载体疫苗奠定了基础。

To construct recombinant adenoviral vector expressing STa-K99 fusion protein of enterotoxigenic Escherichia coli, STa and K99 genes were cloned and fused shuttle plasmid containing STa-K99 fusion gene was constructed. Furthermore, shuttle plasmid pad5 STa-K99 and backbone plasmid （containing the GFP gene） were both digested and linearized with Pac I and co-transfected into 293 cell by LipofectamineTM LTX^PLUS to generate recombinant adenovirus by homologous recombination. Finally, identification of recombinant adenovirus was conducted using PCR and Western blot. The results showed that the recombi- nant adenovirus AdS STa-K99 was successfully constructed and the expressed fusion protein can be recognized by anti-His antibody. Our work lays the foundation for developing novel recombinant adenoviral vector vaccine of the newborn animal diarrhea caused by ETEC.