7型腺病毒E1A基因克隆及真核表达

Cloning and Eukaryotic Expression of Adenovirus Type 7 E1A Gene

为了研究7型腺病毒(Ad7)E1A在感染中的致病机制,分离了Ad7d2病毒株,构建了Ad7E1A基因的真核表达体系。用A549细胞分离培养痰液标本中的腺病毒,应用重叠延伸PCR扩增Ad7E1A基因外显子,产物克隆至真核表达载体pIRES-Neo,构建重组子pIRES-Neo-Ad7E1A并转染A549细胞,利用Western印迹对其表达产物进行鉴定。克隆测序显示扩增的Ad7E1A基因外显子包含了完整的编码区基因,pIRES-Neo-Ad7E1A转染A549细胞的表达产物经Western印迹鉴定与设计相符。成功建立了Ad7E1A基因的真核表达体系。

To provided data and material for the future study on the pathogenic mechanism of the adenovirus type 7 （Ad7） E1A, we isolated the Ad7d2 and constructed eukaryotic expression vector of Ad7 E1A gene. Respiratory specimens were inoculated into A549 cells to isolate adenovirus and extract viral DNA. Overlap extension PCR was applied to amplify the exon of E1A gene. PCR products were cloned into the pIRES-Neo vector to produce the expression vector pIRES-Neo-Ad7E1A. After transfection to A549 cell lines, Western blot was applied to identify the expression products. The correct clone of complete cDNA of Ad7 E1A were confirmed by DNA sequencing. The recombinant E1A products were identified by Western blot. The eukaryotic expression system of adenovirus type 7 E1A gene was constructed.