携带超抗原SEA基因的特异性溶瘤腺病毒载体的构建和鉴定

Construction and identification of oncolytic adenovirus vector expressing SEA gene

目的 构建携带超抗原SEA基因的肿瘤特异性溶瘤腺病毒表达载体。方法采用聚合酶链反应（PCR）技术，从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中获得SEA全长基因序列，酶切后克隆入pCA13质粒，构建重组病毒质粒pCA13-SEA。将鉴定正确的pCA13-SEA与含有腺病毒右臂的质粒pBHGE3通过Lipofectamine2000共转染HEK293细胞，经同源重组产生重组腺病毒Ad-SEA。Ad．SEA在293细胞中大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度。结果克隆得到771bpSEA全长基因。经PCR扩增、酶切鉴定、序列测定证实，SEA基因成功克隆到溶瘤腺病毒载体中，可实现SEA基因的表达，且病毒滴度达6．5×10^9pfu／ml。结论成功构建表达超抗原SEA基因的溶瘤腺病毒载体。

Objective To construct the oncolytic adenovirus vector expressing SEA gene. Methods The SEA gene was amplified from standard staphylococcus aureus ATCC13565 by PCR method and then cloned into adenovirus vector pCA13. Recombinant adenovirus plasmid pCA13-SEA was identified and coransfected together with pBHGE3 into 293 packaging cells. The Ad-SEA recombinant adenovirus was efficiently duplicated in 293 cells and purified by CsCl density centrifugation, and the titer was measured. Results The 771 bp DNA sequencing showed that the cloned SEA gene sequence was the same as that of the published sequence. The evidence of endonuclease digestion, DNA sequencing and PCR analysis confirmed that SEA gene was correctly inserted into the oncolytic adenovirus vector, and the titer was 6.5 × 10^9 pfu/ml. Conclusion Ad-SEA has been successfully constructed.