论文部分内容阅读
目的:构建表达超抗原SEA基因的溶瘤腺病毒载体并鉴定。方法:采用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Lipofectamine 2000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: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。结论:成功构建了表达超抗原SEA基因的溶瘤腺病毒载体,为进一步研究该病毒对膀胱肿瘤靶向治疗的作用奠定了基础。
Objective: To construct and identify oncolytic adenovirus vector expressing superantigen SEA gene. Methods: The full-length SEA gene of SEA was obtained from the staphylococcus aureus standard strain ATCC13565 by SEP. The full-length SEA gene was cloned into pCA13 plasmid to construct the recombinant plasmid pCA13-SEA. The pCA13-SEA and the pBHGE3 containing the right arm of the adenovirus were co-transfected into HEK293 cells by Lipofectamine 2000 to generate the recombinant adenovirus Ad-SEA by homologous recombination. Ad-SEA was extensively expanded in 293 cells and purified by cesium chloride density gradient centrifugation to determine its titer. Results: The results of PCR, restriction enzyme digestion and sequence analysis confirmed that the SEA gene was successfully cloned into oncolytic adenovirus vector to express SEA gene. CONCLUSION: The oncolytic adenovirus vector expressing superantigen SEA gene has been successfully constructed, which lays the foundation for further study on the role of this virus in targeted therapy of bladder cancer.