表达超抗原SEA基因的溶瘤腺病毒载体的构建与鉴定(英文)

Construction and Identification of the Tumor-selective Oncolytic Adenovirus Vector with Superantigen SEA Gene

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 the expression vectorforthe tumor-selective oncolytic adenovirus with superanti-gen SEA gene.Methods：The SEA full-length gene sequence was obtained from the DNA of ATCC13565 genome of the staphylococcus reference strain producing SEA by using PCR technology, then cloned into pCA13 plasmid after restriction enzyme cutting, so as to construct recombinant virus plasmid pCA13-SEA.The pCA13-SEA identified to be correct and the plasmid containing adenovirus right arm passed through the Lipofectamine 2000 cotransfection HEK293 cell and then produced the recombinant adenovirus Ad-SEA through the homologous recombinant.The Ad-SEA was largely amplified in 239 cells and was purified and measured forthe titerthrough the density gradient centrifugation of cesium chloride.Result：It is proved that the successful cloning of SEA gene into the oncolytic adenovirus vectorcould realize the expression of the SEA gene by PCR amplification, restriction enzyme cutting identification, and sequence determination.Conclusion：Here has successfully constructed the expression vector for the oncolytic adenovirus with the superantigen SEA gene, so as to provided foundation forfurther research the targeted treatment effect of this virus vectoron tumor such as blad dertumor.