表达金黄色葡萄球菌肠毒素A基因的条件增殖型溶瘤腺病毒的构建

Construction of conditionally replicating adenovirus expressing staphylococcal enterotoxin A gene

目的构建表达超抗原金黄色葡萄球菌肠毒素A(SEA)基因的条件增殖型溶瘤腺病毒pPE3-SEA。方法将已构建好的携带SEA基因的非增殖溶瘤腺病毒载体质粒pDC318-SEA经SpeⅠ和SalⅠ双酶切后,将酶切下的超抗原SEA基因片段连接到同样经SpeⅠ和SalⅠ双酶切后的增殖型溶瘤腺病毒穿梭质粒pENTR12中,将鉴定正确的携带SEA基因增殖溶瘤腺病毒载体命名为pENTR12-SEA。增殖溶瘤腺病毒载体pENTR12-SEA与病毒骨架质粒pPE3-ccdB重组,通过Lipofectamine2000共转染HEK293细胞,经同源重组产生重组腺病毒pPE3-SEA。结果应用PCR验证、双酶切分析、序列测定表明,成功构建了携带SEA基因增殖型溶瘤腺病毒载体pENTR12-SEA,通过同源重组成功获得携带超抗原SEA基因片段的pPE3-SEA增殖型溶瘤腺病毒,且病毒滴度为2.5×1010pfu/ml。结论成功构建了表达超抗原SEA基因的条件增殖型溶瘤腺病毒pPE3-SEA,为以后进一步研究该病毒对肿瘤靶向治疗的作用提供了实验基础。

Objective To construct the conditionally replicating adenovirus pPE3-SEA which expressing staphylococcal enterotoxin A（SEA）gene.Methods The SEA full-length gene sequence was cloned into pENTR12 plasmids after restriction enzyme cutting,so as to construct recombinant virus plasmids pENTR12-SEA.The plasmid pENTR12-SEA was co-transfected with pPE3-ccdB in 293 cells to generate recombinant adenoviruses pPE3-SEA.The pPE3-SEA was largely amplified in 293 cells and was purified and measured for the titer through the density gradient centrifugation of cesium chloride.Results It is proved that the successful cloning of SEA gene into the two oncolytic adenovirus vectors could realize the expression of the SEA gene,by PCR amplification,restriction enzyme cutting identification.And the virus titer was 2.5×1010 pfu/ml.Conclusions We have successfully construct the conditionally replicating adenovirus pENTR12-SEA.This investigation provides the basis for studying tumor gene therapy by pPE3-SEA.