【摘要】 目的:构建表达超抗原金黄色葡萄球菌肠毒素A基因的双调控选择增殖型溶瘤腺病毒SG502-SEA及对照组携带超抗原SEA基因的非增殖溶瘤腺病毒DC318-SEA,并探讨其潜在的抗膀胱肿瘤作用。方法:将超抗原SEA基因片段经SpeI和SalI双酶切后,克隆入非增殖溶瘤腺病毒载体pSG218中,将鉴定正确的腺病毒载体命名为pDC318-SEA。同样方法将超抗原SEA基因片段克隆入双调控特异性增殖溶瘤腺病毒载体pSG502中,将鉴定正确的腺病毒载体命名为pSG502-SEA。将以上两种携带SEA基因的病毒载体与病毒骨架质粒PPE3-ccdB共转染293细胞,9～14d出现病毒空斑,经过3次病毒空斑纯化,提取腺病毒DNA,应用PCR进行鉴定,经鉴定正确的腺病毒分别命名为DC318-SEA和SG502-SEA。大量扩增后,氯化铯梯度离心纯化腺病毒,测病毒滴度。结果:经PCR及酶切鉴定,SEA基因成功克隆到两病毒载体中,可以表达SEA基因,且病毒滴度为2.5×1010pfu/ml。结论:成功构建表达超抗原SEA基因的双调控增殖型溶瘤腺病毒SG502-SEA及对照组携带超抗原SEA基因的非增殖溶瘤腺病毒DC318-SEA,为下一步抗膀胱肿瘤体内外实验奠定基础。更多还原

【Abstract】 Objective:To construct the expression of superantigen staphylococcus enterotoxin A gene high targeting,dual regulated replication-selective oncolytic adenovirus SG502-SEA and contrasting group the non-proliferation oncolytic adenovirus DC318-SEA of gene,and investigate the potential anti-tumor effect on bladder.Methods:After double-digestion by SpeI and SalI,superantigen SEA gene section was cloned into the vector of non-proliferation oncolytic adenovirus vector pSG218,and named the accurately identified advenovirus vector as pDC318-SEA.By the same method,the superantigen SEA gene segment was cloned into the dual regulated specific proliferous oncolytic adenovirus vector pSG502,and named the accurately identified adenovirus vector as pSG502-SEA.The two virus vectors carrying SEA gene and the virus framework plasmid PPE3-ccdB were cotransfect the above into 293 cells.After cotransaction 9～14d appears virus plaque.After the virus plaque purification for three times,extract the adenovirus DNA and use PCR for identification.The adenovirus,identified to be correct,is named as DC318-SEA and SG502-SEA respectively.After large amplification,the adenovirus was purified through cesium chloride gradient centrifugation,and then the virus titer was measured.Results:Through PCR and enzyme cutting identification,the virus vectors can express the SEA gene,and the virus titer is 2.5×1010 pfu/mL.Conclusion:The dual regulated,high targeting replication-selective oncolytic adenovirus SG502-SEA and non-proliferous oncolytic adenovirus DC318-SEA of contrasting group carrying SEA were construct successfully.更多还原