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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。
OBJECTIVE: To construct a non-proliferating oncolytic adenovirus DC318-SEA, a double-regulated proliferating oncolytic adenovirus SG502-SEA expressing the superantigen Staphylococcal enterotoxin A gene and a control group carrying the superantigen SEA gene and explore its potential Anti-bladder tumor effect. Methods: The superantigen SEA gene fragment was double digested with SpeI and SalI and cloned into the non-proliferating oncolytic adenovirus vector pSG218. The correct adenovirus vector was named pDC318-SEA. In the same way, the superantigen SEA gene fragment was cloned into the double-regulated specific proliferating oncolytic adenovirus vector pSG502, and the correctly identified adenovirus vector was named as pSG502-SEA. 293 cells were co-transfected with the above two viral vectors carrying the SEA gene and the viral backbone plasmid PPE3-ccdB, virus plaques appeared 9 to 14 days, adenovirus DNA was extracted after 3 times of virus plaque purification, and PCR was used to identify the virus. Identification of the correct adenovirus were named DC318-SEA and SG502-SEA. After a large number of amplification, cesium chloride gradient centrifugation of adenovirus, virus titer. Results: The SEA gene was successfully cloned into two viral vectors by PCR and restriction enzyme digestion. The SEA gene was expressed with a titer of 2.5 × 1010 pfu / ml. CONCLUSIONS: The dual-regulatory proliferating oncolytic adenovirus SG502-SEA expressing the superantigen SEA gene and the non-proliferating oncolytic adenovirus DC318-SEA carrying the superantigen SEA gene in the control group were successfully constructed, which laid the foundation for the next in vitro and in vitro experiments against bladder cancer basis.