装载超抗原SEA基因的由前列腺特异性抗原和端粒酶逆转录酶启动子双调控靶向前列腺癌的溶瘤腺病毒载体的构建  

Construction of double-regulated conditionally replicating adenovirus expressing Staphylococcal enteroloxin A gene and targeted anti-tumor on prostate cancer

作　　者：韩从辉[1] 郝林[1] 胡建鹏[1] 王涛[2] 贡震[1] 贺厚光[1] 董秉政[1] 张培影[3] 

机构地区：[1]东南大学附属徐州医院(徐州市中心医院)泌尿外科,江苏徐州221009 [2]首都医科大学肿瘤中心 [3]东南大学肿瘤研究所

出　　处：《中华实验外科杂志》2010年第3期330-332,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30973443）;江苏省自然科学基金资助项目（BK2009085） 本研究得到第二军医大学上海肝胆外科医院病毒与基因治疗中心吴红平和钱其军教授的指导,并在该所完成超抗原病毒复合物的合成和鉴定

摘　　要：目的构建表达超抗原金黄色葡萄球菌肠毒素（SEA）基因的由前列腺特异性抗原（PSA）启动子及端粒酶逆转录酶（hTERT）启动子双调控的特异性增殖溶瘤腺病毒SG504-SEA。方法从前列腺癌组织基因组DNA中获得522bp大小的PSA启动子序列，将PSA启动子克隆到由hTERT启动子调控的特异性增殖溶瘤腺病毒载体SG502中，得到靶向前列腺癌细胞的双调控溶瘤腺病毒载体SG504。将已构建好的含有771bp大小SEA基因片段的病毒骨架质粒PPE3-ccdb—SEA与SGSIM用Lipofectamine 2000共转染至293细胞。共转染后9—14d出现病毒空斑，经过3次病毒空斑纯化，经鉴定正确的腺病毒命名为SG504-SEA，即携带SEA基因的双调控靶向前列腺癌的特异性溶瘤腺病毒。结果经聚合酶链反应（PCR）及酶切鉴定，SEA基因成功克隆到病毒载体中，可以表达SEA基因，且病毒滴度为2．0×10^10pfu／ml。结论成功构建表达超抗原SEA基因的双调控选择增殖型溶瘤腺病毒SG504-SEA。Objective To construct double-regulated conditionally replicating adenovirus SG504- SEA. Methods The prostate-specific antigen （PSA） promoter was amplified from prostate cancer tissues by polymerase chain reaction （PCR） method and the 522 bp PSA promoter gene sequence was cloned into pSG504 plasmids after restriction enzyme cutting. The plasmid pSG504 was co-transfected with PPE3-ccdb- SEA in 293 cells to generate recombinant adenoviruses SG504-SEA. The recombinant adenoviruses were verified by PCR, purified by cesium chloride density purification and propagated in 293 cells. Virus titer was measured by TCID50 methed and its titre was 2.0 ×10^10pfu/ml. Results It was proven that the successful cloning of PSA promoter and SEA gene into the oncolytic adenovirus vector could realize the expression of the SEA gene. And the virus titer was 2.0 ×10^10 pfu/ml. Conclusion We have successfully constructed double-regulated conditionally replicating adenovirus SG504-SEA, which lays the foundation for further research on application of SEA in targeted gene therapy for prostate cancer and the underlying immunological mechanisms.

关 键 词：前列腺癌 前列腺特异性抗原 端粒酶逆转录酶 超抗原SEA 溶瘤腺病毒 

分 类 号：R737[医药卫生—肿瘤]