| 董秉政梁清郝林△ 范涛贺厚光张治国胡建鹏韩从辉.荷载金黄色葡萄球菌肠毒素A 基因的双调控溶瘤腺病毒载体
的构建、鉴定及其抗膀胱肿瘤作用[J].,2012,12(22):4225-4230 |
| 荷载金黄色葡萄球菌肠毒素A 基因的双调控溶瘤腺病毒载体
的构建、鉴定及其抗膀胱肿瘤作用 |
| Construction, Identification and Anti-bladder Tumor Effect of HighTargeting, Dual Regulated Oncolytic Adenovirus Vector Expressingthe Staphylococcus Aureus Enterotoxin A Gene |
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| DOI： |
| 中文关键词: 超抗原 腺病毒 膀胱癌 |
| 英文关键词: Dual regulated Superantigen SEA Oncolytic adenovirus |
| 基金项目:江苏省分子和功能影像重点实验室开放基金资助（PYZX2011001）；徐州市医学科研项目（XWJ2011027） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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