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| 降落和重叠延伸PCR构建靶向肿瘤干细胞腺病毒载体及感染实验 | |
| 引用本文： | 张超,刘国炳,田平阁,周春平,李学农. 降落和重叠延伸PCR构建靶向肿瘤干细胞腺病毒载体及感染实验[J]. 南方医科大学学报, 2011, 0(9) |
| 作者姓名： | 张超 刘国炳 田平阁 周春平 李学农 |
| 作者单位： | 1. 南方医科大学 病理学系/广东省分子肿瘤病理学重点实验室,广东广州,510515 2. 南方医科大学 南方医院妇产科,广东广州,510515 |
| 基金项目： | 国家自然科学基金(30871156); 广东省科技计划项目(2007B031001001,2009B030803041); 广东省自然科学基金(8151051501000057)~~ |
| 摘 要： | 目的应用降落和重叠延伸PCR法构建对肿瘤干细胞有特异靶向性的高效载体-复制缺陷型腺病毒载体,观察其对CD133+人大肠癌细胞株SW480的感染效率。方法利用重叠延伸PCR技术扩增5型腺病毒纤毛球端HI环两端的基因序列,定向插入真核表达质粒pEGFP-N1中,构建重组质粒pEGFP-N1.KNOB△HI,同时在缺失HI环部位引入EcoRV限制性酶切位点。在此基础上,用降落PCR克隆纤毛基因全长编码区序列,构建出腺病毒穿梭质粒pNEB-F5。利用凝胶图像分析和基因测序验证所构建质粒的正确性。再与pAdEasy-1,pShuttle-GFP在E.coli BJ5183中同源重组,得腺病毒载体Ad5FHI-GFP和Ad5-GFP。用Ad5FHI-GFP和Ad5-GFP分别感染CD133+人大肠癌细胞株SW480,通过荧光显微镜观察感染效率。结果降落PCR和重叠延伸PCR都扩增出特异性条带,测序结果与预期相符。与Ad5-GFP相比,Ad5FHI-GFP对CD133+人大肠癌细胞株SW480有显著提高。结论利用降落和重叠延伸PCR成功构建新型靶向肿瘤干细胞的腺病毒载体,利用降落和重叠延伸PCR成功构建新型靶向肿瘤干细胞的... |
| 关 键 词： | 降落PCR 重叠延伸PCR 肿瘤干细胞 复制缺陷型腺病毒 穿梭质粒 |
Touchdown PCR and overlap extension PCR for generateing CD133+ cancer stem cell-selective adenovirus vector |
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| ZHANG Chao,LIU Guo-bing,TIAN Ping-ge,ZHOU Chun-ping,LI Xue-nong. Touchdown PCR and overlap extension PCR for generateing CD133+ cancer stem cell-selective adenovirus vector[J]. Journal of Southern Medical University, 2011, 0(9) | |
| Authors: | ZHANG Chao LIU Guo-bing TIAN Ping-ge ZHOU Chun-ping LI Xue-nong |
| Affiliation: | ZHANG Chao1,LIU Guo-bing2,TIAN Ping-ge1,ZHOU Chun-ping1,LI Xue-nong1 1Department of Pathology/Key Laboratory of Molecule Tumor Pathology of Guangdong Province2,Department of Gynecology and Obstetrics,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China |
| Abstract: | Objective To construct a replication-incompetent adenovirus vector targeting cancer stem cells by modified touchdown PCR and overlap extension PCR and investigate its infection efficiency in CD133+ SW480 cells in vitro.Methods The two portions of the fiber gene encoding the Ad5 fiber knob domain with the HI loop deleted were amplified using two pairs of designed primers and then linked by overlap extension PCR.The product obtained was identified by sequencing and inserted into prokaryotic expression vector ... |
| Keywords: | touchdown PCR overlap extension PCR cancer stem cell adenovirus shuttle plasmid |
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