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腺病毒载体,其对CD133+人大肠癌细胞株SW480实验显示Ad5FHI-GFP组感染效率明显强于Ad5-GFP组。
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 pEGFP-N1.The product,pEGFP-N1·KNOB△HI,contained a unique EcoRV restriction site in the deleted portion of the sequence encoding the HI loop.The gene sequences of the adenovirus fiber were amplified using both common PCR and overlap extension PCR,then identified by sequencing and inserted into pNEB193,resulting in pNEB-F5.CD133＋ SW480 cells were infected with the generated adenovirus vectors Ad5-GFP and Ad5FHI-GFP to investigate the infection efficiency using fluorescent microscope.Results The target fragments of expected sizes were amplified by touchdown PCR and overlap extension PCR,but not by common PCR.Ad5FHI-GFP showed a higher infection efficiency than Ad5-GFP in CD133＋ SW480 cells.Conclusion Compared with common PCR,touchdown PCR and overlap extension PCR can significantly improve the specificity and efficiency of the PCR products for constructing CD133＋ cancer stem cell-selective adenovirus type 5 vector,which provides carriers for tumor-targeted gene therapy.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2011年第9期1513-1517,共5页
Journal of Southern Medical University
基金
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)~~
