含AFP启动子腺病毒载体构建的基础研究

目的]为提高基因治疗载体的靶向性，在现有的溶瘤性腺病毒基础上，应用细菌内同源重组方法构建含AFP启动子和E1A基因的条件复制性腺病毒载体。方法]应用限制性内切酶以及体外重组技术获取目的片段AFP—AP—IRES—E1Am并进行重组质粒的筛选；将目的片段插入Pshuttle穿梭质粒中；重组的Pshuttle质粒应用PmeI线性化，与含腺病毒骨架DNA、删除了腺病毒E1、E3区的pAdEasyl质粒在BJ5183大肠杆菌细胞中同源重组形成新重组体；抽提重组体DNA，用PacI酶切后，转染至人胚肾细胞株HEk293细胞中进行复制包装，扩增纯化；进行病毒检测。结果]经用具有Amp抗性及kan抗性的培养板和多种限制性内切酶酶切筛选，证实应用同源重组方法构建了含AFP启动子和目的基因E1A的腺病毒载体；该重组腺病毒可转染HEk293细胞，并进行有效复制。结论]成功构建了具有特异靶向性的条件复制腺病毒载体，为进一步研究该腺病毒的功能提供了条件。

Construction of conditionally replicating adenoviruses driven by AFP promoter

Objective By using AdEasy system, which is based on the homologous recombination in bacteria, an AP (alkaline phosphatase)-labeled conditionally replicating adenovirus vector containing adenovirus E1A driven by AFP promoter was constructed. Methods] By endonucleases digestion, fragment harboring of AFP promoter-AP-IRES-E1A coding sequence was subcloned into shuttle plasmid, then linearized and cotransferred with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. After positive kanamycin-resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis. By endonuclease Pac-I digestion, it was transfected into HEK293 cells to assembly recombinant adenovirus. Results] Positive ampicillin-resistant and kanamycin-resistant colony screening and further restriction analysis showed the successful insert of AFP promoter-AP-IRES-E1A coding sequence and the successful construction of recombinant adenovirus vector. The vector can transfect into HEK293 cells and replicate effectively. Conclusion] The successful construction of recombinant adenovirus vector makes the further in vivo experiments possible.