中文摘要：

目的构建RGD修饰表达绿色荧光蛋白（eGFP）和肿瘤抑素（tumstatin）的新型腺病毒载体。方法PCR扩增tttrnstatin基因，经克隆后构建pAd5CMV／tumstatin穿梭载体。重叠PCR方法将RGD序列插入到Ad5纤维蛋白的HI环中，获得pMfiber-RGD穿梭载体。穿梭载体与腺病毒骨架载体线性化后共转化EcoliBJ5183，构建pAd-RGD-eGFP骨架载体。最后，使用磷酸钙法将pAd5-CMV／tumstatin穿梭载体和pAd-RGD-eGFP骨架载体共转化HEK293细胞。7-10d后收集细胞裂解物得到Ad—RGD-tumstatin／eGFP病毒。将经过修饰的Ad-RGD-tumstatin／eGFP和未修饰的Ad—WT-tumstatin／eGFP病毒分别感染脑胶质瘤U87MG细胞（病毒剂量分别为1、10、100Vp／细胞），通过观察其荧光强度评估其感染效率。以上两种病毒分别感染HeLa细胞，并分别采用RT-PCR及Western blotting检测tumstatin细胞mRNA及培养上清中蛋白的表达水平。结果病毒感染48h后，可观察到修饰后的腺病毒Ad-RGD-tumstatin／eGFP对U87MG细胞的感染效率明显高于未修饰的腺病毒斛WT-tumstatin／eOFP。RT-PCR及Western blotting可检测到感染Ad-RGD-tumstatin／eGFP后的HeLa细胞中tumstatin的转录和表达。结论成功构建经RGD修饰并表达tumstatin和eGFP基因的新型腺病毒载体，为进一步研究turnstatin在低表达柯萨奇一腺病毒受体的肿瘤中的作用奠定了良好基础。

英文摘要：

Objective To construct a novel RGD modified adenovirus vector expressing enhanced green fluorescent protein （eGFP） and human tumstatin. Methods Turnstatin gene was amplified by PCR, and then sub-cloned into the shuttle plasmid to result in pAd5- CMV/tumstatin. RGD sequence was inserted into the HI-loop region of adenovirus fiber by overlapping PCR to result in pMfiber-RGD shuttle vector. Linearized pMfiber-RGD shuttle vector was co-transfected into E. coli BJ5183 with linearized adenovirus backbone vector to result in pAd-RGD-eGFP backbone vector. At last, pAdS-CMV/tumstatin shuttle vector and pAd RGD-eGFP backbone vector were cotransfected into HEK293 cells using standard calcium phosphate method. The cell lysates were harvested 7 to 10 days after transfection to obtain Ad RGD-tumstatin/eGFP virus. The glioblastoma U87MG cells were infected by the novel RGD modified adenovirus Ad RGD- tumstatin/eGFP and unmodified vector Ad-WT-tumstatin/eGFP with dosage of 1, 10 and 100Vp/cell, respectively. The infection efficiency was evaluated by the fluorescence intensity. HeLa cells were infected by the two viruses mentioned above. The transcription and expression of tumstatin were detected by RT-PCR and Western blotting, respectively. Results After 48hrs virus infection, the infection efficiency of U87 MG cells from the modified adenovirus group was significantly higher than that from the wild-type adenovirus group. The results from RT PCR and Western blotting indicated that the tumstatin had been transcribed and expressed in HeLa cells infected by Ad- RGDtumstatin/eGFP. Conclusion A novel RGD modified adenovirus vector expressing tumstatin and eGFP genes has been successfully constructed, which lays a foundation for studying the effectiveness of tumstatin in the gene therapy of CAR deficient cancers.