中文摘要：

目的：构建经嵌合纤维蛋白Ad5/35修饰的双表达hIL-12和hIFN-α2a的新型腺病毒载体并进行体外实验研究。方法：采用常规基因克隆和同源重组技术,构建在E3区携带hIL-12B（p40）和在纤维蛋白及E4区之间携带hIFN-α2a及eGFP表达框的经嵌合纤维蛋白Ad5/35修饰的嵌合型腺病毒骨架质粒载体pAd5/35-Backbone.E3-CMV-hIL-12B（p40）/E4-hIFN-α2a-eGFP。此外,采用分子克隆手段构建携带hIL-12A（p35）的腺病毒E1穿梭载体pAd5-E1-CMV-hIL-12A（p35）。PCR法鉴定腺病毒骨架载体及穿梭载体质粒。将上述所获得的两种质粒载体经过PacI线性化后共转染HEK293细胞包装新型病毒载体。将纯化的腺病毒感染人恶性胶质瘤细胞U87MG,48小时后,荧光显微镜下观察报告基因eGFP的表达,RT-PCR检测hIL-12及hIFN-α2a目的基因的表达。结果：PCR法检测到所构建的腺病毒骨架载体及穿梭载体质粒成功携带所需的目的基因。成功包装并纯化获得新型腺病毒载体pAd5/35.E1-CMV-hIL-12A（p35）/E3-CMV-hIL-12B（p40）/E4-hIFN-α2a-eGFP。体外感染U87MG细胞,48h后荧光显微镜下可以观察到报告基因eGFP的表达,RT-PCR可以检测到hIL-12及hIFN-α2a目的基因的表达。结论：体外实验结果表明,成功制备的经嵌合纤维蛋白Ad5/35修饰的双表达hIL-12和hIFN-α2a新型腺病毒载体,为进一步探讨其在恶性脑胶质瘤动物模型中的体内治疗研究奠定了基础。

英文摘要：

Objective：To construct a novel chimeric fiber 5/35 modified adenoviral vector dually expressing hIL-12 and hIFN-α2a and its experimental study in vitro.Methods：By using conventional technology of molecular cloning and homologous recombination,we constructed adenoviral backbone plasmid pAd5/35-Backbone.E3-CMV-hIL-12B（p40）/E4-hIFN-α2a-eGFP with the modification of chimeric fiber Ad5/35,which carries human interleukin-12B（p40） in E3 region and an expression cassette inserted between the fiber and E4 region for expressing human interferon α2a and eGFP.In addition,the adenoviral E1 shuttle vector pAd5-E1-CMV-hIL-12A（p35） expressing hIL-12A（p35） was also constructed by molecular cloning technique.Then both the adenoviral backbone and shuttle plasmids were detected by polymerase chain reaction（PCR）.The two plasmids above were linearized with PacI and co-transfected into HEK 293 cells to package the novel adenovirus.Then the U87MG cells were infected by the purified virus,and the expression of eGFP was detected 48 hours post infection by fluorescence microscopy.The expressions of hIL-12 and hIFN-α2a were demonstrated by reverse transcription-polymerase chain reaction（RT-PCR）.Results：The adenoviral backbone vector and the shuttle vector plasmid carrying the target gene were successfully detected by PCR.The novel adenovirus pAd5/35.E1-CMV-hIL-12A（p35）/E3-CMV-hIL-12B（p40） /E4-hIFN-α2a-eGFP was successfully constructed.The U87MG cells were infected by the virus,the expression of eGFP was detected 48 hours post infection by fluorescence microscopy,meanwhile the expression of hIL-12 and hIFN-α2a were demonstrated by RT-PCR.Conclusion：The results in vitro indicated that the fiber chimeric Ad5/35 modified adenoviral vector co-expressing hIL-12 and hIFN-α2a was successfully generated and provided a foundation for its experimental therapy in the model of glioblastoma.