中文摘要：

目的构建4-1BBL基因重组腺病毒载体,为前列腺癌的免疫治疗奠定基础。方法以质粒pcDNA3-m4-1BBL为模板,PCR法扩增出4-1BBL cDNA,并将其插入腺病毒穿梭质粒pAdTrack-CMV中构建成腺病毒穿梭质粒pAdTrackCMV-m4-1BBL,经PmeⅠ酶切线性化,与腺病毒骨架质粒pAdEasy-1共转化大肠杆菌BJ5183,挑选同源重组质粒,PacⅠ酶切线性化后,转染HEK293细胞包装成重组病毒颗粒,荧光显微镜观察绿色荧光表达,RT-PCR和Western blot法检测4-1BBL表达。结果目的基因经酶切分析,测序鉴定正确,RT-PCR和Western blot证实4-1BBL表达。结论成功构建了含4-1BBL基因的腺病毒载体,该载体可用于前列腺癌的免疫治疗。

英文摘要：

Objective To construct recombinant adenovirus vector encoding 4-1BBL gene and pave the way for the immunotherapy of prostate cancer.Methods Amplified the 4-1BBL cDNA fragment from plasmid pcDNA3-m4-1BBL by PCR method,and inserted it into adenovirus shuttle plasmid pAdTrack-CMV resulting pAdTrackCMV-m4-1BBL.Then Pme Ⅰ-linearized plasmid pAdTrackCMV-m4-1BBLand co-transformed into competent BJ5183 with adenovirus backbone plasmid pAdEasy-1.After homologous recombination in BJ5183,pAd-m4-1BBL was linearized by Pac Ⅰ,and transfected into HEK293 cells.Then Ad-m4-1BBL was packaged and amplified.GFP was observed by fluorescenct microscope,and 4-1BBL expression was detected by RT-PCR and Western blot.Results Ad-m4-1BBL was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis.4-1BBL expression was confirmed by RT-PCR and Western blot.Conclusion Recombinant adenovirus vector carrying 4-1BBL is successfully constructed and can be used for the immunotherapy of prostate cancer.