TNFαTumstatin融合基因腺病毒载体的构建及其在人脐带间质干细胞中的表达（临床医学论文）.docVIP

TNFαTumstatin融合基因腺病毒载体的构建及其在人脐带间质干细胞中的表达（临床医学论文） 文档信息 属性： F-00BP2U，doc格式，正文7595字。质优实惠，欢迎下载！ 适用： 作为医学论文、临床医学写作的参考文献，解决如何写好实用应用文、正确编写文案格式、内容摘取等相关工作。 作者： 周忠海 目录 TOC \o 1-9 \h \z \u 目录 1 正文 1 搞要 2 关键字：腺病毒载体 融合基因 同源重组 细胞转染 2 1 材料与方法 4 2 结 果 6 3 讨 论 8 【参考文献】 9 论文原创声明（模板） 10 论文致谢（模板） 10 正文 TNFαTumstatin融合基因腺病毒载体的构建及其在人脐带间质干细胞中的表达（临床医学论文） 搞要 摘要：构建携带绿色荧光蛋白的TNFα?Tumstatin融合基因腺病毒表达载体，转染人脐带间质干细胞(humanumbilicalcordmesenchymalstemcells,hUCMSCs)。方法：设计含有GM?CSF信号肽序列和BglⅡ及HindⅢ酶切位点的引物，PCR扩增TNFα?Tumstatin，将扩增产物亚克隆到穿梭质粒pAdTrack?CMV上，重组穿梭质粒经PmeⅠ线性化后转化含有腺病毒骨架质粒pAdEasy?1的BJ5183中同源重组。筛选获得含有融合基因的重组腺病毒质粒，酶切鉴定并测序。重组病毒质粒用PacⅠ酶切线性化后转染293A细胞，经过包装、扩增后感染hUCMSCs并检测细胞内融合基因的表达。结果：重组腺病毒质粒经PCR和PacⅠ酶切鉴定，证实含有TNFα?Tumstatin融合基因，测序结果和设计片段的序列一致。重组腺病毒感染的hUCMSCs表达绿色荧光蛋白和融合基因。结论：成功构建了TNFα?Tumstatin融合基因的腺病毒表达载体，能高效率感染hUCMSCs，为进一步研究融合基因修饰的hUCMSCs抗肿瘤效应奠定了基础。快速论文发表 关键字：腺病毒载体 融合基因 同源重组 细胞转染 ［Abstract］ Objective: To construct green fluorescence protein (GFP)labeled recombinant adenovirus vector carrying TNFα?Tumstatin fusion gene and transfect it into hUCMSCs. Methods： The amplification products of TNFα?Tumstatin by PCR with a pair of primers with BglⅡand HindⅢ restriction endonuclease sites and signal peptide sequence of GM?CSF were subcloned into shuttle plasmid pAdtrack?CMV after digesting with BglⅡand HindⅢ. The resultant plasmid, after linearized by digesting with restriction endonuclease PmeⅠ, was transformed into that had been transformed by adenoviral backbone plasmid pAdEasy?1. Recombinant plasmid were obtained by alternation of kanamycin and then confirmed by PCR and restriction endonuclease analysis. The adenovirus was packaged and propagated in human embryonal kidney cells (293A cells)after being linearized by digesting with restriction endonuclease PacⅠand then was transfected into hUCMSCs. Fusion gene expression in infected hUCMSCs was detected by RT?PCR. Results： The results of PCR and restriction endonuclease assay indicated that target gene was inserted into recombin