TNFα-Tumstatin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  

Construction of adenovirus vector carrying TNFα-Tumstatin fusion gene and its expression in human umbilical cord mesenchymal stem cells

作　　者：周忠海[1] 许文荣[1] 朱伟[1] 乔纯[1] 王兴忠[1] 陈圆[1] 司煜安[1] 徐静[1] 周洪兴[1] 张蕾蕾[1] 李继刚[1] 钱晖[1] 

机构地区：[1]江苏大学基础医学与医学技术学院

出　　处：《江苏大学学报（医学版）》2008年第3期185-188,192,I0001,共6页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30471938);卫生部科研基金资助项目(WKJ2005-2-024);江苏省自然科学基金重点项目(BK2007705);江苏省自然科学基金项目(BK2007092);江苏省医学领军人才资助项目(LJ200614);镇江市重点实验室项目(2006066)

摘　　要：目的: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(human umbilical cord mesenchymal stem cells,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。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 Bg1 Ⅱ and Hind m restriction endonuclease sites and signal peptide sequence of GM-CSF were subcloned into shuttle plasmid pAdtrack-CMV after digesting with Bgl Ⅱ and Hind m. The resultant plasmid, after linearized by digesting with restriction endonuclease Pme Ⅰ , was transformed into E. coll. BJ5183 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 Pac I and then was transfected into hUCMSCs. Fusion gene expression in infected hUCMSCs was detected by RTPCR. Results ： The results of PCR and restriction endonuclease assay indicated that target gene was inserted into recombinant adenovirus vector successfully. The sequence of fusion gene was the same as that of de- signed fragments, hUCMSCs infected by the recombinant adenovirus expressed GFP and fusion gene which could be demonstrated by fluorescence microscope and RT-PCR respectively. Conclusion： Recombinant adenovirus vector containing TNFα-Tumstatin has been constructed successfully and could transfected hUC- MSCs efficiently, which laid a foundation for further investigation of anti-tumor effect of hUCMSCs modified with TNFα-Tumstatin.

关 键 词：腺病毒载体 融合基因 同源重组 细胞转染 

分 类 号：R392.12[医药卫生—免疫学]