Si Shao-yan, Sui Yan-fang, Hu Pei-zhen, Zhang Xiu-min, Ge Wei, Li Zeng-shan, Huang Yang
Department of Pathology, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Sep;22(5):613-6.
To construct hepatoma-targeting recombinant co-expression adenovirus vector of Staphylococcal enterotoxin A (SEA) and CD80 gene.
Us-ing the adenovirus transfer plasmids pShuttle and pShuttle-CMV, we constructed a new transfer plasmid pShuttle2 with polyA signal sequence instead of CMV enhancer/promoter. AFP enhancer, promoter, SEA or CD80 gene was subcloned into pShuttle2 from the vectors pKS-EP or pMD18-T-BIS respectively, and then the constructed plasmid pShuttle2-BIS containing AFP enhancer, promoter, SEA or CD80 gene was cotransformed into E.coli BJ5183 with backbone vector pAdEasy-1 to obtain recombinant adenovirus DNA. The recombinant adenovirus DNA was transfected into 293 cells to prepare adenovirus. After AFP-producing cell line Hepa1-6 and AFP-nonproducing cell lines B16 and NIH3T3 were infected by recombinant adenovirus, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining, laser confocal microscope and flow cytometry (FCM).
SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on B16 and NIH3T3 cells.
Hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene was successfully constructed, which lays the foundation for further research on application of SEA and CD80 in targeted gene therapy for hepatoma and the underlying immunological mechanisms.
构建葡萄球菌肠毒素A（SEA）与CD80基因的肝癌靶向重组共表达腺病毒载体。
利用腺病毒转移质粒pShuttle和pShuttle-CMV，构建一种带有polyA信号序列而非CMV增强子/启动子的新转移质粒pShuttle2。将甲胎蛋白（AFP）增强子、启动子、SEA或CD80基因分别从载体pKS-EP或pMD18-T-BIS亚克隆至pShuttle2，然后将构建好的含有AFP增强子、启动子、SEA或CD80基因的质粒pShuttle2-BIS与骨架载体pAdEasy-1共转化至大肠杆菌BJ5183中，以获得重组腺病毒DNA。将重组腺病毒DNA转染至293细胞中制备腺病毒。用重组腺病毒感染产AFP细胞系Hepa1-6以及不产AFP细胞系B16和NIH3T3后，通过间接免疫荧光染色、激光共聚焦显微镜和流式细胞术（FCM）检测细胞表面SEA和CD80的表达情况。
SEA和CD80在受感染的Hepa1-6细胞表面特异性共表达，而在B16和NIH3T3细胞表面未表达。
成功构建了SEA与CD80基因的肝癌靶向重组共表达腺病毒载体，为进一步研究SEA和CD80在肝癌靶向基因治疗中的应用及其潜在免疫机制奠定了基础。
