Her2/neu膜外及跨膜区蛋白基因重组腺病毒的构建和鉴定  

Construction and identification of recombinant adenoviruses containing the gene fragments encoding Her2/neu extracellular and transmembrane domains

作　　者：马树东[1] 罗荣城[1] 丁振华[2] 袁长青[2] 丁雪梅[1] 

机构地区：[1]南方医科大学南方医院肿瘤科,广东广州510515 [2]南方医科大学热带医学研究所放射医学教研室,广东广州510515

出　　处：《南方医科大学学报》2006年第11期1641-1643,1647,共4页Journal of Southern Medical University

摘　　要：目的构建编码Her2/neu膜外第一受体区、全长膜外区、膜外及跨膜区3个蛋白的重组腺病毒真核表达载体,为信号传导和免疫研究建立实验材料。方法RT-PCR扩增出3个目的片段的cDNA,克隆进pAdTrack-CMV穿梭质粒,在BJ5183细菌内同源重组形成腺病毒质粒,脂质体法转染293细胞后,荧光显微镜观察病毒噬斑形成和绿色荧光蛋白表达,Westernblot检测目的蛋白的表达。结果PCR扩增出了预定大小的目的基因cDNA片段,测序结果和GenBank完全相符。病毒质粒转染293细胞后形成了病毒噬斑,绿色荧光蛋白和目的蛋白均获得表达。目的蛋白的表达量随着病毒感染复数的升高而增加。结论成功地构建了在真核细胞中表达Her2/neu膜外及跨膜区蛋白的腺病毒载体,为Her2/neu膜外及跨膜区蛋白的功能研究提供了材料基础。Objective To construct eukaryotic expression vectors using recombinant adenovirus containing the gene fragments encoding Her2/neu extracellular first ligand-binding domain （Her2-ECD）, full-length extracellular domain （Her2-ECD）, and extracellular and transmembrane domain （Her2-TM）, Methods The cDNAs were amplified by RT-PCR and inserted into shuttle pAdTrack-CMV plasmids. Viral plasmids were obtained from homologous recombination in E.coli B J5183, and transfected into 293 cells via liposome. Formation of viral plaque and expression of green fluorescent protein were observed by fluorescence microscopy, and the target proteins were detected by Western blotting. Results The target cDNA fragments were amplified by PCR with expected lengths and the DNA sequences were confirmed against Genbank. Formation of viral plaque, expression of green fluorescent protein and the target proteins were detected in 293 cells transfected by the viral plasmids, which showed elevated expression of Her2/neu protein with the increase of multiplicity of infection （MOD, Conclusion The eukaryotic expression vectors using recombinant adenovirus have been successfully constructed for expression of Her2/neu extracellular and transmembrane domains.

关 键 词：HER2/NEU基因 载体 基因克隆 重组腺病毒 

分 类 号：Q78[生物学—分子生物学]