CD133慢病毒表达载体和SGC7901-CD133细胞株的构建  

Construction of a CD133 lentiviral expression vector and establishment of its stably transfected SGC7901 cell line

作　　者：朱优龙[1] 姜波健[1] 王守练[1] 吴巨钢[1] 俞继卫[1] 

机构地区：[1]上海交通大学医学院附属第三人民医院普外一科,201999

出　　处：《中华实验外科杂志》2014年第5期1012-1015,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81101850）;上海市教委基金资助项目（12YZ047）

摘　　要：目的构建CD133慢病毒表达载体，建立稳定过表达CD133的人胃腺癌SGC7901细胞株。方法聚合酶链反应法扩增CD133基因全长序列，将序列插入pGMLV—PB1—1载体，构建pGMLV-PBI-1-CD133慢病毒表达载体，随后转入293T细胞中对慢病毒进行包装，用获得的慢病毒毒液感染人胃腺癌细胞株SGC7901。建立稳定过表达CD133的SGC7901细胞株。荧光显微镜下观察pGMLV—PB1—1-CD133慢病毒表达载体的转染效果，逆转录-聚合酶链反应（RT-PCR）和Westernblot法检测了SGC7901、pGMLV—PB1-1-SGC7901和pGMLV—PB1—1-CD133-SGC79013种细胞中CD133mRNA及蛋白的表达水平。结果经限制性内切酶鉴定及测序分析，我们成功构建了pGMLV—PB1—1-CD133慢病毒表达载体质粒。Westernblot结果显示，pGMLV—PB1—1-CD133慢病毒表达载体质粒成功转染293T工具细胞，pGMLV-PB1—1-CD133慢病毒表达载体有较好的过表达效果。pGMLV—PB1-1-SGC7901和pGMLV—PB1—1-CD133-SGC7901细胞在荧光显微镜下均发出绿色荧光，并且转染效率高。RT—PCR和Western blot检测显示，与对照组比较，转染pGMLV—PB1—1-CD133慢病毒表达载体组的细胞，CD133的表达在mRNA和蛋白两个水平均显著提高（1．160±0．051、0．835±0．077），差异有统计学意义（P〈0．01）。结论成功构建CD133慢病毒表达载体和SGC7901-CD133细胞株。Objective To construct a CD133 lentiviral expression vector and to establish its stably transfected SGC7901 cell line. Methods Full-length CD133 gene amplified by reverse transcription-poly- rnerase chain reaction （RT-PCR） was inserted into a pGMLV-PBI-1 vector to construct pGMLV-PBl-1- CD133 and pGMLV-PBI-1 vectors, and then transfected into 293T cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the SGC7901 cells and establish a stably overexpressed cell line named SGC7901-CD133. Results A CD133 lentiviral expression vector （pGMLV- PBl-1-CD133） was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blotting revealed the increased mRNA and protein expression of CD133 gene in cells transfected with pGMLV-PB1-1-CD133 （ 1. 160 ± 0. 051,0. 835 ± 0. 077 ） as compared with the control groups. Therefore the SGC7901-CD133 cell line was successfully established through the experiment. Conclusion A lentiviral CD133 expression vector and its SGC7901 cell line were successfully constructed.

关 键 词：胃腺癌 CD133 慢病毒载体 转染 

分 类 号：R735.2[医药卫生—肿瘤]