Egr-1基因反义干扰表达载体的构建与鉴定  

The Construction and Identification of Egr-1 Gene Antisence Interfering Expression Vector

作　　者：刘锐锐 李聪慧 虞游[1] 陈建明[1] 

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州310036

出　　处：《杭州师范大学学报（自然科学版）》2017年第6期618-622,共5页Journal of Hangzhou Normal University(Natural Science Edition)

基　　金：杭州市社会发展自主申报项目(20160533B01)

摘　　要：为构建可在人肝癌细胞中稳定表达Egr-1基因短发夹RNA(shRNA)的表达载体,设计合成Egr-1基因shRNA片段,连接到经BamHⅠ和EcoRⅠ双酶切的pGreenPuro^(TM)shRNA真核表达载体中,连接产物转化大肠杆菌后挑取阳性菌落扩大培养,经菌液PCR法初步鉴定为pGreenPuro-Egr-1shRNA重组子的重组子DNA,用测序法进一步鉴定,结果显示成功构建了Egr-1基因shRNA的反义干扰表达载体pGreenPuro-Egr-1shRNA.To construct the vector stably expressing Egr-1 shRNA in human hepatic carcinoma cells, the well designed and correctly synthesized Egr-1 shRNA oligonucleotides were used to ligate into pGreenPuroTM shRNA eukaryotic expressing vector predigested with BamH I and EcoR I enzymes. The ligation product was then used to transform the E. coli and several positive colonies were picked out for further culture. And subsequently the positive colonies were checked with bacteria liquid PCR method to identify the pGreenPuro-Egr-1 shRNA DNA recombinant. The PCR preliminary identified DNA recombinant was further checked with DNA sequencing method. And finally, the sequencing results showed the Egr-1 shRNA recombinant expression vector termed pGreenPuro-Egr-1 shRNA was successfully and correctly constructed.

关 键 词：EGR-1基因 SHRNA 反义干扰 载体构建 

分 类 号：Q253[生物学—细胞生物学]