人BRAF野生型及V600E突变型真核表达载体的构建及表达  

Construction and expression of eukaryotic expression vector for human wild-type and V600E mutant BRAF gene

作　　者：程张军[1] 石欣[1] 汤永辉[1] 高乃荣[1] 

机构地区：[1]东南大学附属中大医院普外科,江苏省南京市210009

出　　处：《世界华人消化杂志》2008年第21期2395-2398,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金面上项目;No.30500491~~

摘　　要：目的:构建BRAF野生型和V600E突变型真核表达载体;转染并筛选稳定表达BRAF的胰腺癌细胞株,为探讨BRAF V600E突变在胰腺癌发生发展中的作用提供合适的细胞模型.方法:将BRAF野生型及V600E突变型cDNA克隆于真核表达载体pCMV-Myc中,构建pCMV-Myc-BRAFW及pCMV-Myc-BRAF V600E重组质粒,酶切鉴定、测序证实序列正确后,利用脂质体将重组质粒转染胰腺癌细胞Panc-1,经G418培养筛选获得抗性细胞克隆,用RT-PCR和Western blot鉴定野生型和V600E突变型BRAF基因在Panc-1细胞中的表达.结果:酶切鉴定和序列分析证实,重组克隆pCMV-Myc-BRAFW和pCMV-Myc-BRAF V600E序列正确,RT-PCR和Western blot结果显示G418筛选获得的转基因Panc-1细胞稳定表达BRAF和BRAF V600E.结论:成功构建了pCMV-Myc-BRAFW和pCMV-Myc-BR AFV600E真核表达载体,建立了稳定表达BRAF和BRAF V600E的胰腺癌细胞株.AIM： To construct eukaryotic expression vectors for human wild-type and V600E mutant BRAF （v-raf murine sarcoma viral oncogene homolog B1） gene respectively, and to provide suitable cell models for evaluating the potential role of BRAF V600E in pancreatic carcinoma. METHODS： Human fulMength BRAF and BRAF V600E cDNA were subcloned to pCMV-Myc to construct recombinant eukaryotic expression vectors pCMV-Myc-BRAFw and pCMV-Myc- BRAF^V600E, The recombinant plasmids were transfected into Panc-1 cells by lipofectamin method and the positive cell clones were screened with G418. The expression of human wild-type and V600E mutant BRAF gene in Panc-1 cells was tested by reverse transciptase-polymerase chain reaction （RT-PCR） and Western blot.RESULTS： Enzyme digestion and sequencing analysis showed that the target gene was cloned into recombinant vectors successfully, and the expression of human wild-type and V600E BRAF gene was identified in the transfected Panc-1 cells by RT-PCR and Western blot. CONCLUSION： The eukaryotic expression vectors containing human wild-type and V600E mutant BRAF gene are successfully constructed. The positive Panc-1 cell clones expressing human wild-type and V600E-mutant BRAF gene stably are obtained,

关 键 词：BRAF 胰腺癌 逆转录聚合酶链式反应 WESTERN BLOT 克隆 真核表达 

分 类 号：R735.9[医药卫生—肿瘤]