脑胶质瘤组织中R132H突变型hIDH1基因双顺反子真核表达载体的构建及鉴定

Construction and identification of a bicistronic eukaryotic expressional vector carrying the R132H mutated hIDH1 gene found in gliomas

目的以pcDNA3.1(+)为骨架,构建并鉴定人脑胶质瘤组织中含R132H突变的人源异柠檬酸脱氢酶1基因(hIDH1R132H)和增强型绿色荧光蛋白(EGFP)序列的双顺反子真核表达载体。方法以内部核糖体进入位点(IRES)/EGFP片段为模板,扩增出IRES/EGFP片段,并在5'末端引入3×Flag标签;通过PCR介导的定点突变法,以pCMV-sports6-hIDH1为模板,扩增hIDH1R132H基因片段,并在3'末端引入3×Flag标签;通过PCR连接两种产物后经双酶切定向克隆入pcDNA3.1(+)骨架;转化大肠杆菌后挑取阳性克隆,提取质粒进行PCR检测及基因序列测定。结果 PCR检测7个菌落中有6个阳性克隆,DNA测序发现引入了hIDH1基因。结论成功构建了双顺反子真核表达载体pcDNA3.1(+)-hIDH1R132H/3×Flag/IRES/EGFP,为研究人胶质瘤组织中R132H突变的作用机制奠定了基础。

Objective To construct a bicistronic eukaryotic expressional vector encoding both human R132H mutated isocitrate dehydrogenase 1 （hIDH1 R132H） gene found in glioma and enhanced green fluorescent protein （EGFP） sequence, using pcDNA3.1 （ ＋ ） as the frame. Methods Internal ribosome entry site（IRES）/EGFP DNA fragments with a 3 x Flag tag introduced to the 5＇-terminus were obtained by PCR using the bought IRES/EGFP DNA fragments as the template. R132H mutated human IDH1 gene fragments with a 3 x Flag tag introduced to the 3 ＇-terminus were obtained from pCMV- sports6-hIDH1 template using PCR site-directed mutagenesis （PCR-SDM） procedures. The 3 x Flag tagged IRES/EGFP and hIDHIR132H were fused together by PCR. The purified fused PCR product was digested with two restriction enzymes and forced cloned into the plasmid frame pcDNA3.1 （ ＋ ） to get pcDNA3.1 （ ＋ ） -hIDH1R132H/3 x Flag/IRES/EGFP. The Stbl3 E. coli was transformed with the constructed vector. Plasmids extracted from the positive E. coli clones were screened by PCR and gene sequencing finally. Results There were 6 positive clones in 7 colonies by PCR, and the hIDH1 gene was successfully introduced in bying DNA sequencing. Conclusions The successful construction of pcDNA 3.1 （ ＋ ） -hIDH1R132H/3 x Flag/IRES/EGFP lays a foundation for better comprehension of the mechanisms of the R132H mutation in glioma＇s forming and progressing.