Objective To construct a bicistronic eukaryotic expressional rector encoding both human R132H mutated isocitrate dehydrogenase l(hIDHlR132H) 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 a3 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-hIDHl template using PCR site-directed mutagenesis (PCR-SDM) procedures. The 3 x Flag tagged IRES/EGFP and hIDHI R132H 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 ( + )-hIDHlR132H/3 x Flag/IRES/EGFP. The StbB 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 hIDHI gene was successfully introduced in bying DNA sequencing. Conclusions The successful construction of pcDNA 3. 1 ( + ) -hIDHI R132H/3 x Flag/IRES/EGFP lays a foundation for better comprehension of the mechanisms of the R132H mutation in glioma s forming and progressing.%[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(hIDHIR132H)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h1DH1基因.[结论]成功构建了双顺反子真核表达载体pcDNA3.1(+)-hIDH1 R132H/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.
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