pHIF-1α/EGFP-C2质粒的构建与鉴定  

Construction and identification of pHIF-1α/EGFP

作　　者：马玲[1] 卢晓梅[1] 金玉楠[1] 魏文娟[1] 马海英[1] 于艳秋[1] 

机构地区：[1]中国医科大学基础医学院病理生理教研室,辽宁沈阳110001

出　　处：《解剖科学进展》2011年第2期121-123,共3页Progress of Anatomical Sciences

基　　金：辽宁省科技厅资助项目(2009225010-25)

摘　　要：目的构建pHIF-1α/EGFP-C2真核细胞表达质粒载体。方法缺氧培养与化学性缺氧两种方法诱导A498人肾癌细胞中HIF-αmRNA水平升高。RT-PCR法扩增HIF-1α的cDNA片段,经T-A克隆扩增后,连接入pEGFP-C2质粒。结果缺氧培养的A498人肾癌细胞组出现HIF-1α的cDNA扩增条带,DNA测序结果Genebank所公布的HIF-1α序列(NM001530)相同,无突变和移码。转染pHIF-1α/EGFP-C2的细胞中检测到HIF-1α蛋白的表达。结论 pHIF-1α/EGFP-C2真核细胞表达质粒构建成功。Objective To construct and identify the pHIF-1α/EGFP plasmid. Methods A498 human renal carcinoma cells were cultured by application of CoCl2（200μM） and 3% O2 to elevate the level of HIF-1α mRNA. HIF-1α cDNA was amplified by RT-PCR and linked into pEGFP-C2 vector after amplification through T-A colon. Results Fragment of HIF-1α cDNA can be detected in the A498 human renal carcinoma cells cultured in 3% O2. The cDNA sequence was same to the sequence of HIF-1α（NM001530） announced by Genebank,no mutation and no frame shift were found. The expression of HIF-1αprotein was detected in cells transfected by pHIF-1α/EGFP plasmid by Western blot. Conclusion The plasmid of pHIF-1α/EGFP-C2 was constructed successfully.

关 键 词：缺氧诱导因子-1 绿色荧光蛋白 质粒构建 肾癌细胞 

分 类 号：R363[医药卫生—病理学]