【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。更多还原

【Abstract】 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.更多还原