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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 pHIF-1α / EGFP-C2 eukaryotic expression plasmid vector. Methods Both hypoxia and chemical hypoxia induced HIF-α mRNA expression in A498 human renal cell carcinoma. The cDNA fragment of HIF-1α was amplified by RT-PCR and cloned into pEGFP-C2 plasmid by T-A cloning. Results HIF-1α cDNA was amplified by hypoxia in A498 human renal cell carcinoma cell line. The DNA sequencing results showed that HIF-1α sequence (NM001530) released by Genebank was the same without mutation and frameshift. HIF-1α protein expression was detected in cells transfected with pHIF-1α / EGFP-C2. Conclusion The eukaryotic expression plasmid pHIF-1α / EGFP-C2 was constructed successfully.