低氧诱导因子基因重组腺病毒载体的构建及其在内皮细胞中的表达

目的：构建携带低氧诱导因子-1α（HIF-1α）基因的腺病毒载体（pAdxsi-GFP-HIF），观察其在内皮细胞中的表达。 方法：低氧处理A549细胞后提取总RNA并逆转录为cDNA，以之作为模板，依据基因库公布的HIF-1α cDNA 设计引物，分别引入KpnI和BamHI酶切位点，PCR扩增后将目的基因HIF-1α连接到载体pShuttle-CMV-EGFP上，构建穿梭质粒pShuttle-GFP-HIF。采用细菌内重组方法将目的序列重组到pAdxsi病毒骨架载体上构建携带HIF-1α基因的重组腺病毒载体。检测重组腺病毒效价后，转染人脐静脉内皮细胞ECV304，检测目的基因的转染表达。 结果：通过对构建质粒克隆进行测序及酶切，证实携带HIF-lα基因的重组腺病毒载体pAdxsi-GFP-HIF构建成功，且构建的重组腺病毒纯度好、效价高。以100 MOI转染ECV304细胞24 h后在荧光显微镜下可观察到细胞有较强的绿色荧光表达，转染48 h时荧光表达更强，且培养上清液中HIF-1蛋白表达水平为（48.93±3.86）ng/mL。 结论：本实验构建的携带HIF-1α基因的腺病毒载体pAdxsi-GFP-HIF转染效率及目的基因的蛋白表达水平均较高，有望应用于缺血缺氧组织局部。

Construction of recombinant adenovirus vector pAdxsi-GFP-HIF containing hypoxia inducible factor gene and its expression in endothelial cells

Objective： To construct a recombinant adenovirus (pAdxsi-GFP-HIF) encoding human hypoxia inducible factor 1αgene (HIF-1α) and to express it in endothelial cells. Methods： HIF-1αgene was obtained from human lung cancer cell line A549，which was cultured in hypoxia condition，by RT-PCR.The HIF-1αgene was subcloned into shuttle vector pShuttle-CMV-EGFP at KpnI and BamHI sites.After identified with restriction enzymes，plasmid pShuttle-GFP-HIF was linearized by digestion with restriction endonuclease I-CeuI and I-SceI，and subsequently cotransformed into E.coli DH5a with adenoviral backbone plasmid pAdxsi to make homologous recombination.After linearized by PacI，the homologous recombinant adenovirus plasmid was transfected into 293 cells to package and amplify.The recombinant adenovirus was infected with human umbilical vein endothelial cells (ECV304)，and the expression level of HIF-1αprotein was evaluated by ELISA. Results： The recombinant adenovirus vector containing HIF-1αgene (pAdxsi-GFP-HIF) was successfully constructed and amplified with titer of 3.38×1010 pfu/mL.The green fluorescence protein was detected under fluorescent microscope in ECV304 at 24h after transfection and with a stronger degree after 48h.The concentration of HIF-1 protein was (48.93±3.86)ng/mL in supernatant at 48 h after transfection. Conclusion： A recombinant adenovirus vector pAdxsi-GFP-HIF，encoding human hypoxia inducible factor 1αgene，has been constructed in vitro and expressed successfully in ECV304 cells.