表达HIF1α与EGFP融合蛋白的重组逆转录病毒载体构建及鉴定  

Construction and identification of retroviral vector expressing hypoxia-inducible factor-1α and EGFP fusions

作　　者：段小军[1] 周跃[2] 杨柳[1] 董世武[3] 唐康来[1] 

机构地区：[1]第三军医大学西南医院关节外科,重庆400038 [2]第三军医大学新桥医院骨科,重庆400037 [3]第三军医大学基础部解剖学教研室,重庆400038

出　　处：《重庆医学》2005年第3期373-374,377,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(30270375)

摘　　要：目的　构建表达人缺氧诱导因子 1α(HIF 1α)与EGFP融合蛋白的重组逆转录病毒载体,并观察其对 NIH3T3 细胞感染效率。方法　采用基因工程技术,将HIF 1α基因片段克隆至逆转录病毒载体 pLEGFP N1上,鉴定后用脂质体法转染 PT67细胞包装、扩增,最后用重组逆转录病毒感染NIH3T3细胞。其中采用PCR方法对重组病毒进行鉴定,利用绿色荧光蛋白作为报告基因,对病毒滴度和感染效率进行监测。结果　酶切、测序及 PCR结果与 HIF 1α基因重组逆转录病毒载体的预期结果一致,病毒滴度达1.2×106 pfu/ml,并对NIH3T3细胞有强感染能力。结论　应用基因工程技术,成功构建了表达HIF 1α与EGFP融合蛋白的重组逆转录病毒载体,为应用于治疗性血管生成创造了条件。Objective To construct the recombinant retrovirus of human hypoxia-inducible factor-1α(HIF-1α) gene and to observe its ability to infect NIH3T3 fibroblasts.Methods The full-length human HIF-1α cDNA was cloned into the retroviral vector pLEGFP-N1 to express an amino-terminal fusion protein of HIF-1α and enhanced green fluorescent protein (EGFP) by the method of gene engineering. Then the recombinant retrovirus was transfected into NIH3T3 cells using Lipofectine DOTAP. The target gene was detected by polymerase chain reaction (PCR). The titer and its infection rate were determined using the EGFP expression with fluorescent microscope.Results Restriction endonuclease, sequencing and PCR analyses confirmed that the human HIF-1α cDNA was successfully inserted into the retroviral vector. The titer of recombinant retrovirus with HIF-1α gene was 1.2×10 6 pfu/ml and the retrovirus had a strong effect on NIH3T3 cells.Conclusion The recombinant retrovirus containing HIF-1α gene was successfully constructed by the method of gene engineering and it established a base for therapeutic angiogenesis.

关 键 词：逆转录病毒 缺氧诱导因子-1 基因克隆 

分 类 号：Q782[生物学—分子生物学]