携EGFP的人低氧诱导因子-1α腺病毒载体的构建及鉴定  

Construction and identification of adenovirus vector of human hypoxia-inducible factor-1α fused with enhanced green fluorescent protein

作　　者：谢宜军[1] 王月刚[1] 郭寿贵[1] 童锴[1] 吴平生[1] 

机构地区：[1]南方医科大学南方医院心内科,广州510515

出　　处：《第三军医大学学报》2007年第2期117-120,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30370587)~~

摘　　要：目的构建携EGFP的人低氧诱导因子(hypoxia-induciblefactor1alpha,HIF-1α)腺病毒表达载体,为研究人HIF-1α基因对冠心病的血管新生作用奠定基础。方法采用分子克隆技术,以KpnⅠ、ApaⅠ双酶切pcDNA3.1(+)-HIF-1α质粒获得HIF-1αcDNA,克隆到质粒pEGFP-C1,以NheⅠ、ApaⅠ双酶切得到EGFP-HIF-1αcDNA,重组到穿梭质粒pShuttle2,再以PI-SceⅠ和I-CeuⅠ双酶切重组穿梭质粒,获得含有EGFP-HIF-1αcDNA的表达盒,与线性化的腺病毒骨架质粒Adeno-XViralDNA连接,重组成pAdeno-EGFP-HIF-1α腺病毒质粒,脂质体法转染HEK293细胞,荧光显微镜下观察结果,在HEK293细胞中包装成为重组Adeno-EGFP-HIF-1α腺病毒,并进行PCR鉴定及滴度测定。结果经酶切鉴定及PCR证实重组腺病毒质粒构建成功,包装后冻融细胞的上清PCR检测重组腺病毒包装成功,病毒滴度为2×109nfu/ml。结论成功构建重组腺病毒Adeno-EGFP-HIF-1α,为冠心病的HIF-1α基因治疗研究奠定更直观的基础。Objective To construct adenovirus vector containing the enhanced green fluorescent protein （EGFP）-HIF-1 alpha gene for studying therapeutic angiogenesis of coronary heart disease. Methods Human HIF-1α cDNA obtained from the plasmid pcDNA3.1 （ ＋ ）-HIF-1 alpha with double digestion of Kpn Ⅰ and Apa Ⅰ was cloned into plasmid pEGFP-C1. EGFP-HIF-1 alpha cDNA obtained from pEGFP-C1 with double digestion of Nhe Ⅰ and ApaⅠ was cloned into plasmid pShuttle2. The expression cassette containing EGFP-HIF-1 alpha cDNA was obtained from the recombinant pShuttle2-EGFP-HIF-1 alpha with double digestion of PI-Sce Ⅰ and I-Ceu Ⅰ , then ligated to Adeno-X Viral DNA. The recombinant adenoviral plasmid was identified and transfected into the adenoviral packaging cell HEK293 by lipofectamine2000. The recombinant adenovirus was confirmed by polymerase chain reaction （PCR） and the titer was determined. Results The recombinant pAdeno-EGFP-HIF-1 alpha was correctly constructed and confirmed by restriction endonuclease analysis and PCR amplification. The transfected HEK293 cells were lysed by freeze-thawing to obtain the recombinant adenovirus in the lysate. The PCR product of the lysate confirmed the presence of recombinant adenovirus. The viral titer was 2×10^9 nfu/ml. Conclusion The recombinant adenovirus containing the EGFP-HIF-1 alpha gene was successfully constructed. It provides the further appreciable foundation of HIF-1 alpha gene therapy for coronary heart disease.

关 键 词：低氧诱导因子-1Α 绿色荧光蛋白 基因治疗 

分 类 号：Q503[生物学—生物化学] Q513.1