人突变型低氧诱导因子1α腺病毒载体的构建及鉴定论文.docVIP

　　人突变型低氧诱导因子1α腺病毒载体的构建及鉴定论文 郭寿贵，吴平生，王月刚，傅锐斌 【关键词】 腺病毒 Construction and identification of human mutant hypoxia inducible factor1α adenovirus vector 【Abstract】 AIM: To construct adenovirus vector containing the mutant HIF1α gene and to study the effect of mutant hypoxia inducible factor1α on the angiogenesis of coronary heart disease. METHODS: Human mutant HIF1α cDNA obtained from the plasmid pcDNA3.1(+)HIF1α id pShuttle2. The expression cassette containing mutant HIF1α cDNA the rebinant pShuttle2 id ine2000 mediated gene transfer method to pack the virus. The rebinant adenovius ed by polymerase chain reaction (PCR) and the titer ined. RESULTS: The rebinant pAdenoHIF1α ed by restriction endonuclease analysis and DNA sequencing analysis. The transfected HEK293 cells ed the presence of rebinant adenovirus. The viral titer utant HIF1α gene has been successfully constructed, utant HIF1α gene therapy of coronary heart disease. 【Keyutant; adenovirus vector; gene therapy 【摘要】 目的: 构建人突变型低氧诱导因子1(HIF1)α腺病毒表达载体，研究人突变型HIF1α基因对冠心病的血管新生作用. 方法: 采用分子克隆技术，由pcDNA3.1(+)HIF1α（突变型）质粒获得突变型HIF1α cDNA，克隆到穿梭质粒pShuttle2，以PISce I和ICeu I双酶切重组穿梭质粒.freelent，HRE）结合调控血管内皮生长因子（vascular endothelial groain,ODDD）内564位脯氨酸残基Pro564发生羟基化，迅速被降解. 为此我们在已定点突变其Pro564为丙氨酸（Ala）基础上，构建重组人突变型HIF1α腺病毒载体，以进一步研究常氧条件下该突变型基因表达及对冠心病的血管新生作用. 1材料和方法 1.1材料 1.1.1试剂、试剂盒各种限制性内切酶PacⅠ，PISce I，ICeu I等及T4 DNA连接酶、Taq DNA聚合酶、XGal分别购自Takara，NEB等公司；DMEM培养基(Gibco)，胎牛血清(PAA)，转染试剂Lipofectamine 2000(Invitrogen)，超纯质粒提取试剂盒（Qiagen）. 1.1.2质粒、菌株、细胞系等AdenoXTM Adenoviral Expression Systems(BD.Clontech):包括有pShuttle2, pShuttle2laZ, AdenoXTM Viral DNA等，重组质粒pcDNA3.1（+）HIF1α(突变型)(本室构建)，大肠杆菌DH5α（本室保存），HEK293细胞（中科院上海细胞所）. 1.1.3引物HIF1αcDNA片段引物（由博亚公司设计并合成），上游引物：5′GACACAGAAGCAAAGAACCC3′，下游引物：5′TCAAAGCGACAGATAACACG3′，PCR产物片段长460 bp. BD.Clontech所提供引物（两个引物分别与穿梭质粒表达盒及骨架质粒的部分序列结合），上游引物：5′TAGTGTGGCGGAAGTGTGATGTTGC3′，下游引物：5′AGATCTGAGCTTTCGCTACC3′，PCR产物片段长287 bp. 1.2方法 1.2.1重组穿梭质粒构建以NheⅠ，ApaⅠ双酶切pcDNA3.1（+）HIF1α（突变型，下同）及pShuttle2，胶回收突变型HIF1α片段及pShuttle2