携带内皮型一氧化氮合酶基因重组腺病毒载体的构建

Construction of recombinant adenovirus vector coding endothelial nitric oxide synthase gene

目的：构建带有内皮型一氧化氮合酶（ｅＮＯＳ）基因的重组腺病毒载体。方法：将ｅＮＯＳ ｃＤＮＡ克隆于腺病毒穿梭质粒ｐＣＡ１４，得到重组质粒ｐＣＡ１４－ＣＭＶ－ｅＮＯＳ。采用磷酸钙ＤＮＡ沉淀法将质粒ｐＣＡ１４－ＣＭＶ－ｅＮＯＳ与腺病毒拯救质粒ｐＢＨＧ１０共转染２９３细胞，通过同源重组，生成带有ｅＮＯＳ基因的复制缺陷型重组腺病毒载体（ＡｄＣＭＶｅＮＯＳ）。ｅＮＯＳｃＤＮＡ重组进入腺病毒Ｅ１区并受ＣＭＶ启动子控制。结果：经形态学、病毒ＤＮＡ酶切、ＰＣＲ和ＲＴ－ＰＣＲ等方法的鉴定，证实了载体构建的正确性。结论：带有ｅＮＯＳ基因的重组腺病毒载体的成功构建，为心血管等疾病的基因治疗打下了基础。

Objective:With the aim of constructing recombinan t adenovirus vec tor that codes endothelial nitric oxide synthase(eNOS) gene.Methods:eNOS cDNA was inserted into adenovirus shuttle plasmid pCA14 to generate a recombinant plasmid pCA14 CMV eNOS. The plasmid, pCA14 CMV eNOS, together with adenovirus rescue plasmid, pBHG10, was cotransfected into 293 cells by c alcium phosphate DNA coprecipitate method, and then a replication defective re combinant adenovirus that codes eNOS gene AdCMVeNOS was propagated in 293 cells via homologous recombinant. The eNOS cDNA was inserted into the early 1 region i n the adenovirus genome and driven by the human cytomegalovirus (CMV) promoter. Results:The methods of morphology, restriction analysis and PCR/RT PCR were employed and proved the soundness of the construction.Conclus ion:This work lay a foundation in gene therapy of cardiovascular diseases.