【摘要】 腺病毒具有免疫原性,可被机体快速免疫清除,导致目的基因失去表达,这是其作为基因治疗载体的主要缺陷。一些特定的细胞,如角膜上皮细胞和睾丸Sertoli细胞能表达和分泌Fas配体（FasL）,诱导带有Fas分子的免疫细胞发生凋亡。目的:构建FasL和诱导型一氧化氮合酶（iNOS）共表达PCA₁₃质粒用于腺病毒的包装,以进一步观察FasL对腺病毒载体的免疫保护作用。方法:利用内源性核糖体进驻位点（IRES）,通过感受态细胞的制备和转化、质粒抽提、琼脂糖凝胶电泳以及限制性内切酶等多种基因工程技术,经多步亚克隆后完成能同时表达FasL和iNOS基因的多顺反子腺病毒表达载体PCA₁₂/FasL-IRES-iNOS的构建。结果:新质粒PCA₁₃/FasL-IRES-iNOS经限制性内切酶酶切后得到1600 bp（600 bp FasL,1000 bp IRES）和4000 bp（iNOS）的相应基因片断,基因序列经测定与基因文库报道相符。结论:成功构建了多顺反子腺病毒表达载体PCA₁₃/FasL-IRES-iNOS,为门脉高压症的基因治疗打下了基础。更多还原

【Abstract】 The immunogenicity of adenovirus that causes the clearance by the host, is the major barrier to successful gene transfer. Some special cells such as cornea-epithelial cells and testicular Sertoli cells can induce apoptosis in immune cells with Fas molecule through expression and secretion of Fas ligand （FasL）. Aims: To construct FasL and inducible nitric-oxide synthase （iNOS） co-expressed PCA₁₃ plasmid used for packing of adenovirus, so as to observe the immunoprotective effect of FasL on adenovirus vector. Methods: By way of internal ribosome entry site （IRES）, genetic engineering techniques as preparation and transformation of competent cells, plasmid extraction, agarose gel electrophoresis, restriction enzymolysis were used for the construction of polycistron adenoviral expression vector PCA₁₃/FasL-IRES-iNOS, which could co-express with FasL and iNOS gene after multi-subcloning steps. Results: FasL and iNOS gene connected with IRES were successfully cloned to PCA₁₃ plasmid and verified by enzymolysis （600 bp FasL, 1000 bp IRES and 4000 bp iNOS）, and the gene sequence was concordant with the Genebank. Conclusions: It is one of the crucial steps to construct successfully the polycistron adenoviral expression vector PCA₁₃/FasL-IRES-iNOS for the gene therapy of portal hypertension.更多还原