内皮型一氧化氮合酶腺病毒表达载体的构建与鉴定

Construction and identification of adenovirus expression vector for endothelial nitric oxide synthase

目的 构建重组内皮型一氧化氮合酶(eNOS)腺病毒表达系统载体。方法 eNOS的cDNA插入pcDNA3.1(-)中,进行DNA测序、酶切鉴定;插入片段用Xbal和AflⅡ双酶切后连接到pshuttle中,并予酶切鉴定;用P1-sce Ⅰ和Ⅰ-ceu Ⅰ双酶切下pshuttle中eNOS的cDNA插入腺病毒载体,通过PCR鉴定。结果 正确构建重组eNOS腺病毒表达系统,并被目的基因的PCR鉴定证实。结论 重组腺病毒表达载体构建正确,为其在预防经皮腔内动脉成形术术后再狭窄基因转染奠定基础。

Objective To construct recombinant endothelial nitric oxide synthase ( eNOS) adenovirus for evaluating the possibility of gene therapy in restenosis. Methods eNOS cDNA was inserted to pcDNA3. 1( - ) vector, identified by DNA analysis and restriction endonuclease analysis . The inserted cDNA was excised with Xba Ⅰ and Afl Ⅱ , cloned to pshuttle vector. Pshuttle-eNOS was identified by restriction endonuclease analysis, excised with Pl-sce Ⅰ and Ⅰ-ceu Ⅰ , then cloned into adenoviral DNA. An adenovirus expression vector for endothelial NO synthase (AdCMV-eNOS) was identified by PCR. Results The recombinant adenoviral vector for endothelial nitric oxide synthase was correctly constructed and confirmed by PCR method. Conclusion The recombinant vector can be constructed correctly, which establishes the base for eNOS transfection in the prevention of postangioplasty restenosis.