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目的:克隆人PKGⅠa基因,构建其重组腺病毒载体。方法:用RT-PCR方法从人肺动脉平滑肌组织中扩增PKGⅠa基因全长,经T/A克隆后,亚克隆至腺病毒穿梭质粒pAdTrack-CMV上,构建穿梭质粒pAdTrack-PKGⅠα。PmeⅠ酶切pAdTrack-PKGⅠα,然后分别将腺病毒骨架质粒pAdEasy-1和穿梭质粒pAdTrack-PKGⅠα转化至BJ5183感受态细菌中进行同源重组,PacⅠ酶切线性化重组质粒AdCMV-PKGⅠα后转染Ad293细胞进行病毒包装和扩增。检测PKGⅠα基因的表达,并用绿色荧光蛋白(GFP)法测定其滴度。结果:用RT-PCR方法,从人肺动脉中层扩增出PKGⅠα,测序证实为人PKGⅠα基因。构建了PKGⅠα基因的重组腺病毒载体并制备出高滴度重组病毒保存液。结论:成功地克隆了人PKGⅠα基因,并构建其重组腺病毒载体,为进一步研究PKGⅠα基因在低氧肺血管重建中的作用提供了有效的基因转移载体。
Objective: To clone human PKGⅠa gene and construct its recombinant adenovirus vector. Methods: The full length of PKGⅠa gene was amplified from human pulmonary artery smooth muscle by RT-PCR. After T / A cloning, the full length of PKGⅠa gene was subcloned into the adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-PKGⅠα. PAdⅠwas used to digest pAdTrack-PKGⅠα, then the adenoviral backbone plasmid pAdEasy-1 and shuttle plasmid pAdTrack-PKGⅠα were transformed into BJ5183 competent cells for homologous recombination respectively. The recombinant plasmid AdCMV-PKGⅠα was digested with PacⅠ and then transfected into Ad293 cells Virus packaging and amplification. The expression of PKGⅠα gene was detected and its titer was determined by green fluorescent protein (GFP) assay. Results: PKGⅠα was amplified from human pulmonary artery by RT-PCR and confirmed to be human PKGⅠα gene by sequencing. The recombinant adenoviral vector containing PKGⅠα gene was constructed and a high titer recombinant virus stock solution was prepared. CONCLUSION: The human PKGⅠα gene was cloned successfully and its recombinant adenovirus vector was constructed. It provides an effective gene transfer vector for further studying the role of PKGⅠα gene in hypoxic pulmonary vascular remodeling.