携带人野生型PKGⅠα基因的重组腺病毒载体的构建及鉴定  

Construction and identification of recombinant adenovirus containing wild-type human PKGⅠα gene

作　　者：易斌[1] 陆俊羽[2] 白莉[2] 王关嵩[2] 钱桂生[2] 

机构地区：[1]第三军医大学西南医院麻醉科,重庆400038 [2]第三军医大学新桥医院呼吸科,重庆400037

出　　处：《第二军医大学学报》2009年第1期69-72,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30700347);重庆市自然科学基金(2007BB5045)~~

摘　　要：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 PKGⅠα gene and construct a recombinant adenovirus vector containing wild-type PKGⅠα.Methods：RT-PCR was used to amplify the full-length PKGⅠα gene from human pulmonary arterial smooth muscle.After T/A cloning,PKGⅠα cDNA was cloned into shuttle plasmid pAdTrack-CMV to construct pAdTrack-PKGⅠα.The plasmid was linearized by PmeⅠ and transformed into BJ5183 E.coli,where the plasmid was recombined with pAdEasy-1 by homologous recombination.The recombinants were then transfected into Ad293 cells by Lipofectamine2000 for packaging the adenovirus,the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope to determine the titer.Results：PKGⅠα was successfully amplified from human pulmonary arterial smooth muscle by RT-RCR.After 3 cycles of amplification,the titer of adenovirus containing wild-type PKGⅠα reached the indicated level.Conclusion：We have successfully constructed PKGⅠα gene and constructed the PKGⅠα recombinant adenovirus,which provides a foundation for the study of PKGⅠα function and its role in hypoxia pulmonary vessel remodeling.

关 键 词：PKGⅠα基因 腺病毒载体 基因克隆 DNA重组 

分 类 号：R349.65[医药卫生—基础医学]