| 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。 |
| 关键词: PKGⅠα基因 腺病毒载体 基因克隆 DNA重组 |
| DOI：10.3724/SP.J.1008.2009.00069 |
| 投稿时间：2008-07-31修订日期：2008-09-06 |
| 基金项目:国家自然科学基金(30700347),重庆市自然科学基金(2007BB5045). |
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| Construction and identification of recombinant adenovirus containing wild-type human PKGⅠα gene |
| YI Bin1, LU Jun-yu2, BAI Li2, WANG Guan-song2, QIAN Gui-sheng2* |
| (1.Department of Anesthesiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China*2.Department of Respiratory Medicine Medicine,Xinqiao Hospital,Third Military Medical University,Chongqing 400037) |
| Abstract: |
| 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. |
| Key words: PKGⅠα gene adenovirus vector gene cloning DNA recombination |
