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| 过氧化物酶体特异性绿色荧光蛋白腺病毒载体的构建 |
| 江天行1△,刘育鹏2△,李阳1,宋晓伟1,赵仙先1,郭志福1,史承勇3,吴弘1* |
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(1. 海军军医大学(第二军医大学)第一附属医院心血管内科, 上海 200433;
2. 中国人民解放军总医院京中医疗区, 北京 100141;
3. 中国人民解放军联勤保障部队第九〇三医院心内科, 杭州 310013
△共同第一作者
*通信作者) |
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| 摘要: |
| 目的 构建一种特异性标记过氧化物酶体的GFP腺病毒载体。方法 设计含过氧化物酶体信号肽的GFP序列，PCR法扩增该序列片段，并将其与腺病毒穿梭质粒载体pAdTrack连接，然后将连接产物转化到大肠埃希菌DH5α感受态细胞中获得重组质粒并测序鉴定。重组质粒经限制性内切酶PmeⅠ酶切线性化后，转化到含腺病毒骨架质粒pAdEasy-1的BJ5183感受态细胞中进行同源重组。重组腺病毒质粒经限制性内切酶PacⅠ酶切线性化后感染293A细胞，进行病毒包装，得到含过氧化物酶体信号肽的Ad-GFP-Peroxi重组腺病毒颗粒。用腺病毒 Ad-GFP-Peroxi和不含过氧化物酶体信号肽的腺病毒Ad-GFP感染培养的大鼠心肌细胞H9C2，48 h后在荧光显微镜下观察绿色荧光强弱。将用Ad-GFP-Peroxi腺病毒感染的H9C2细胞分为正常培养组和1% O2浓度培养组，培养24 h后于共聚焦显微镜下观察，并进行荧光信号聚集情况分析。结果 含过氧化物酶体信号肽的GFP腺病毒载体构建成功。与不含过氧化物酶体信号肽的腺病毒Ad-GFP相比，含过氧化物酶体信号肽的腺病毒Ad-GFP-Peroxi能够特异性显示大鼠心肌细胞H9C2内过氧化物酶体的分布，且缺氧培养的H9C2细胞内过氧化物酶体分布出现聚集现象。结论 利用同源重组方法成功构建了过氧化物酶体特异性GFP腺病毒，其对大鼠心肌细胞H9C2有较高的感染效率，共聚焦显微镜下可以明确显示心肌细胞内过氧化物酶体的分布情况。 |
| 关键词: 心肌细胞 腺病毒 绿色荧光蛋白 过氧化物酶体 共聚焦显微镜 |
| DOI：10.16781/j.CN31-2187/R.20230011 |
| 投稿时间：2023-01-16修订日期：2023-06-05 |
| 基金项目:国家自然科学基金（82070419）. |
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| Construction of adenovirus vector carrying peroxisome specific green fluorescence protein |
| JIANG Tianxing1△,LIU Yupeng2△,LI Yang1,SONG Xiaowei1,ZHAO Xianxian1,GUO Zhifu1,SHI Chengyong3,WU Hong1* |
(1. Department of Cardiovasology, The First Affiliated Hospital of Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Central Beijing Medical Sector, PLA General Hospital, Beijing 100141, China;
3. Department of Cardiology, No. 903 Hospital of Joint Logistics Support Force of PLA, Hangzhou 310013, Zhejiang, China
△Co-first authors.
* Corresponding author) |
| Abstract: |
| Objective To construct a green fluorescence protein (GFP) adenovirus vector specifically labeled with peroxisomes (Ad-GFP-Peroxi). Methods The GFP sequence modified with peroxisome signal peptide sequence was designed. The sequence fragment was amplified by polymerase chain reaction and linked with adenovirus shuttle plasmid vector (pAdTrack); then the connecting product was transformed into E. coli DH5α. The recombinant plasmids were obtained from DH5α cells and sequenced for identification. The recombinant plasmid was linearized by PmeⅠ, and homologous recombination was carried out in BJ5183 competent cells containing pAdEasy-1. The recombinant plasmid was linearized by PacⅠ, and then used to infect 293A cells for virus packaging to obtain Ad-GFP-Peroxi particles. Rat cardiomocytes H9C2 were infected with Ad-GFP-Peroxi adenovirus or Ad-GFP adenovirus, and the green fluorescence intensity was observed under fluorescence microscope after 48 h. H9C2 cells infected with Ad-GFP-Peroxi adenovirus were divided into 2 groups (normal culture group and 1% oxygen concentration group), observed under confocal microscope after 24 h, and the fluorescence signal aggregation was analyzed. Results The recombinant Ad-GFP-Peroxi was successfully constructed. Compared with Ad-GFP, Ad-GFP-Peroxi could specifically display the distribution of peroxisomes in H9C2 cells. Peroxisomes accumulated in H9C2 cells after hypoxia stimulation. Conclusion Ad-GFP-Peroxi is successfully constructed by homologous recombination, and it can highly infect rat cardiomyocytes H9C2, clearly showing the distribution of peroxisomes in cardiomyocytes under confocal microscope. |
| Key words: cardiomyocytes adenovirus green fluorescence protein peroxisomes confocal microscope |
