| 曹珊珊,白翔宇,吉晓莹,万绍贵,肖东斌,鲍登克.Ratiometric-pericam-mt慢病毒的构建及其线粒体钙检测功能的验证[J].,2020,(8):1441-1445 |
| Ratiometric-pericam-mt慢病毒的构建及其线粒体钙检测功能的验证 |
| Construction of Ratiometric-pericam-mt-lentivirus and Validation of the Function in Mitochondrial Calcium Detection |
| 投稿时间：2019-09-24 修订日期：2019-10-19 |
| DOI：10.13241/j.cnki.pmb.2020.08.009 |
| 中文关键词: 慢病毒载体 线粒体定位荧光蛋白 线粒体钙 |
| 英文关键词: Lentiviral vector Ratiometric-pericam-mt Mitochondrial calcium |
| 基金项目:河南省重点研发与推广项目（182102310120） |
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| 中文摘要: |
| 摘要 目的：基于由环状异构黄色荧光蛋白(Circularly Permuted EYFP)改造的具有线粒体定位功能的Ratiometric-pericam-mt(rp-mt)荧光蛋白构建携带有rp-mt基因的慢病毒载体，验证其在线粒体钙检测中的功能，为进一步研究线粒体钙稳态在细胞生命活动中发挥的作用提供参考依据。方法：采用慢病毒载体EF-1αF-puro和rp-mt质粒构建携带有rp-mt基因的重组慢病毒载体，经脂质体法在293T细胞中与包装质粒共转染，收集构建好的重组慢病毒EF-1αF-puro-rp-mt并体外感染人肝癌细胞SNU-739，验证重组慢病毒EF-1αF-puro-rp-mt的活性功能。结果：以慢病毒载体EF-1αF-puro为骨架质粒，成功构建了携带有rp-mt基因的重组慢病毒载体(EF-1αF-puro-rp-mt)，收集获得的重组慢病毒滴度为4×106TU/mL。将携带有rp-mt基因的慢病毒EF-1αF-puro-rp-mt感染SNU-739细胞48小时后，在荧光显微镜下观察到有超过85%的细胞发出绿色荧光，定位于细胞线粒体内，且对SNU-739细胞进行Ru360和CGP37157处理后，细胞荧光强度随线粒体内钙水平改变而增强或减弱。结论：采用脂质体包装法成功构建了携带有rp-mt基因的重组慢病毒EF-1αF-puro-rp-mt，细胞经该慢病毒转染后可稳定表达具有线粒体定位功能的荧光蛋白rp-mt，准确地检测细胞线粒体钙离子水平。 |
| 英文摘要: |
| ABSTRACT Objective: To construct lentiviral vector carrying the rp-mt gene based on a ratiometric-pericam-mt (rp-mt) fluorescent protein modified by a circularly permuted yellow fluorescent protein (cpEYFP), verify the function of lentiviral vector in the mitochondrial calcium detection, and provide a reference for the further study on the role of mitochondrial calcium homeostasis in the cell life. Methods: A recombinant lentivirus vector carrying the rp-mt gene was constructed with the lentivirus vector EF-1αF-puro and the rp-mt plasmid, and was co-transfection with package plasmid in 293T cells. The constructed recombinant lentivirus was collected and infected the HCC cells SNU-739 in vitro. The function of recombinant lentivirus was validated. Results: Are combinant lentivirus vector carrying rp-mt gene with the lentivirus vector EF-1αF-puro was successfully constructed, the titer of recombinant lentivirus was 4×106TU/mL. More than 85% of the SNU-739 cells were observed to emit green fluorescence under a fluorescence microscope after infected by the recombinant lentivirus vector for 48 hours. The fluorescence located in the mitochondria of cells and was brighter or weaker as the variation of the concentration of calcium in mitochondria with Ru360 or CGP37157 treated of the cells. Conclusion: Are combinant lentivirus vector carrying rp-mt gene with the liposomes packing method was successfully constructed. The cell can stably express the fluorescigenic protein which located in the mitochondrial after infected by the recombinant lentivirus, and the calcium of the mitochondrial can be accurately detected. |
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