| 李航1 赵振广1 卢晓朝2 刘芳3 李青3 潘树义1△.Perilipin5 基因功能结构域载体的构建及亚细胞定位分析*[J].,2014,14(5):805-810 |
| Perilipin5 基因功能结构域载体的构建及亚细胞定位分析* |
| Construction and Subcellular Localization of Perilipin5Functional Domain |
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| DOI： |
| 中文关键词: perilipin5 功能结构域 脂滴 亚细胞定位 |
| 英文关键词: Perilipin5 Functional domain Lipid droplet Subcellular location |
| 基金项目:国家自然科学基金项目（81000171；51203185） |
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| 中文摘要: |
| 摘要目的：依据perilipin5 的功能结构域，构建含perilipin5 截断体的真核表达载体，并研究它们的亚细胞定位。方法：以小鼠肝脏
cDNA 文库为模板，PCR 扩增出perilipin5 的全长及功能结构域，将之分别装载入真核表达载体PCMV5中，并引入HA标签。酶
切和测序鉴定，脂质体法将构建的质粒转染293T细胞，Western blot 验证表达，免疫荧光检测标记HA，于荧光显微镜下观察perilipin5
各结构域的亚细胞定位。结果：构建的质粒序列正确，转染细胞后可检测到HA- perilipin5 融合蛋白的表达，免疫荧光显示
含有1-188aa 结构域的perilipin5 截断体可定位于脂滴表面，1-188aa 一旦缺失perilipin5 的截断体则弥散于胞内。结论：包含perilipin5
功能结构域的真核表达载体构建成功, perilipin5 的1-188aa 与其脂滴定位密切相关。 |
| 英文摘要: |
| ABSTRACT Objective:To constructe the truncations of perilipin5 according to the sequence analysis and observe the subcellular
location of truncated perilipin5 in mammalian cells. Methods: The full length and truncations of perilipin5 gene were PCR-amplified
from mouse liver cDNA library, followed by subcloning into the PCMV5 eukaryotic expression vector, containing a HA-tagged
fragment. Obtained recombination plasmids were digested by restriction enzyme and sequencing, then transfected into 293T cells. The
expression of fusion protein of perilipin5 with HA was confirmed by western blot. The subcellular location of perilipin5 in transfected
cells was observed under fluorescence micro scope after immunofluorescence. Results:The validity of all the constructs was confirmed
by DNA sequencing. Distribution of perilipin5 truncations was discrepant obviously, the truncations with 1-188aa was localized
predominantly on the surface of lipid droplets. In contrast, the loss of 1-188aa residues showed a diffused pattern on nucleus and
cytoplasm. Conclusion:The eukaryotic expression vector containing functional domain of perilipin5 were constructed successfully.
Localization of perilipin5 on lipid droplet may be related to the 1-188aa domain. |
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