Perilipin5基因功能结构域载体的构建及亚细胞定位分析

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。

Construction and Subcellular Localization of Perilipin5 Functional Domain

Objective： To constructe the truncations ofperilipin5 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.