Rab GDP解离抑制因子的定位和表达

Localization and Expression of Rab GDP Dissociation Inhibitor

目的:构建含人GDI1和GDI2基因的真核表达载体进行定位和蛋白表达研究。方法:用PCR从U251细胞cDNA克隆GDI1和GDI2基因,构建真核表达载体pEGFP-N2-GDI1和pEGFP-N2-GDI2,转染HEK293T细胞。荧光显微镜观察GDI1和GDI2蛋白的细胞内定位,再通过SDS-PAGE和Western blotting鉴定蛋白的表达。结果:成功克隆到1 341bp和1 335bp的人源GDI1和GDI2基因,并准确插入真核表达载体pEGFP-N2中,荧光观察这两个蛋白定位到细胞浆中,并能利用标签抗体检测到GDI1和GDI2的表达。结论:GDI1和GDI2能够定位到细胞浆,并能通过Western blotting检测,为进一步研究GDI1和GDI2的功能奠定了基础。

Objective： To study the localization and expression of Rab GDP dissociation inhibitor construct eukaryotic expression vector con- taining the human GDI1 and GDI2 gene. Method ：GDI1 and GDI2 gene were amplified from U251 cell cDNA by PCR, and subeloned into eukaryotic expression vector pEGFP - N2. Recombinant plasmid were transfected into HEK293T cells. Localization of GDII and GDI2 were observed through fluorescence microscopy. SDS - PAGE and Western blotting were used to identify the expression of recombinant GDI1 and GDI2. Result：Human GDI1 gene fragment of 1 341 bp and GDI2 gene fragment of 1 335bp were obtained, and was subeloned into eukaryot- ic expression vector pEGFP - N2. GDI1 and GDI2 were found localized cytoplasm, and 76kDa recombinant GDI1 and GDI2 were success- fully expression in HEK293T cells. Conelusion：GDI1 and GDI2 localized to cytoplasm and can be detected by Western blotting,laid the foundation to further study the function of GDI1 and GDI2.