BTBD10基因的真核表达载体构建和亚细胞定位

目的构建BTBD10重组真核表达载体，观察BTBD10在HEK293细胞中的定位。方法利用PCR方法扩增BTBD10全长，经HindIII和BamHI酶切后连接入pGFP-C3真核表达载体，构建pGFP—C3-BTBD10重组表达质粒，转染HEK293细胞，利用激光共聚焦显微镜观察BTBD10在细胞中的定位。结果经双酶切和核酸序列分析证实重组质粒包含有正确编码的BTBD10读码框。激光共聚焦显微镜观察到空质粒pGFP—C3转染后，整个细胞内弥散绿色荧光，而转染pGFP-C3-BTBD10重组载体后，可见绿色荧光分布于HEK293细胞膜一侧的细胞浆中，提示BTBD10定位于细胞浆中。结论成功构建人BTBD10真核表达载体，BTBD10定位于哺乳细胞的细胞浆中，可能发挥蛋白之间的相互作用。

Construction of Eukaryotic Expression Plasmid of BTBD10 and its Subcellular Localiztion

AIM To induce the expression of BTBD10 in E. coli a nd preparethe rabbit polyc lonal antibody against it. Methods The full length BTBD10 gene was amplified by PCR, and cloned into green fluorescence protein vector pEGFP-C3 to construct recombinant expression plasmid pEGFP-C3-BTBD10. After recombinant plasmids were transfected into HEK293 cells by lipofectimane 2000, the expression of BTBD10 gene and its subce llular localization were observed by fluorescent microscope and laser confocal microscope. Results Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid was correct. Detected by the laser confocal microscope, the BTBD10 fusion protein was localized in cytoplasm of HEK293 cell . As control, GFP was distributing in the whole cell. Conclusion The recombinant expres- sion plasmid pEGFP-C3-BTBD10 was constructed successfully. BTBD10 protein was located in cytoplasm of HEK293 cell, which may play important role in mutual interaction among proteins.