FAM92A1-289基因的真核表达载体构建和亚细胞定位

利用PCR方法扩增FAM92A1-289全长,经BamH I和Xho I酶切后连接入pEGFP-N1真核表达载体,构建pEGFP-N1-FAM92A1-289重组表达质粒,转染Hela细胞,利用荧光显微镜观察FAM92A1-289在细胞中的定位。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FAM92A1-289读码框。荧光显微镜观察到空质粒pEGFP-N1转染后,整个细胞内弥散绿色荧光,而转染pEGFP-N1-FAM92A1-289重组载体后,可见绿色荧光分布于Hela细胞核中,显示FAM92A1-289定位于细胞核。成功构建人FAM92A1-289真核表达载体,FAM92A1-289定位于哺乳细胞的细胞核中。

Construction of eukaryotic expression plasmid of FAM92A1-289 and its subcellular localization

The full length FAM92A1-289 gene was amplified by PCR, and cloned into green fluorescence protein vector pEGFP-N1 to construct recombinant expression plasmid pEGFP-N1-FAM92A1-289. After recombinant plasmids were transfected into Hela cells by lipofectimane 2000, the expression of FAM92A1-289 gene and its subcellular localization were observed by fluorescent microscope. Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid was correct. Detected by fluorescence microscope, the FAM92A1-289 fusion protein was localized in nucleus of Hela cell. As control, GFP was distributing in the whole cell. The recombinant expression plasmid pEGFP-N1-FAM92A1-289 was constructed successfully. FAM92A1-289 protein was located in nucleus of Hela cell, it may play imoortant role in mutual interaction among nroteins.