FAT10真核质粒的构建、转染及其蛋白的表达

Construction of human FAT10 eukaryotic plasmid and its expression in human embryonic kidney cell line 293T

目的构建类泛素基因FAT10的真核表达质粒,观察其蛋白在转染人胚肾细胞系293T中的表达。方法采用RT-PCR法扩增FAT10全长基因片段后,克隆至pMD18-T载体进行测序分析,将FAT10克隆至pcDNA5-FRT表达载体并行酶切鉴定;用脂质体LipofectamineTM2000分别介导空质粒(空载组)及重组质粒(FAT10质粒组)转染293T细胞,以脂质体混合PBS(PBS组)及未加入脂质体(未转染组)作为对照。转染48 h后,收集细胞。采用Western blot法检测293T细胞中的FAT10蛋白。结果 pMD18-FAT10重组质粒经双酶切获得498 bp片段,对PCR产物及498 bp片段进行测序,结果与GenBank报道序列完全一致。FAT10质粒组、未转染组、空载体组和PBS组FAT10蛋白表达量分别为1.556±0.072、0.334±0.051、0.425±0.095、0.382±0.063,FAT10质粒组与未转染组、空载体组和PBS组比较,P均<0.05。结论成功构建了FAT10的真核表达质粒pcDNA5-FRT-FAT10,FAT10蛋白在293T细胞中高表达。

Objective To construct the human FAT10 eukaryotic plasmid and analyze its expression in human human embryonic kidney cell line 293T.Methods The full-length FAT10 cDNA was obtained by reverse transcription-polymerase chain reaction（RT-PCR） and cloned into pMD18-T simple vector for sequence analysis.The FAT10 gene was subcloned into pEGFP-C1 plasmid.The resulting recombinant vector pcDNA5-FRT-FAT10 was identified by digestion with restriction endonucleases and transfected into 293T cells.The expression of FAT10 protein was examined by semi-quantitative western blot.Results The full-length human FAT10 cDNA was successfully obtained,and the recombinant plasmid pcDNA5-FRT-FAT10 was successfully constructed.After transfection into 293Tcells,western blot analysis showed that FAT10 was highly expressed in 293T cells.Conclusion The FAT10 eukaryotic expression vector pcDNA5-FRT-FAT10 is constructed successfully FAT10 protein express highly in 293T cells.