激活转录因子5的克隆及其在真核细胞中的表达定位

Cloning the activating transcription factor 5 gene and detection of its expressional localization in cultivated cells

目的克隆激活转录因子(ATF)5,构建其真核表达载体,观察其在细胞中的表达定位。方法根据人ATF5的cDNA序列设计引物,通过巢式PCR扩增ATF5全长及其N端(ATF5/N)和C端(ATF5/C),构建重组表达质粒pCS2MT-ATF5、pCS2MT-ATF5/N和pCS2MT-ATF5/C及融合绿色荧光蛋白(GFP)基因的质粒pEGFP-ATF5。将表达质粒转染293T和Cos7细胞,Westernblot检测其表达情况,荧光显微镜观察ATF5在293T和Hela细胞的定位。结果巢式PCR产物与预期大小一致,重组质粒pCS2MT-ATF5、pCS2MT-ATF5/N和pCS2MT-ATF5/C能表达携带Myc标签的蛋白Myc-ATF5、Myc-ATF5/N和Myc-ATF5/C,分子量分别为52、35和43×103。荧光显微镜观察发现融合了GFP的ATF5蛋白定位在细胞核。结论成功的克隆和表达ATF5基因为进一步研究其在肾肿瘤发生、发展过程中的作用奠定了基础。

Objective To clone the gene activating transcription factor 5 (ATF5),construct the expression plasmid and detect the localization of ATF5 in cultivated cells.Methods The nest-PCR primers for the full length of ATF5,the N-terminal of ATF5 (ATF5/N) and the C-terminal of ATF5 (ATF5/C) were designed and synthetized according to the cDNA sequence of human ATF5 in GenBank,then PCR amplication was carried out and the recombinant plasmids of pCS2MT-ATF5,pCS2MT-ATF5/N and pCS2MT-ATF5/C were obtained.The co-expression plasmid fused ATF5 with GFP named pEGFP-ATF5 was also successfully constructed.The constructed plasmids were transfected to 293T and COS7 cells with liposome and the expression was detected by Western blot.The localization of GFP-ATF5 in 293T and Hela cells were examined by fluorescence microscopy.Results Nest-PCR showed that amplified products of ATF5,ATF5/N and ATF5/C were the estimated basepair.Western blot of the cells transfected with pCS2MT-ATF5,pCS2MT-ATF5/N and pCS2MT-ATF5/C detected the expression of the Myc-tag fusion protein Myc-ATF5,Myc-ATF5/N and Myc-ATF5/C with molecular weight 52 KD,35 KD and 43 KD respectively.Fluorescence microscopy revealed that the GFP fusion protein ATF5 localized in nucleus of 293T and Hela cells.Conclusion The ATF5 gene was successfully cloned and expressed,which laid a foundation for future study on its molecular functions in renal carcinogenesis.