PA28γ及其胞浆定位突变体在HepG2中的表达

Expression of PA28γ and Its Cytoplasm Localized Mutant in HepG2 Cells

本研究采用RT-PCR法从293T细胞中克隆得到PA28γcDNA全长序列,并将该片段亚克隆到pMD-18T载体中,利用定点突变获得核定位序列缺失突变的PA28γ突变体,分别将野生型(WT)和突变型(MT)PA28γ基因克隆到真核表达载体pDsRed1-C3中,脂质体法转染293T细胞,荧光显微镜观察发现,在293T细胞中,PA28γWT定位在胞核中,而PA28γMT定位在胞浆中;再将野生型和突变型PA28γ基因分别克隆到真核表达载体pcDNA3.1(-)Flag中,用脂质体法將其转染HepG2細胞.Western印迹检测结果表明,PA28γWT和PA28γMT蛋白在HepG2细胞中获得了高效表达,建立了高表达PA28γWT和PA28γMT蛋白的HepG2细胞系.这些结果的获得,为进一步研究人类PA28γ基因的功能奠定了基础.

Full length coding sequence of PA28γ cDNA was cloned from 293T cells using RT-PCR,and the segment was inserted into pMD-18T vector,and then a nuclear locating sequence deficient mutant was obtained by site-directed mutagenesis.The wild type（WT） PA28γ and nuclear locating sequence mutated type（MT） PA28γ were inserted into eukaryotic expression vector pDsRed1-C3 separately,and either WT PA28γ or MT PA28γ expression vectors were transfected into 293T cells using liposome method.Observation of fluorescence microscope showed that DsRed1-PA28γ WT was mainly localized in nucleus while DsRed1-PA28γ MT was localized in cytoplasm.WT and MT PA28γ genes were also cloned into eukaryotic expression vector pcDNA3.1（-） Flag,separately,and then were transfected into HepG2 cells by liposome method.Western blot indicated that the recombinant PA28γWT and PA28γMT protein were high-levelly expressed in HepG2 cells respectively,and recombinant HepG2 cells expressed PA28γ WT and PA28γ MT proteins were constructed authentically.These results provided a basis for further investigating the function of PA28γ gene.