pEGFP-N1-PP1α真核表达载体的构建及其在骨肉瘤细胞中的表达

Construction of pEGFP-N1-PP1α eukaryotic expression vector and its expression in human osteosarcoma cell line

目的构建人PP1α基因真核表达载体,并观察其在人骨肉瘤细胞株(U-2OS)中的表达,为进一步研究PP1对骨肉瘤细胞凋亡影响提供基础。方法提取U-2OS细胞中总RNA,RT-PCR扩增PP1α并克隆至pEGFP-N1载体,鉴定出阳性克隆送测序,以重组质粒转染U-2OS细胞,通过West-ernblot检测转染细胞中PP1α的表达。结果成功扩增PP1α基因大小片段,重组质粒pEGFP-N1-PP1α的酶切鉴定及测序结果均证明PP1α基因成功克隆到真核表达载体中,Westernblot结果证实转染重组质粒后的U-2OS细胞PP1α蛋白表达水平增强。结论实验将pEGFP-N1-PP1α质粒转染到U-2OS细胞,可以在U-2OS细胞中获得PP1α蛋白增强表达。

Objective To construct eukaryotic expression vector of protein phosphatase 1α（PP1α）gene and observe its expression in human osteosarcoma cell line（U-2OS）.Methods Total RNA was extracted from U-2OS cells and the coding sequence of PP1α gene was amplified by RT-PCR.After its purification,the fragment and eukaryotic expression vector pEGFP-N1 plasmid were ligated.The recombinant plasmid was verified by agarose gel electrophoresis and sequencing.pEGFP-N1-PP1α was transfected into U-2OS cells and its expression was evaluated by Western blot.Results The length of specific fragment amplified by PCR PP1α was 993 bp,the recombinant plasmids pEGFP-N1-PP1α was verified by digestion using restriction endonuclease and sequencing results.Western blot revealed that U-2OS cells transfected with recombinant plasmids expressed higher level of PP1α protein.Conclusion The recombinant eukaryotic expression vectors of pEGFP-N1-PP1α is successfully constructed and expressed in U-2OS cells.The level of PP1α protein in the transfected cells are elevated.