【摘要】 目的构建真核表达质粒pCAG-IRES-SHIP-GFP,并观察其在K562细胞中的表达。方法将SHIP基因的RT-PCR产物克隆入载体pCAG-IRES-GFP,经酶切、鉴定及测序分析,构建pCAG-IRES-SHIP-GFP重组真核表达质粒。实验设置3个组:K562组(未转染)、K562/IRES组(转染空载体pCAG-IRES-GFP),K562/SHIP组(转染重组质粒pCAG-IRES-SHIP-GFP)。荧光显微镜和流式术检测重组质粒的转染效率;Western blotting检测各组SHIP蛋白的表达及Akt磷酸化水平变化;MTT法和流式术分析SHIP基因转染对细胞增殖的影响。结果重组质粒pCAG-IRES-SHIP-GFP已成功载入SHIP的全长编码基因,序列测定的结果与预期设计完全一致。荧光显微镜在K562/SHIP组可见绿色荧光,转染效率为48.2%±6.6%。Western blotting显示K562/SHIP组有明显的SHIP蛋白表达,且p-Akt-308和p-Akt-473的水平(分别为0.182和0.381)较K562/IRES组(0.361和0.716)和K562组(0.365和0.725)明显下降(P<0.01)。流式术分析显示K562/SHIP组细胞生长减慢,G0/G1期细胞增多、G2/M期细胞减少,增殖指数降低。结论成功构建了3 585bp的SHIP基因真核表达质粒pCAG-IRES-SHIP-GFP,并在K562细胞中表达。外源性SHIP基因转染能使K562细胞增殖受抑,原因可能与Akt的磷酸化下调有关。更多还原

【Abstract】 Objective To construct and identify the mammalian eukaryotic expression vector of pCAG-IRES-SHIP-GFP,and to investigate whether it could express in human acute leukemia cell line K562.Methods The cDNA fragments of SH2 domain containing inositol 5’-phosphatase(SHIP) gene obtained by RT-PCR were inserted into empty vector,pCAG-IRES-GFP,to construct pCAG-IRES-SHIP-GFP plasmid.The recombinant plasmid was confirmed by restriction enzyme digestion,PCR and DNA sequencing.K562 cells were divided into 3 groups: control group(untransfected cells),K562/IRES group(transfected with empty vector pCAG-IRES-GFP) and K562/SHIP group(transfected with recombinant plasmid,pCAG-IRES-SHIP-GFP).The transfection efficiency of recombinant plasmid was determined by fluoresence microscope and flow cytometry.The expression of SHIP protein and phospha-Akt were detected by Western blotting.The effects of exogenous SHIP transfection on the proliferation of K562 cells were tested by MTT and flow cytometry.Results Sequencing confirmed that the recombinant plasmid pCAG-IRES-SHIP-GFP had been inserted SHIP full-length coding gene successfully.Green fluorescence was observed under fluorescence microscope in K562/SHIP group,and the transfection efficiency was 48.2%±6.6%.Western blotting indicated that SHIP protein was highly expressed in K562/SHIP group,the expressive levels of p-Akt-308(0.182) and p-Akt-473(0.381) in K562/SHIP group were obviously lower compared with that in K562/IRES group(0.361 and 0.716,respectively,P<0.01) and K562 group(0.365 and 0.725,respectively,P<0.01).Flow cytometry analysis also showed that the cells in K562/SHIP group became slower in proliferation,and the cell growth was arrested in G0/G1 phase,resulting in a declination in G2/M phase,thus the proliferation index was decreased.Conclusions A 3585bp recombinant eukaryotic expression vector,pCAG-IRES-SHIP-GFP,has been successfully constructed,and expressed in K562 cells.The transfection of exogenous SHIP gene may inhibit proliferation of K562 cells,which may be associated with down-regulation of Akt phosphorylation.更多还原