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构建真核表达载体pCAG-IRES-SHIP-GFP,并观察其在K562细胞中的表达。将SHIP逆转录聚合酶链反应(RT-PCR)产物克隆入pCAG-IRES-GFP,经酶切、PCR鉴定及测序分析,构建pCAG-IRES-SHIP-GFP重组真核表达载体;采用脂质体转染法将pCAG-IRES-SHIP-GFP及空载体转入K562细胞,实时荧光定量PCR(FQ-PCR)、Westernblot方法检测转染前后K562细胞中SHIPmRNA和蛋白的表达及Akt磷酸化水平改变,MTT、流式细胞仪检测等方法观察野生型SHIP基因表达对白血病细胞K562凋亡的影响。结果显示重组后的pCAG-IRES-SHIP-GFP质粒已成功载入SHIP的全长编码基因,序列测定的结果与预期设计完全一致。荧光显微镜下可见在转染pCAG-IRES-SHIP-GFP的K562细胞中存在荧光分布。外源性SHIP基因表达能使K562细胞增殖受抑;Westernblot检测提示K562细胞Akt308和Akt473的磷酸化水平较转染前显著下调,分别为转染前的38.7%和36.8%(P<0.01)。上述结果表明基因全长3.5kb的人野生型SHIP基因真核表达载体质粒pCAG-IRES-SHIP-GFP构建成功,并在K562细胞中表达;外源性SHIP基因表达能使K562细胞凋亡增加;这些改变可能与p-Akt表达下调有关。
The eukaryotic expression vector pCAG-IRES-SHIP-GFP was constructed and its expression in K562 cells was observed. Recombinant eukaryotic expression vector pCAG-IRES-SHIP-GFP was constructed by restriction endonuclease digestion, PCR identification and sequencing analysis. The recombinant plasmid pCAG-IRES-SHIP- The transfected pCAG-IRES-SHIP-GFP and empty vector were transfected into K562 cells. The expression of SHIP mRNA and protein and the level of Akt phosphorylation in K562 cells before and after transfection were detected by real-time fluorescence quantitative PCR (FQ-PCR) MTT, flow cytometry and other methods to observe the expression of wild-type SHIP gene leukemic cells K562 apoptosis. The results showed that the recombined pCAG-IRES-SHIP-GFP plasmid successfully loaded the SHIP full-length coding gene, and the sequence determination results are in good agreement with the expected design. Fluorescence microscopy revealed the presence of fluorescence in K562 cells transfected with pCAG-IRES-SHIP-GFP. Exogenous SHIP gene expression inhibited the proliferation of K562 cells. Western blot analysis showed that phosphorylation levels of Akt308 and Akt473 in K562 cells were significantly down-regulated compared with that before transfection (38.7% and 36.8%, respectively) (P <0.01). The above results showed that the eukaryotic expression vector plasmid pCAG-IRES-SHIP-GFP of human wild-type SHIP gene with a full-length gene of 3.5kb was successfully constructed and expressed in K562 cells; exogenous SHIP gene expression increased the apoptosis of K562 cells; These changes may be related to the down-regulation of p-Akt.