SHIP基因真核表达载体的构建、鉴定及其表达抑制白血病细胞增殖的实验研究

Construction,Identification and Expression of Recombinant Eukaryotic Vector pCAG-IRES-SHIP-GFP on Porliferation of Leukemia Cell Line K562

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 aim was to construct and identify the mammalian expression vector of pCAG-IRES-SHIP- GFP and to detect whether it could express in human acute leukemia cell line K562. The cDNA fragment of SHIP obtained by RT-PCR was inserted into pCAG-IRES-GFP. The recombinant plasmid was confirmed by restriction enzyme digesiton, PCR and DNA sequecing, pCAG-IRES-SHIP-GFP was transfected into K562 cells with lipofectamine 2000. The expression of SHIP was determined by GFP fluorescence and Western blot analysis. FQ -PCR was used to quantitate SHIP mRNA. The expression of p-Akt,Akt were determined by Western blot. PI were tested by flow cytometry and MTT to verify whether exogenous SHIP could inhibit proliferation of K562 cells. The results showed that the correct constrution of the recombinant plasmid pCAG-IRES-SHIP-GFP has been shown by restriction enzyme digestion, PCR and DNA sequencing, pCAG-IRES-SHIP-GFP could express SHIP protein in K562 cells. The K562 cells viability after transfected with SHIP gene droped down. Western blot analysis showed that phospha-Akt308 and Akt473 were reduced to 38. 7% and 68% respectively. It was concluded that the vector of pCAG-IRES-SHIP-GFP has been successfully constructed and it can be expressed in K562 cells. The expression of exogenous SHIP gene can lead to apoptosis of K562 cells by down-regulating the p -Akt expression. What found here might be one of the mechanisms involved in the pathogenesis of leukemia.