Construction,Identification and Expression of Recombinant Eukaryotic Vector pCAG-IRES-SHIP-GFP on Porliferation of Leukemia Cell Line K562 / 中国生物工程杂志

ABSTRACT

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.