Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2009, Vol. 29 Issue (06): 14-19    DOI:
    
Construction, Identification and Expression of Recombinant Eukaryotic Vector pCAG-IRES-SHIP-GFP on Porliferation of Leukemia Cell Line K562
Download: HTML   PDF(847KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Objective This study was aimed 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. Methods 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. Results 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. Conclusions 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.



Key wordsK562 cell;pCAG-IRES-SHIP-GFP;SIHP;gene transfection;Leukemia;p-Akt;proliferation     
Received: 06 October 2008      Published: 02 July 2009
Cite this article:

YANG Lin- Luo-Jian-Min- Liu-Xiao-Jun- Cheng-Zhi-Yong- Wen-Shu-Feng- Du-Hang-Yan- Yang-Xiao-Yang- Wu-Hua-Wen. Construction, Identification and Expression of Recombinant Eukaryotic Vector pCAG-IRES-SHIP-GFP on Porliferation of Leukemia Cell Line K562. China Biotechnology, 2009, 29(06): 14-19.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2009/V29/I06/14

[1] Geier S J, Algate P A, Carlberg K, et al. The human SHIP gene is differentially expressed in cell lineages of the bone marrow and blood. Blood, 1997,89(6):1876~1885 [2] Helgason C D, Antonchuk J, Bodner C, et al. Homeostasis and regeneration of the hematopoietic stem cell pool are altered in SHIP-deficient mice. Blood, 2003,102(4):1541~1547 [3] Luo J M,Yoshida H,Komura S, et al. Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia. Leukemia, 2003,17(1):1~8 [4] 罗建民,刘泽林,郝红领,等.急性白血病细胞SHIP基因的突变分析.中华血液学杂志,2004,25(7):385~388 Luo J M, Liu Z L, Hao H L,et al.Chin J Hematol, 2004, 25(7):385~388 [5] 张苏江,李建勇,施静艺,等. 急性髓系白血病患者PDGFβ和SHIP基因突变及其单核苷酸多态性研究. 中华血液学杂志,2006,27(6):383~385 Zhang S J,Li J Y,Shi J Y,et al.Chin J Hematol, 2006,27(6):383~385 [6] 刘晓军, 罗建民, 杨琳, 等. 慢性粒细胞白血病中SHIP基因的表达变化及机制探讨. 解放军医学杂志, 2007,33:701~703 Liu X J, Luo J M, Yang L, et al.Medical Journal of Chinese People’s Liberation Army, 2007,33:701~703 [7] 任金海,罗建民,杨敬慈,等. 白血病细胞内SHIP基因表达及其对AKT磷酸化的影响. 中华血液学杂志,2007,28(12):844~845 Ren J H,Luo J M,Yang J C,et al.Chin J Hematol, 2007,28(12):844~845 [8] Kisseleva M V, Cao L, Majerus P W, et al. Phosphoinositide-specific inositol phlyphosphate 5-phosphatase Ⅳ inhibits Akt/protein kinase B phosphorylation and leads to apoptotic cell death. J Biol Chem, 2002, 277(8):6266~6272 [9] Aggerholm A, Gronbaek K, Guldberg P, et al. Mutational analysis of the tumor suppressor gene MMAC1/PTEN in malignant myeloid disorders. Eur J Haematol, 2000, 65(2):109~113 [10] Yang J, Liu J, Zheng J, et al. A reappraisal by quantitative flow cytometry analysis of PTEN expression in acute leukemia. Leukemia. 2007,21(9):2072~2074 [11] Liu Q, Sasaki T, Kozieradzki I, et al. SHIP is negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev, 1999,13(7):786~791

No related articles found!