Construction of GPI-anchored bcr/abl and its expression on COS-7 cells membrane
- VernacularTitle:慢性粒细胞白血病bcr/abl的糖基化磷脂酰肌醇锚定修饰及在COS-7细胞膜上的表达
- Author:
Kun TAO
;
Dong WANG
;
Jianming ZENG
;
Shifeng HUANG
;
Xinmin CHEN
;
Zonggan HUANG
;
Wenli FENG
- Publication Type:Journal Article
- Keywords:
bcr/abl fusion gene;
glycosyl-phosphatidylinositol;
eukaryotic expression;
chronic myelogenous leukemia
- From:Journal of Third Military Medical University
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant eukaryotic expression plasmid of glycosylphosphatidyl inositol(GPI)-anchored bcr/abl and explore its expression at mRNA and protein level.Methods The gene fragment encoding bcr/abl was amplified by PCR using the plasmid containing the cDNA sequence of P210 as template and then inserted into a eukaryotic expression vector pBudCE4.1.The constructed recombinant plasmid pBudCE4.1-bcr/abl was identified by restriction analysis and DNA sequencing.Lymphocytes were isolated from human peripheral blood and their total RNA was extracted.The gene fragment encoding GPI was amplified by RT-PCR using the obtained RNA as template and was inserted into the constructed recombinant plasmid pBudCE4.1-bcr/abl in order to anchor GPI and bcr/abl.The constructed recombinant plasmid pBudCE4.1-bcr/abl-GPI was transfected into COS-7 cells,and the expressions of objective fragment were detected by RT-PCR and Western blotting.Results The results of restriction analysis,PCR and DNA sequencing proved that GPI-anchored bcr/abl fusion fragment was correctly inserted into vector pBudCE4.1.The expression of bcr/abl fusion gene and fusion protein were identified in transfected COS-7 cells and on their membrane.Conclusion The recombinant plasmid pBudCE4.1-bcr/abl-GPI was successfully constructed and expressed on the membrane of COS-7 cells,which found a basis of cell immunity with GPI-anchored bcr/abl fusion gene.
