TAO Kun,WANG Dong,ZENG Jian-ming,HUANG Shi-feng,CHEN Xin-min,HUANG Zong-gan,FENG Wen-li.Construction of GPI-anchored bcr/abl and its expression on COS-7 cells membrane[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(08):717-720.
TAO Kun,WANG Dong,ZENG Jian-ming,HUANG Shi-feng,CHEN Xin-min,HUANG Zong-gan,FENG Wen-li.Construction of GPI-anchored bcr/abl and its expression on COS-7 cells membrane[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(08):717-720.DOI:
Construction of GPI-anchored bcr/abl and its expression on COS-7 cells membrane
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.