Construction of mammalian cell expression vector for HLA-A~*0201 and EGFP fusion protein and its expression and localization in K562 cells / 中国病理生理杂志

ABSTRACT

AIM: To construct the mammalian cell expression vector for enhanced green fluorescent protein (EGFP) and HLA-A*0201 fusion protein and analyze its expression and subcellular localization in the transfected K562 cells. METHODS: The HLA-A*0201 cDNA was cloned by RT-PCR and the gene was inserted into pEGFP-N1 to construct a vector for the fusion protein. The expression of the fusion protein in K562 cells transfected with the vector was evaluated by flow cytometry and its subcellular localization was investigated by confocal microscopy. RESULTS: The full-length encoding region of HLA-A*0201 cDNA was cloned from two HLA-A2 positive donors and the expression vector for the HLA-A*0201-EGFP fusion protein was constructed by PCR using a primer pair to introduce a Kozak sequence before ATG and the stop codon was deleted. Five hours after K562 cells was transfected with the vector, the expression percentages of HLA-A*0201 and EGFP were 25.12?2.26 and 27.37?3.59, respectively and no significant increase was observed after 24 h. The fusion protein was predominantly located on the membrane with low level distribution within the cells. In contrast, no HLA-A*0201 but only EGFP was detected in the empty vector transfected K562 cells and the EGFP was dispersed within the cells. CONCLUSIONS: The expression vector for HLA-A*0201-EGFP fusion protein was constructed and the fusion protein expressed in K562 cells was primarily distributed on the membrane. The results suggest that the transfected K562 cells are potential antigen-presenting cells.