Construction and targeted expression of CEA promoter positively controlled IL-15 expression pissmid vectors

Objective To construct carcincembryonic antigen(CEA)promoter positively controlled human interlukin-15(IL-15)eukaryotic expression plasmids and aim to achieve high level and targeted IL-15 expression in CEA positive tumor cells.Methods CMV promoter positively controlled gene expression plasmid vector pHi2-IL-15-CMV-TAT and CEA promoter positively controlled gene expression plasmid vector pHi2-IL-15-CEA-TAT were constructed through genetic cloning.The IL-15 signal peptide (SP)sequence was then replaced by IL-2SP sequence through site-directed mutagenesis,resulting plasmids pHi2-IL-2SP-IL-15-CMV-TAT and pHi2-IL-2SP-IL-15-CEA-TAT.These plasmids were transfected into CEA-positive colon cancer SW480 cells and CEA-negative breast carcinoma MCF-7 cells by eationic lipid mediated gene transfer.IL-15 expression in culture supernatants 48 h after transfection was detected by enzyme-linked immunosorbent assay(EUSA).Results All plasmids were successfully constructed.EUSA results showed:(1)Replacing IL-15SP with IL-2SP increased IL-15 expression for 4.8-5.9 fold by transfected cells(P＜0.01):(2)There was no significant difference in the IL-15 expression in transfected SW480 cells between the CEA promoter positively controlled plasmids.pHi2-IL-15-CEA-TAT and pHi2-IL-2SP-IL-15-CEA-TAT,and the CMV promoter positively controlled plasmids.pHi2-IL-15-CMV-TAT and pHi2-IL-2SP-IL-15-CMV-TAT,respectively(P＞0.05);(3)After being transfected into MCF-7 cells,CEA promoter positively controlled plasmids gave rise to significantly lower IL-15 expression than that of CMV promoter positively controlled plasmids(P＜0.01).Conclusion CEA promoter positively controlled IL-15 expression plasmids,especially plasmid pHi2-IL-2SP-IL-15-CEA-TAT,express high level of IL-15 in CEA positive cells.whereas low level of IL-15 in CEA negative cells.Efficient and targeted IL-15 expression is achieved. Key words: Gene therapy;  Interleukin-15(IL-15);  Carcincembryonie antigen; Promoter; Gene expression