Construction And In Vitro Characterization Of CEA Promoter Positively Controlled IL-15 Expression Plasmid Vectors

In this study,CEA promoter positively controlled IL-15 expression plasmid vectors were constructed.SW480,a CEA-positive human colon cancer cell line,and MCF-7,a CEA-negative human breast cancer cell line,were transfected by these vectors in vitro.Results showed that CEA-specific positively controlled IL-15 expression plasmid vectors express IL-15 efficiently in SW480 cells but low expression in MCF-7 cells,indicating targeted gene expression was achieved by CEA promoter controlled plasmid vectors.This research lays a foundation for further study to develop IL-15 based immunogene therapies for cancer in vivo.Part One Construction of CEA Promoter Positively Controlled IL-15 Expression Plasmid VectorsObjective:To construct CEA promoter positively controlled IL-15 expression plasmid.Methods:?Construction of CMV promoter positively controlled gene expression plasmid vectors:Sequence encoding human IL-15 was cloned into themultiple cloning sites behind the HIV2 promoter in the empty plasmid pHi2-MCS-CMV-TAT(M7),resulting plasmid pHi2-IL-15-CMV-TAT(L1).Further,for enhance IL-15 expression in tumor cells,the IL-15 signal sequence(144bp)was replaced by IL-2 signal sequence(72bp,derived from plasmid pHi-IL-2-CMV-TAT)through site-directed mutagenesis,resulting plasmid pHi2-IL-2SP-IL-15-CMV-TAT(L3).In addition,for evaluating the transfection efficiency,gene encoding enhanced green fluorescence protein(EGFP)was cloned into the same backbone plasmid behind the HIV2 promoter,resulting EGFP expression plasmid pHi2-EGFP-CMV-TAT(L6).? Construction of CEA promoter positively controlled gene expression plasmid vector:The CEA promoter was cloned into plasmid pHi2-MCS-CMV-TAT(M7)and replaced CMV promoter to construct plasmid pHi2-MCS-CEA-TAT(L7).In addition,the encoding sequence of IL-15(LSP-IL-15),IL-2SP-IL-15,EGFP gene were inserted into the L7 plasmid behind the HIV2 promoter respectively,resulting in plasmid pHi2-IL-15-CEA-TAT(L2),pHi2-IL-2SP-IL-15-CEA-TAT(L4),pHi2-EGFP-CEA-TAT(L5).Results:The succussful construction of plasmid L1,L2,L3,L4,L5,L6,L7 were confirmed by restriction enzyme digestion,polymerase chain reaction(PCR)and sequence analysis.Conclusions:Double enzyme digestion and oriented cloning is the best choice for inserting a DNA segment into a plasmid vector when there is suitable restriction site in the target position.If not,site-directed mutagenesis could be a simple,fast and effecient method for molecular cloning.Part Two In vitro Charaterization of the CEA Promoter Positively Controlled IL-15 Expression Plasmid VectorsObjective:To analysis the gene expression of CEA promoter positively controlled IL-15 expression plasmid in CEA positive tumor cells after transfection,to investigate the feasibility of for cancer gene therapy using targeted cytokine expression vectors.Methods:?Plasmid grouping:CMV promoter controlled plasmids(L1,L3,L6,M7),CEA promoter controlled plasmids(L2,L4,L5,L7),and null transfection.?The mentioned plasmids were transfected into CEA-positive colon cancer SW480 cells and CEA-negative breast carcinoma MCF-7 cells by cationic lipid mediated gene transfer.The transfection efficiency were determinated by fluorescence microscope and flow cytometry.IL-15 expression were detected by ELISA.? Data were analysis by SPSS 13.0 for Windows.Results:? Both plasmid L6 and L5 could be transfected into CEA-positive SW480 cells and CEA-negative MCF-7 cells.The transfection efficiency were about 21.82%;?ELISA result:IL-15 production was not detected from empty vectors(M7 and L7)transfected cells or null transfected cells.At 24hr and 48hr posttransfection into SW480 cells,the IL-15 expression in culture supernatant was significantly deferent between L1 and L3,and between L2 and L4(P<0.01).The ratios of IL-15 expression for L3 to L1 were 4.3/5.9(24hr/48hr)folds,for L4 to L2 were 4.8/5.3 folds.There was no significant deference between L1 and L2,L3 and L4(P>0.05).After transfected into MCF-7 cells,the IL-15 expression in culture supernatant was significantly deferent between L1 and L3 at 24hr and 48hr after transfection(P<0.01).The ratios of IL-15 expression for L3 to L1 were 5.4/4.8 folds.However,there was no significant deference between L2 and L4(P>0.05).The IL-15 expression between L1 and L2?L1 and L4 were significantly deferent at 48hr after transfection(P<0.05).In the supernatant of plasmid L4 transfected into SW480 and MCF-7 cells,the IL-15 expressions were significantly deferent at 24hr and 48hr after transfection(P<0.01).The L4 plasmid produced 7.0-fold and 9.8-fold higher levels of IL-15 at 24hr and 48hr after transfection into SW480 cells,respectively,than transfection into MCF-7 cells.Conclusions:All of the plasmid constructs can be transfected into tumor cells and result transgene expression.IL-2 signal peptide can significantly elevate IL-15 expression after it replaces IL-15 signal peptide.CEA promoter positively controlled IL-15 expression plasmid give rise similar amount of IL-15 expression after transfected into CEA positive cells as that of the CMV promoter positively controlled plasmids.The IL-15 expression is low in CEA-negative cells for the CEA promoter positively controlled plasmid.These results indicate that efficient and targeted IL-15 expression could be achieved in CEA positive tumor cells by our plasmid constructs.