Construction And In Vitro Characterization Of Interleukin-15 Lentiviral Expression Vector

Objective:Construct and package IL-15-expression lentiviral vector, infect tumor cells in vitro for effective transgene expression for enhancing immunity.Methods:(1) Construction of lentiviral IL-15 gene expression vector:the GFP coding sequence was removed by restriction enzymes from lentiviral plasmid CS-CDF-EG-PRE (L0), Using the plasmid pHi2-spIL15-CMV-TAT (L3) as a template, sequence encoding IL-2SP-IL-15 was amplified by PCR and cloned to the plasmid CS-CDF-EG-PRE (L0) to construct CS-CDF-EF-1-spIL15-PRE (LT15). (2) The resulting plasmid and control plasmid (CS-CDF-EG-PRE) were transfected into embryonic kidney epithelial 293T cells, colon cancer SW480 cells and breast carcinoma MCF-7 cells by liposome-mediated transfection. (3) CS-CDF-EG-PRE or CS-CDF-EF-1-spIL15-PRE was transfected into 293T cells with the other three helper plasmids pMDL/pRRE(L7), pRSV-Rev(L8) and pMD.G(L9) to package GFP or IL-15-expression lentivirus,and infect cervical cancer Hela cells. (4) The transfection efficiency was determinated by fluorescence microscope and flow cytometry(FCM). IL-15 was detected by enzyme-linked immunosorbent assay(ELISA). Data analysis was completed by SPSS 17.0.Results:(1) The succusful construction of plasmid CS-CDF-EF-1-spIL15-PRE(LT15) was verified by restriction enzyme digestion, polymerase chain reaction (PCR)and sequence analysis. (2) Both plasmids (L0 and LT15) were transfected into 293T cells, SW480 cells and MCF-7 cells successfully, and the transfection efficiency was about 35.3%. (3) GFP or IL-15-expression lentivirus vector through co-transfection 293T cells with CS-C-DF-EG-PRE or CS-CDF-EF-1-spIL15-PRE and the other three helper plasmids pMDL/pRRE (L7), pRSV-Rev(L8), pMD.G(L9).The resulted product infected Hela cells effectively. (4) ELISA result:IL-15 was not detected in the culture supernatant of tumor cells which were transfected by PBS, CS-CDF-EG-PRE(L0),or GFP lentiviral vector; IL-15 was detected in the culture supernatant of tumor cells 293T cells, SW480 cells and MCF-7 cells which were transfected by CS-CDF-EF-1-spIL-15-PRE(LT15). There were significantly deference for the amount of IL-15 expressed among these cells (P<0.01).The ratios of IL-15 expression in the culture supernatant of 293T cells to SW480 cells were 1.6/1.5 folds(24h/48h),293T cells to SW480 cells 1.8/1.6 folds(24h/48h), and SW480 cells to MCF-71.2/1.1 folds(24h/48h). Hela cells infected with IL-15-expression lentivirus expressed IL-15 efficiently. The infection efficiency of lentiviral vector was different according to doses of virus.Conclusion:IL-15-expression lentiviral vector was successfully constructed, and tumor cells transfected with IL-15-expression plasmid by liposome-mediated transfection expressed different amount of IL-15. The IL-15-expression lentivirus was prepared successfully and can infect tumor cells efficiently,resulting efficient IL-15 expression. The lentiviral vector contructed in this study will be useful for gene transfer of primary cells, such as T cells.