Construction And Characterization Of AFP Antigen Epitope Modified IL-15Vector

Objective:Based on previously constructed plasmid pHi2-spIL15-CMV-TAT (L3), to modify of cytokine signal peptide through genetic engineering technology, construct plasmid vecotor co-exprssing AFP peptide (AFP158-166) and IL-15.Method:①Design AFP peptide gene-containing the plasmid vector:predicted the replacing site of AFP158-166in the signal peptide of IL-2using SignalP3.0server.②Construct plasmid pUC19-spIL-15using site-directed mutagenesis technique, of which part of the IL-2signal peptide were replaced with AFP epitope. Then the AFP158-166modified spIL-15into the corresponding site of L3, and DNA sequencing.③In vitro cationic liposome-mediated plasmid transfection of colon cancer SW480cells and breast cancer MCF-7cells, assessing cell transfection by inverted fluorescence microscope, determing transfection efficiency by flow cytometry, and detect IL-15expression by enzyme-linked immunosorbent assay.Result:①The successful contruction of AFP158-166expression plasmid pHi2-afpILl5-CMV-TAT (LA-15) were confirmed by restrictive digestion and sequencing.②AFP158-166-expression plasmid pHi2-afpIL15-CMV-TAT (LA-15), L3and L6can be transfected into SW480cells and MCF-7cells, transfection efficiency was35.30%.③ELISA results:Twenty-four and48hr posttransfection, IL-15expression in the supernatants of SW480cells were significantly different between LA-15and L3transfectant (P<0.05), and the IL-15expression level of LA-15transfectant was1.58/3.54(24hr/48hr)-fold higher than that of L3. Similiarly, for MCF-7cells, the expression of IL-15between LA-15and L3transfectant was significantly different, IL-15expression of LA-15is1.26/2.20-fold higher than that of L3.Conclutions:①In gene cloning technology, DNA fragment is cloned into plasmid vector, the most critical part is the choice of restriction sites, if there is an appropriate restriction enzyme sites, the double digestion and directional cloning is the best of choice; without the appropriate restriction enzyme sites, site-directed mutagenesis technique is a simple, rapid and effective method.②The constructed plasmid can be transfected into tumor cells and express target gene products IL-15.③AFP158-166 -modified IL-15signal peptide did not compromise the expression of IL-15. This AFP antigen-expressing vector could be used for liver cancer immunotherapy.