The optimized construction and cell transfection assay of new MUC1 DNA vaccine for pancreatic cancer

Abstract

Objective To construct new MUC1 DNA vaccine for pancreatic cancer and investigate whether the target genes are expressed in eukaryotic cells.Methods Three strategies were combined to optimize new MUC1 DNA vaccine.Variable number of tandem repeat (VNTR) number was increased to facilitate target antigen to form epitope.The HBeAg gene (C1-144) and interleukin (IL)-18 gene were also inserted alone or in combination.All the recombinant plasmids were successfully constructed and further confirmed by restricting enzyme digestion and DNA sequencing.Four recombinant plasmids were all transfected into Panc-02 and 293T (2 × 106) cells by using the Liperfectamine 2000.After incubation for 48 h at 37℃ in 5％ CO2,cells were observed under fluorescent microscope and harvested in 100 μL of lysis buffer for Western blotting assay.Results The pIRES2-EGFP-A-B clone was digested with Sac I and EcoR Irestriction enzymes and a 1100 bp.fragment could be seen in the 1％ argrose gel.The pIRES2-EGFP-A-C and pIRES2-EGFP-A-B-C clones were both digested with EcoR I and Sal I restriction enzymes and 1200 bp fragments were generated in the argrose gel.Green fluoresces could be seen under the fluorescent microscope 48 h after transfection.The expression of VNTR from the four recomhinant plasmids was examined in 293T cells,and HBV C1-144 and mouse IL-18 (mIL-18) were detectable in panc-02 cells.Conclusion The new MUC1 DNA vaccine for pancreatic cancer were successfully constructed to express the target genes in eukaryotic cells. Key words: Pancreatic cancer; DNA vaccine; Cell transfection