Design and Construction of Multi-Epitope Fusion Gene Vaccine of Wilm Tumor Gene WT1 and Stimulating Epitope of Mycobacterium Tuberculosis Heat Shock Protein 70

Abstract

AbstractAbstract 4713 ObjectiveDNA vaccination respresents a novel therapeutic strategy for cancer. Researchs have showed DNA vaccines encoding Wilms tumor gene WT1-Derived HLA *0201-restricted epitopes effectively induce epitope-specific human cytotoxic T-lymphocytes (CTLs) and kill human leukemia cells in vitro. Nevertheless, there is still a need to increase the potency of DNA vaccine and span the HLA diversity of a target population. In this study, we designed polytpoe WT1 DNA vaccine encoding multiple CTL and helper T-lymphocyte (HTL) epitopes and containing HSP70407-426 as immunoadjuvants for immunotherapy. MethodsOn the basis published data, To design a Multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted, one HLA *2402-restricted epitopes, two HTL epitopes and one universal Th Pan- DR epitope (PADRE) sequence. The construct also included IgG K chain leader sequence as signal peptide to facilitate transport into the endoplasmic reticulum and Kozak sequence at the N-terminus to support translation of the gene transcript. DNA-coding sequesces was used computer-based modeling to optimize proteasome mediated epitope processing through the introdcution of amino acid spacer sequences, then cloned into the eukaryotic vector to construct the plasmid PcDNA3.1(+). Mycobacterial HSP70 fragment including stimulatory domain P407-426 as immunotherapy was fused to the C-terminal of Multi-WT1 for enhancing CTL activity and then inserted to PcDNA3.1(+). Transfection HEK-293T cells with the Multi -WT1-HSP70 and identify the expressed product by Western bolt. ResultsMost of Multi -WT1 epitopes can be cleaved by software Net Chop 3.1 and PAPROC± analysis. Western blot showed correct expression of target gene in HEK293T cells. ConclusionMulti-WT1-mtHSP70 fusion gene eukaryotic expression vector was successfully designed, which prepared the materials for the research of DNA vaccine. Disclosures:No relevant conflicts of interest to declare.