CYTOKINE-BASED MULTI-EPITOPE PROTEIN FOR BINDING TO CCR7-POSITIVE CELLS

Abstract

AbstractDrugs based on cytokine genes increase the body’s immunity against cancer and several viral diseases. Cytokines such as CCL21 and CCL19 specifically bind to the CCR7 receptor and have anti-tumor properties and a prognosis of tumorigenesis. An epitope of IL-Iβ is also involved in various cellular activities such as neutrophil activation, T and B lymphocyte cell production, antibody production, and fibroblast proliferation. They bind to secretory proteins without any inflammatory response. GM-CSF adjuvant is one of the growth factors of white blood cells (WBC) that stimulates stem cells to produce granulocytes and myocytes. In our project, we designed and synthesized codon-optimized multiepitope genes construct from human genes. The molecular dynamics (MD) simulation and binding affinity of this recombinant protein and CCR7 receptor were examined through in silico analyses. This construct was introduced into the pET-28a vector that was cloned in Ecoli to explore this recombinant protein. The purified multiepitope protein produced a strong signal in Dot-blot, SDS-PAGE, and Western-blot assays comparable to the positive control. The assessments of FTIR measurement and MALDI-TOF MS displayed that synthetic gene constructs correctly be expressed in E. coli. We also investigated the potential activity of the purified multiepitope protein in stimulating migration and proliferation of MCF7CCR7+ cancer cell line using wound healing assay. Also, in this study, the MTT method was used to determine the half-maximum inhibitory concentration (IC50) of all multiepitope protein concentrations. We used Agarose assay on PBMCsCCR7+ to observe whether PBMC cells and chemoattractants attract to each other. Also, we found that intraperitoneal injection of recombinant antigen affected the level of WBC in BALB/c mice, and the level of WBC in tumor mice increased significantly compared to healthy mice. Our project aims to produce the first multi-epitope vaccine with many beneficial advantages such as low-cost price and any major or significant complications that can be used as biomarkers for cancer screening and prognosis tests to immunize the patient before chemotherapy.