Protein Expression and DNA Transport of Chicken CD40 Ligand Fusion Cytokines

• Main Authors: Hsiao-Wen Chiang, 姜曉雯
• Other Authors: Shiow-Her Chiou
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/uvcq9u

碩士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 106 === Stromal cell derived factor-1 (SDF-1) is a chemotactic cytokine, which binds to its receptor and guides various leukocytes to target locations. CD40 ligand (CD40L) is a cytokine that regulates immune responses, such as macrophage activation. The specific aims of this study were to express and transport chicken CD40L fusion cytokines in prokaryotic and avian systems for future veterinary applications. Chicken SDF-1 and CD40L-derived cDNAs were linked by a peptide linker. The resultant construct was cloned into pET32a to express SDF1-CD40L-derived fusion protein in Escherichia coli BL21 (DE3). We measured the chemotactic and macrophage activating activities of recombinant SDF1-CD40L-derived protein based on a chemotaxis assay and a nitric oxide production assay, respectively. SDF1-CD40L-derived protein presented with chemotactic activities on peripheral blood mononuclear cell and macrophage activating activities on macrophages. To express such chicken fusion cytokine in avian system, we performed a feasibility test by using a cell-penetrating peptide (CPP) to carry pEGFP-N1 into the chicken fibroblast cell line, DF-1. The result confirmed that CPP was able to transport pEGFP-N1 into DF-1 cells and successfully expressed enhanced green fluorescent protein encoded by pEGFP-N1. We further introduced DNA constructs encoding SDF1-CD40L-derived protein into pEGFP-N1 or pVAX1 eukaryotic expression vector and delivered them into DF-1 cells. The results showed that CPP was able to transport these constructs and express bioactive SDF1-CD40L-derived cytokines. In the future, these fusion cytokines may be applied as immunomodulatory molecules for poultry industry.