Expression and Transport of Chicken Interleukin-2 Fusion Cytokines in Prokaryotic and Avian Systems

• Main Authors: Yin-Chuan Huang, 黃胤銓
• Other Authors: Shiow-Her Chiou
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/22kjzw

碩士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 106 === Cytokines are natural proteins which can serve as principal mediators for communication between cells of the immune system. The specific aims of this study were to express and transport chicken interleukin-2 (IL-2) fusion cytokines in prokaryotic and avian systems. IL-2, is an important cytokine mainly produced by T helper cells to modulate immune responses, in particular, to stimulate the proliferation of a variety of leukocytes. In this study, chicken stromal-derived factor-1 (SDF-1) that can attract leukocytes to target locations was fused to IL-2 with a peptide linker by employing recombinant polymerase chain reaction (PCR) technique. Subsequently, the resultant SDF1-5PA-IL2 DNA fragment was cloned to a prokaryotic expression vector, pET32a, to express recombinant chicken fusion cytokine in Escherichia coli BL21 (DE3). After induction, we harvested SDF1-IL2 fusion cytokine and purified the protein by nickel (Ni2+) chelation affinity chromatography. We evaluated the bioactivities of SDF1-IL2 based on chemotactic and proliferating activities of SDF-1 and IL-2, respectively. On the other hand, we endeavored to establish an eukaryotic fusion cytokine expression and transport system by using cell penetrating peptides (CPPs). CPPs have been used to introduce biomolecules into a number of human cell lines. However, chicken system has not been tested yet. As a feasibility test, we confirmed that CPP could carry pEGFP-N1 into a human cell line, CNE2. Subsequently, we constructed eukaryotic chicken IL-2 fusion protein expression plasmids (pVAX- and pEGFP-SDF1-IL2) and verified that CPP could transport these plasmids into chicken cell line, DF-1, and express fusion cytokines in avian system. In the future, these chicken fusion cytokines can be applied in veterinary medicine.