Structural and Functional Studies of Cytokine IL-3 and GM-CSF Fusion Proteins

• Main Author: 胡志煌
• Other Authors: T.H. Hseu
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/58570647179355151983

碩士 === 國立清華大學 === 生命科學系 === 90 === Cytokines assist in regulating the development of immune effector cells, and some cytokines possess direct effector functions of their own. Just as hormones serve as messengers of the endocrine system, so cytokines serve as messengers of the immune system. Unlike endocrine hormones, though, which exert their effects over large distances, cytokines generally act locally. Clinical studies suggested that cytokines IL-3 and GM-CSF might have significant potential in gene therapy. Additionally IL-3 and GM-CSF share several common biological activities and structure features. Theirαβ receptors are almost alike and are both expressed in activated T cells. Consequently, we have constructed two genes one is IL-3─GM-CSF, the other is GM-CSF─IL-3. By way of DNA transfection, the PA137 cell used G418 to select the gene that we had transfectd. We pick up the clones and cultured them for 2 to 3 days. the fusion protein was released to the medium and then harvested the culture medium. Using ELSIA, we can detect the concentration of IL-3 and GM-CSF from two fusion protein (IL-3─GM-CSF and GM-CSF─L-3). To determine the molecular weigh of those two fusion protein, we used immunoprecipitation and wertern blotting. Finally, we check the growth curve by culture TF-1 cell in two fusion protein and cytokine (IL-3 and GM-CSF). The result indicated that we get the IL-3─GM-CSF and GM-CSF─IL-3 fuion protein consisted of cytokines IL-3 and GM-CSF. With only a few exceptions, in biological activity test, we have suggested that IL-3─GM-CSF and GM-CSF─IL-3 fuion protein really have a double effect. By predicting the structure of two fusion protein, we demonstrated a highly consistent structure of IL-3─GM-CSF and GM-CSF─IL-3 fusion protein by using “Insight II” program.