Cloning and functional assay of a G-protein-coupled receptor membrane protein from chicken phagocytic mononuclear cells
| Autor: | Yu-San Chen, 陳裕森 |
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| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Mononuclear phagocyte system, MPS, refers to, generally speaking, macrophages with the phagocytosis function in the endothelial layer. It plays the important role of fighting foreign pathogens. It is believed that the functions of avian macrophages are similar to those in the mammals. Having surveyed the latest researches on signal transduction, however, we found that studies of avian macrophages mostly are confined to avian diseases because of limited avian applications and the lack of research tools. The avian macrophage culture is mostly of the HD11 cell line. Studies on macrophages isolated from chicken and their signal transduction are even rarer. In this paper we cultured, in vitro, adherent macrophages from chicken peripheral blood mononuclear cells, PBMCs, and selected a new gene of chicken 7 transmembrane (Ch-7TM) receptor. Next, we constructed the HeLa stable cell line E5E8, which is capable of continuously expressing Ch-7TM on plasma membrane. We used this to select clone that can secrete anti-Ch-7TM antibody from hybridoma in mice. The clone we selected, hybridoma B28D5, is able to identify Ch-7TM in western blot and IFA. Having secured the mono clone of Ch-7TM, we adopted the immuno-florescent staining method and examined it with the confocal microscope. We verified that the receptor expressed on monocytes from chicken and macrophages. We further examined the distribution of the Ch-7TM gene in various organs with flow cytometry, and found a higher distribution in a particular group of cecal tonsil cells. Having verified Ch-7TM did express on the plasma membrane of macrophages, we took a step further to explore the functions of Ch-7TM: siRNA is a common tool to investigate the functions of genes. We used the siRNA to interfere Ch-7TM on the chicken monocytes/macrophages, and then we analyzed them with MTT. The results showed a significantly lower survival rate of the cells; moreover, we discovered that the chicken serum we used when culturing monocytes/macrophages could lower the death rate of the cells after siRNA interference. This proves that there is something in the serum that is able to activate Ch-7TM. During the process of purifying this particular protein(s), we actually observed that a certain fraction exhibited the activity that suppressed the death rate after the siRNA interference. Although we have not identified the particular protein(s), we succeed in constructing a useful research model which facilitates the identification of the protein(s) that help the monocytes/macrophages survival. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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