| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 93 === A new immunomodulatory protein (PCP) is purified from the dried sclerotium of an therapeutic fungi, Poria cocos (Schw.) Wolf, by extraction using 5 % cold acetic acid in the presence of 0.1 % 2-mercaptoethanol, followed by ammonium sulfate fractionation, DE-52 anion-exchange and FPLC chromatography. Gel filtration chromatography shows that PCP has a molecular mass of 35.6 kDa. SDS-PAGE electrophoresis reveals that PCP contains two subunits with molecular mass of 14.3 and 21.3 kDa, respectively. Based on these results, we suggest that PCP is a heterodimer protein. Additionally, isoelectric focusing electrophoresis shows that the pI value of PCP is around 5.2. The amino acid composition and N-terminal amino acid sequences of these two subunits of PCP are also obtained.
Three hybridoma clones, which secrete monoclonal antibodies recognizing PCP, are obtained. The specificity and isotype of these monoclonal antibodies are also confirmed. Immunocytochemical analysis suggests that PCP mainly expressed on cell wall, cell membrane, and nucleus of Poria cocos sclerotium.
PCP directly activates RAW 264.7 macrophages and enhances the production of both nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) by LPS-induced cells, and increases the mRNA expression of iNOS, TNF-a, IL-1b, IL-6, IL-12, and IL-18 of the cells. Polymyxin B (PMB) and LAL tests show that the capability of PCP activating macrophages is not due to LPS contamination.
Furthermore, PCP activates murine splenocytes, markedly enhances cell proliferation and metabolization of the cells, and also increases their secretion of gamma-interferon (IFN-g) in vitro. Flow cytometry analysis reveals that PCP significantly promotes cell proliferation in both T cells and B cells by increasing S and G2/M phases of DNA content. Additionally, PCP up-regulates the mRNA expression of IL-2, IFN-g, IL-4, and IL-5. However, PCP stimulates IFN-g but not IL-4 secretion in murine splenocytes. Taken together, these findings suggest that PCP can activate murine splenocytes and drive their Th1 development.
This study suggests that PCP is an immune stimulant and can strengthen the immune response of its host and having medicinal capability.
|
|---|
