| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 95 === English abstract
Human granulocyte macrophage-colony stimulating factor (hGM-CSF) is a highly glycosylated protein proved to stimulate proliferation and differentiation of the hematopoietic cells. This study has aimed to express rhgm-csf gene in Aspergillus niger isolate BCRC 31512. Using the template, pUC35I-csf plasmid, and the PCR primers to obtain the hgm-csf gene fragment which had the EcoRⅤ restriction site at the 5’ end and the HindⅢ restriction site at the 3’ end, respectively. The pAN56-8 vector and the hgm-csf fragment, was double-digested with EcoRⅤ and HindⅢ, and subsequently ligated to construct a new recombinant plasmid, pAN-CSF. The protoplasts were prepared form the mycelia of A. niger, the plasmid pAN-CSF was then introduced into the protoplasts via the PEG/CaCl2 mediated transformation protocol. The protoplasts were subsequently regenerated and selected on the AMM plates supplemented with 50 µg/ml hygromycin B to recover the putative transformants. The likely integration of the rhgm-csf gene into the genomes of A. niger transformant AGT were supported by the PCR technique and the Southern dot blot analysis. In addition, the successful translations of rhGM-CSF were confirmed by the Western immunoblot assay, the Western immuno-hybridization test and the ELISA (enzyme-link immunosorbent assay) test. The amount of rhGM-CSF protein in the crude protein extracts prepared from the mycelia harvested after 12, 24, 36 and 48 h of culture, respectively, were determined by the Western immunoassay, and the ELISA. The yield of rhGM-CSF protein topped at 24 h after incubation and declined thereafter. The rhGM-CSF protein content in the mycelia was 657 ng g-1 mycelium dry weight (approximately 0.002628% of the total protein) for the transformant T2 and 331.5 ng g-1 (approximately 0.001326% of the total protein) for another transformant AGT.
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