Expression of recombinant human granulocyte macrophage colony-stimulating factor in Aspergillus niger

• Main Authors: Lin chih-yun, 林芷芸
• Other Authors: Lan, Ching-Long
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/82331855565591071791

碩士 === 輔仁大學 === 生命科學系碩士班 === 93 === 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 attempted to express rhGM-CSF gene in Aspergillus niger BCRC 31512. A 450 bp rhGM-CSF fragment excised from a pUC35I-csf plasmid was cloned into a fungal expression vector pBARMTE1 obtained from the FGSC (Fungal Genetics Stock Center) to construct a new plasmid, pFGM-csf. When the mycelia of A. niger were treated with the lysing enzyme for 3-4 hr, the yield of the released protoplasts was the most abundant and the quality of the protoplasts was the best. The plasmid pFGM-csf was introduced into the protoplasts via the PEG(polyethylene glycol)-mediated transformation protocol, and the treated protoplasts were subsequently regenerated and selected on the PDAS (potato dextrose sorbitol agar) plates supplemented with 100 µg/ml glufosinate. Ten putative transformants were recovered and named T1-T10, respectively. The likely integration of the rhGM-CSF gene into the genomes of A. niger transformants were supported by the PCR technique, the Southern dot blot analysis, the Southern hybridization analysis, and their subsequent transcriptions into mRNAs were further verified by the RT-PCR(reverse transcription-polymerase chain reaction) approach. In addition, the successful translations of rhGM-CSF were supported by the Western dot immunoblot assay and the ELISA(enzyme- link immunosorbent assay) test. The amount of rhGM-CSF in the crude protein extracts prepared from the mycelia harvested after 12, 24, and 36 hours of culture, respectively, were determined by the ELISA and the yield of rhGM-CSF topped at 24 hr after incubation. The yield of rhGM-CSF, as determined by the ELISA, was estimated to 535 pg/ml, approximately 1/100000 of the total protein (5.35 ng rhGM-CSF/1 g mycelial dry weight).