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The Elementary Study Of Recombinant Expression And Biological Characteristics Of Fungal Immunomodulatory Protein From Flammulina Velutipes

Posted on:2010-02-21Degree:MasterType:Thesis
Country:ChinaCandidate:G X XuFull Text:PDF
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A fungal immunomodulatory protein, FIP-fve is a small protein extracted from Flammulina velutipes, named golden needle mushroom in China, which has immunomodulatory activities. It obviously takes a part in physiology as antiallergy decreasing the growth of cancer cells, intensifying lots of cytokines from T lymphocytes and so on.In this study, the FIP-fve gene was cloned from the genome of local golden needle mushroom (Flammulina velutipes). We reconstructed the recombinant expression vectors pPIC9K-FIP-fve and pET-28(+)-FIP-fve and transformed the target gene into the yeast Pichia pastoris GS115 and E. coli BL21 by electroporation and thermal-transformation. The yeast transformants were identified by culture media selection. The yields of the recombinant FIP-fve were about 152 mg/L in yeast and 30 mg/L in bacteria respectively. The purification of the recombinant protein includes ion-exchange chromatograph DEAE Sepharose Fast Flow, dialysis and lyophilization. The purity of the reFIP-fve was probably 80%.The amino acid composition analysis showed that the recombinant FIP had the same amino acid compose as the nature one.The Circular Dichroism analysis result indicated that the secondary structure form was detected and the relative proportion of these structure units was similar to the natural FIP which might mean that the similar nature and bioactive, reFIP-fve were correctly folded and formed. And the phenol-sulfuric acid method showed that the sugar content of the reFIP-fve was 4.2% implying the glycosylation in the yeast expression system. The biological activities of the purified reFIP-fve were test. The reFIP-fve exhibited similar hemagglutinating activity towards red blood cells to nature protein. And the reFIP-fve can induce G1/G0 to S phase of the cell cycle in peripheral blood mononuclear cells which showed that reFIP-fve seemed to be a new mitogen.In this paper, we cloned the FIP-fve gene which was re-expressed in Pichia pastoris and E. coli effectively. We proved that the recombinant FIP-fve had very similar biological activities as nature FIP-fve, which would supply some basis for fermentation by a large scale and the applications of reFIP-fve in the anti-tumor, anti-allergic, health or supplementary foods and medicine in the future.
Keywords/Search Tags:FIP-fve, Recombinant expression, Pichia pastoris, E. coli, Bioactivity
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