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Expression And Bioactivity Study Of Recombinant Fungal Immunomodulatory Protein (FIP) In Pichia Pastoris GS115

Posted on:2010-11-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:J W LinFull Text:PDF
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The fungal immunomodulatory proteins (FIPs) are a new family of micro -proteins with immunomodulatory activities extracted from several species of medical or edible mushrooms. Seven FIPs have been found and identified up to date from Ganoderma lucidium, Ganoderma tsugae, Flammulina velutipes, Volvariella volvacea, Ganoderma japoncium, Ganoderma microsporum and Ganoderma sinense. These FIPs have similar structure and biological functions to mushroom lectins. The immunomodulating capacity of the FIPs mainly includes the stimulatory proliferatin towards mouse splenocytes and human peripheral blood lymphocytes in vitro. They also can enhance the release of several cytokines such as interleukin-2, tumor necrosis factor-αand interferon-γand so on from T lymphocytes and macrophage cells. In addition, the FIPs exhibit anti-tumor activity to some extent.In the current study, two genes enconding proteins of LZ-8 and FIP-fve were amplified from the genome of Lingzhi (Ganoderma lucidium) mycelia and fruit bodies of golden needle mushroom (Flammulina velutipes). We constructed the recombinant expression vectors pPIC9-LZ-8 and pPIC9-FIP-fve and transformed the target genes into the yeast Pichia pastoris GS115 by electroporation. The correct yeast transformants were identified by Histamine-absent plate cultivation selection and PCR, which were later inoculated into liquid media for flask cultivation. The recombinant proteins were induced expression by adding methanol. The yield of the recombinant LZ-8 and FIP-fve was 191 and 158 mg L-1, respectively. The purification of the recombinant proteins included hyper-filtration, DEAE-A25 ion-exchange chromatography, HPLC, dialysis and lyophilization. The purity of the recombinant proteins was over 85%. The amino acid composition analysis showed that the recombinant FIPs had similar amino acid composition to the nature ones. The Circular Dichroism analysis result indicated that the secondary structure forms could be detected and the relative proportion of these structure units was accordant with the natural FIPs, which might mean that the putative mature and bioactive reFIPs were correctly folded and formed. And the phenol-sulfuric acid method showed that the sugar content of the reLZ-8 and reFIP-fve was 1.8% and 1.2%, which was a little high than the natural FIPs. That maybe implied the probable glycosylation in the yeast expression system.The biological activities of the purified reFIPs were tested after identification and purification. The reFIPs exhibited the same hemagglutinating activity towards red blood cells. The MTT method was used to check the mitogenic effect of the reFIPs on the mouse splenocytes and the result showed that both the recombinant proteins could apparently stimulate the proliferation of the splenocytes in vitro. Also, the immunomodulatory capacity of the reFIPs was demonstrated by enhancing the expression level of interleukin-2 release from the mouse splenocytes evaluated by indirect ELISA method. Furthermore, the anti-tumor activity of the reFIPs was tested using Flowcytometry, and the result exhibited that the reLZ-8 showed notable killing activity towards human leukemia NB4 by inducing carcinoma apoptosis, while the reFIP-fve's effect was weak.In this paper, we successfully obtained highly-effective recombinant fungal immunomodulatory proteins expressed in P. pastoris GS115 and proved the reFIPs possessed the similar biological functions to natural proteins. That would supply some theriotical basis and proof for further and deeper research in the function, mechanism and recombinant expression, as well as the potential application in the anti-allergic, anti-tumor, phamarceuticals and supplementary agents. Futher study on the bio-functions, the interation and mechanism of the reFIPs with the lymphocytes and tumor cells are in scheme that will be carried out soon in the future.
Keywords/Search Tags:Fungal immunomodulatory protein, Recombinant expression, Pichia pastoris, Immunomodulatory actvities
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