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Volvariella Volvacea Recombinant Immunomodulatory Protein (reFIP-vvo) Expression In Pichia Pastoris X-33and The Elementary Study Of Its Bioactivity

Posted on:2014-01-13Degree:MasterType:Thesis
Country:ChinaCandidate:S J XiaoFull Text:PDF
GTID:2230330395497977Subject:Biomedical engineering
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Fungal immunomodulatory proteins are small molecular weight proteins extractedfrom macrofungi with immunomodulatory activity. So far, at least nine fungalimmunomodulatory proteins were characterized. They are LZ-8, FIP-fve, FIP-vvo,FIP-gts, FIP-gja, FIP-gsi, FIP-tvc, FIP-aca and FIP-gmi from Ganoderma lucidum,Flammulina velutipes, Volvariella volvacea, Ganoderma tsugae, Ganodermajaponcium, Ganoderma sinense, Trametes versicolor, Antrodia camphorate andGanoderma microsporum respectively. The fungal immunomodulatory proteins havesimilar structures and biological function. Their immunomodulatory activity is mainlyto stimulate lymphocyte proliferation and induce immune cells to secrete a variety ofcytokines. In addition, the fungal immunomodulatory proteins have some anti-tumoractivity.This paper redesigned gene coding FIP-vvo according to the Pichia pastorispreferred codons, so that reFIP-vvo can be highly expressed in Pichia pastorisexpression system. We constructed the recombinant vector pPICZαA-reFIP-vvo andintroduced it into P. pastoris X-33by electroporation. Transformants were initiallyscreened for their resistance to Zeocin and then the positive recombinants wereidentified by PCR. The expression level of the recombinant protein is410mg/L bymethanol induction of recombinant protein expression. Western blot showedreFIP-vvo was correctly expressed. Pichia expressed reFIP-vvo was correctly foldedto be a biologically active form. After dialysis and cation column chromatography,reFIP-vvo fermentation broth was purified to purity90%. After purification thebiological activity of reFIP-vvo was measured. Haemagglutination experimentsshowed that, the hemagglutination activity and specificity of reFIP-vvo consistents with that of the natural protein. WST-1method for the determination of lymphocyteproliferation activity showed that reFIP-vvo promote lymphocyte proliferationactivity.The papers expressed reFIP-vvo efficiently using the recombinant expressionsystem Pichia pastoris X-33, and confirmed reFIP-vvo has a similar biologicalactivity to the native protein. Moreover, to the best of our knowledge, this is the firstsuccessful heterologous expression and production of FIP-vvo in a pichia expressionsystem to be reported. In this paper, the following were concluded:(1) Since Hsu forthe first time isolated FIP-vvo from the fruiting bodies of Volvariella volvacea andanalyzed its biological activity in1997, this is the first time eukaryotic expression ofreFIP-vvo, which has identical amino acid sequence to the natural FIP-vvo;(2) wefound a gene sequence coding the immunomodulatory protein FIP-vvo;(3) thehaemagglutination of blood cells and lymphocyte proliferation experimental indicatesreFIP-vvo which was expressed in the yeast expression system has biological activityThe results of these studies will be the basis for subsequent in-depth study into thebiological activity of the protein and structure-activity relationship between membersof the fungal immunomodulatory protein family.
Keywords/Search Tags:FIP-vvo, Pichia pastoris, recombinant expression, bioactivity
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