| bFGF was first obtainded from bovine pituitary and nerve tissue byGospodarowicz in 1974. It was denominated fibroblast growth factor because itcan stimulate 3T3 cells proliferation .bFGF locates 4q25 of being chromosomeand it was made up of two intron and three exon .The distribution of basic fibroblast growth factor(bFGF) in vivo is verybroad., they can be found in brain, heart, bone, eyes, adrenal gland and placenta .Cells cultivated in vitro, such as vascular smooth muscle cells ,vascularendothelial cells, oligodendrocyte and some tumor cells includingchondrosarcoma cells, neurospongiom cells can also generate bFGF. It has beenproved that bFGF is a cytokine with multi-biological activities in vivo and invitro.bFCF is a broad-spectrum mitogen and inducing factor of morphogenesiaand differentiation, having evident chemotaxis to cells derived frommesoblastic. Studies have proved that bFCF is a angiogenesis factor, with thefuction of promoting wound healing and tissue repair. It can promote cellregeneration too.The praeparatum which are used in the market are proteins expressed fromEscherichia coli . Pichia Pastoris express system which widely used in geneengineering has the properties of simple operation, distinct genetic background,lower cost price. The system has eukaryote's folding and secrete mechanism,can accomplish the post-translational modification process which is theproperties of eukaryote including proteolysis , folding, glycosylation, has rapidgrowth,simple nutritional ingredient,and can accommodate the needs oflarge-scale industrialization .So we build an expression system of recombinantbfgf and seek to produce recombinant protein with gene-tic engineeringtechnology.1,Cloning of hbfgf cDNA sequenceWe first obtained the cDNA sequence encoding hbFGF by RT-PCR, inwhich the template RNA derives from glioma cell U251. Then the purified PCRproduction was cloned to pMD18-T vector. The results of sequencing showedthat the recombinant cloning vector pMD18-T-hbfgf was constructedcorrectly,so we can continue to do the expression of recombinant hbFGF.2,Construction of recombinant hbFGF expression system(1)Construction of hbFGF expression vector pPICZα-hbfgfWith the recombinant plasmid of pMD18-T-hbfgf as template, the sequenceencoding hbFGF was obtained by PCR. The endonuclease sites of XhoI andEcoRI were added into the two ends of interested sequence. Then it was clonedto pPICZαvector after being digested with these two endonucleases. The resultsof sequencing demonstrated that the construct was correct.(2)Transformation pPICZα-hbfgf into Pichia pastoriswe transformed purified pPICZα-hbfgf vector into Pichia pastoris andscreened on YPD plates containing 100μg/ml Zeocin. After 72 hours, dozens oftransforming colonies appeared .(3)Expression of recombinant hbFGF in Pichia pastorisSelected several clones from YPD/Zeocin plates, and induced them by0.5% methanol in BMMY media. Analyzed the expression product by activitydetermination and SDS-PAGE.Results:①Through being detected the time course expression of positiveclone, it was found that there was a cumulative effect of production during thefirst 3 days and reach the peak at the end;In the next two days, the expression ofrecombinant protein was steady;Then the production of recombinant proteinbegan reduce. ②Analyzed the purified recombinant protein by SDS-PAGE, aprotein of 18 kDa molecular weight appeared. These results indicated that thefunctional recombinant hbFGF could be induced in methylotropic yeastexpression system and the secreting of recombinant protein had cumulativeeffect, which increased depending on time change. ③When the positive clonewas incubated in BMMY media with different pH varing from 3.0 to 6.0, thesecretion of recombinant protein had a highest yield at about pH 5.0.The results indicated that the effective expression system of recombinanthbFGF had been constructed. The variant Pichia pastoris can secret recombinanthuman basic fibroblast growth factor , which can exhibits activity and has amolecular weight of 18kD.
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