【作者】 牟旭鹏；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 碱性成纤维细胞生长因子(basic fibroblast growth factor , bFGF)是FGF家族中具有代表性的成员,分子中无糖基化位点,为一单链阳离子多肽,与肝素有很强的亲和能力。它广泛存在于机体的各种组织,是一种具有广泛生物活性的肽类物质,对酸和热敏感,在体内分布极为广泛,是一种重要的神经营养因子和血管生成因子,能促进创伤愈合与组织修复,也能促进某些细胞再生,在胚胎生长、发育及血管形成等多种生理过程中起重要作用。本研究从人神经胶质瘤细胞总RNA中钓取hbFGF cDNA序列,利用基因重组技术构建人bFGF cDNA克隆载体pMD18-T-hbfgf及hbFGF表达载体pPICZα-hbfgf。将hbFGF表达载体转化X-33毕赤酵母,建立兼具原核系统良好操作性和真核系统后加工性的重组hbFGF毕赤酵母真核表达体系。通过PCR和SDS-PAGE的鉴定,筛选出高表达重组hbFGF的酵母菌株,为进一步实验奠定了基础。结果表明,本研究正确克隆了人bFGF cDNA序列,构建的重组hbFGF毕赤酵母真核表达体系经甲醇诱导能够分泌具有活性的重组hbFGF蛋白,相对分子量为18KD;重组hbFGF的表达具有累积效应,产量在一段时间内随表达时间延长而增加。本研究为选用毕赤酵母作为生物反应器生产hbFGF基因工程药物提供了实验依据。更多还原

【Abstract】 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:① Through 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.更多还原