【摘要】 以人胎肝cDNA文库为模板 ,用PCR和DNA重组技术 ,将TPOcDNA克隆到pGEM○R T载体并测序 ,再将目的片段亚克隆到分泌型表达载体上获得重组质粒pPIC9K/TPO ,并通过PCR引物设计、扩增和DNA连接使毕赤酵母α 因子的信号肽序列取代TPO固有的信号肽序列 ,使之与毕赤酵母的α 交配因子(MFα)融合 ,该表达载体受AOX1强启动子的调控 .经电转化和G4 1 8筛选获重组酵母KM71 /pPIC9K/TPO ,并用甲醇进行诱导表达 .经甲醇诱导后 ,酵母重组子可高效分泌表达TPO .经SDS PAGE和WesternBlot分析 ,显示重组人TPO(rhTPO)分子量约为 65ku ,与文献报道的 68～ 85ku基本一致 ,表明该蛋白是经过正确加工和糖基化的蛋白 .薄层扫描分析显示 ,重组蛋白占菌体分泌总蛋白的1 0 .1 4% .由此表明我们用毕赤酵母 (Pichiapastoris ,Pp)表达体系成功地表达了TPO .更多还原

【Abstract】 By using the cDNA library from fetus liver and the technique of PCR and DNA recombinant,TPO cDNAs was cloned into the pGEM D○R -T vector and sequenced,then the target fragment was subcloned into a secretory expression vector pPIC9K to obtain the recombinant plasmid pPIC9K/TPO.We designed the TPO PCR primer to make the TPO cDNAs under the control of the methanol inducible alcohol oxidase promoter and make it be fused to the alpha mating factor (MFα) of Saccharomyces cerevisiae.The MFα can give signal for the secretion of protein.We got recombinant yeast KM71/pPIC9K/TPO by electrotransformation and secreened with G418.Then Pichia pastoris cells with pPIC9K/TPO were induced to express TPO.The molecular weight of approximately 65kD,as determined by SDS-PAGE and Western blot,is the exopected molecular weight for correctly processed and glycosylated protein.Scanning analysis indicated the expressed protein accounted up to 10.14% of total secreted proteins.The result indicates that TPO can be successfully expressed in the methylotrophic yeast Pichia pastoris.更多还原