采用PCR技术,以含有TFP(Tenebrio Fibrinolyric Proteins, TFP) cDNA质粒作为模板,扩增出TFP基因片段.将克隆获得的目的基因插入到毕赤酵母表达载体pPIC9K中,经测序无误后,获得重组表达质粒pPIC9K-TFP.将重组表达质粒线性化后电击转化到毕赤酵母GS115中,经筛选并PCR验证获得多拷贝整合型酵母工程菌,对工程菌进行发酵和甲醇诱导表达.阳性样品经纤溶平板实验验证,表明该表达蛋白具有生物活性.%In this paper, TFP fragment was firstly obtained by PCR amplification using a plasmid which contains TFP cDNA as a template. The amplified fragment wascloned intoP. Pastoris expressing vector pPIC9K. After confirming through sequencing, a recombinant expression plasmid pPIC9K-TFPisobtained. The plasmid islinearized and then transformed intoP. Pastoris cell GS115 by electroporation. The positive recombinantis screened and confirmed by PCR. A single colony is used fermented which grew at 30℃ in a shaking incubator. TFP gene is expressed in yeast after induced with 0.5% methano and the specificity of expressed proteinidentified by SDS-PAGE. TFP fibrinolytic activity is detected by fibrin-plate method. The results showthat TFP gene has been successfully expressed in yeastPichia pastoris, and thatthe expressed protein has fibrinolytic activity.
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