【摘要】 目的:在毕赤酵母SMD1168中用3-磷酸甘油醛脱氢酶基因启动子(glyceraldehyde-3-phosphate dehygrogenase gene promoter,pGAP)表达黑曲霉葡萄糖氧化酶(glucose oxidase,GOD)。方法:将黑曲霉accc30161的GOD基因插入具有pGAP的pGAPZαA质粒中,构建黑曲霉GOD毕赤酵母表达载体,并用菌落PCR、重组质粒琼脂糖凝胶电泳、限制性酶切分析及测序等方法对其进行验证。然后用重组质粒电转化毕赤酵母SMD1168,并用PCR扩增分析观察转化效果,用SDS-PAGE及酶活检测观察重组酵母黑曲霉GOD的表达和活性。结果:用黑曲霉accc30161的GOD基因成功构建了GOD表达载体pGAPZαA-GOD。转化后,pGAPZαA-GOD相关DNA片段已整合进重组毕赤酵母SMD1168-GOD基因组中。SMD1168-GOD可高表达具有活性的GOD,在30℃、pH 6的条件下,其培养液上清GOD酶活可达107.18 u/mL。结论:重组蛋白缺陷性毕赤酵母SMD1168-GOD可以利用pGAP高效表达黑曲霉GOD。更多还原

【Abstract】 Objective:Aspergillus niger glucose oxidase( GOD) was expressed in Pichia pastoris SMD1168 with the glyceraldehyde-3-phosphate dehygrogenase gene promoter( pGAP).Method:The Aspergillus niger GOD gene was cloned into the pGAPZαA plasmid containing pGAP to construct a Pichia pastoris expression vector pGAPZαA-GOD.After identified by colony PCR,agarose gel electrophoresis,restriction analysis and sequencing,the expression vector was electroporated into Pichia pastoris SMD1168.Then,PCR amplification assay was applied to observe the result of electroporation,followed by detecting the expression of the GOD gene with SDS-PAGE and enzyme activity assay.Result:The results indicated that the expression vector of the Aspergillus niger GOD gene,pGAPZαA-GOD,was successfully constructed.After electroporation with pGAPZαA-GOD,the GOD gene was integrated into genome of Pichia pastoris SMD1168.The recombinant Pichia pastoris SMD1168-GOD could express GOD at high level.The activity of GOD in the culture supernatant of SMD1168-GOD could be up to 107.18 u/mL at the condition of 30℃ and pH 6.Conclusion:The recombinant protease deficient Pichia pastoris SMD1168-GOD could efficiently express Aspergillus niger GOD with pGAP.更多还原