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目的:构建以绿色荧光蛋白eGFP为报告基因的毕赤酵母表达载体,为后续致病真菌功能基因的eGFP融合基因过表达作铺垫。方法:利用In-Fusion系统构建eGFP的表达载体PPICZαB-eGFP,氯化锂转化毕赤酵母X33,通过含Zeocin(100μg/mL)抗性的YPD培养基筛选,将得到的菌落X33-eGFP以引物3’-AOX/5’-AOX及eGFP-F/eGFP-R进行PCR,产物行琼脂糖凝胶电泳、测序验证eGFP片段是否成功插入,最后甲醛诱导表达。结果:以X33-eGFP为模板的PCR产物琼脂糖凝胶电泳结果显示片段eGFP成功插入至载体中,测序验证无突变。经甲醛诱导后的毕赤酵母X33-eGFP在蓝色荧光激发下,可观察到发出绿色荧光。结论:成功建立了毕赤酵母X33-eGFP,且证明了eGFP片段可以在毕赤酵母AOX启动子中得到表达。
OBJECTIVE: To construct a Pichia pastoris expression vector using green fluorescent protein eGFP as a reporter gene, to pave the way for eGFP fusion gene over-expression in functional genes of pathogenic fungi. Methods: The eGFP expression vector PPICZαB-eGFP was constructed by In-Fusion system. Pichia pastoris X33 was transformed with lithium chloride, and was screened by YPD medium containing Zeocin (100μg / mL). The obtained colonies X33- 3’-AOX / 5’-AOX and eGFP-F / eGFP-R. The product was subjected to agarose gel electrophoresis. Sequencing was performed to confirm whether the eGFP fragment was inserted successfully. Finally, formaldehyde was induced to express. Results: The results of agarose gel electrophoresis using PCR products of X33-eGFP as a template showed that the fragment eGFP was successfully inserted into the vector and no mutation was confirmed by sequencing. After the formaldehyde-induced Pichia pastoris X33-eGFP under the blue fluorescence excitation, green fluorescence was observed. Conclusions: Pichia pastoris X33-eGFP was successfully established and it was demonstrated that the eGFP fragment can be expressed in the Pichia pastoris AOX promoter.