里氏木霉细胞表面表达系统的构建

摘要

[Objective] AfMp1p is a glycosylphosphatidylinositol (GPI) anchored cell wall protein (GPI-CWP) identified in filamentous fungus Aspergillus fumigatus, which contains a specific C terminal signal for cell wall localization. Trichoderma reesei is known as a safe fungal species ( GRAS) and widely used in the industry. Thus, developing of the cell-surface expression systems in T. Reesei is of industrial interest. [Methods] The CPI signal from the A. Fumigatus Affiplp was fused to the C-terminal of green fluorescent protein ( GFP) and transformed into T. Reesei. The optimization of T. Reesei transformation and the expression profiles of the CFP were investigated in detail. The cellular location of the CFP fusion protein was detected. [Results] Fluorescent image analysis and Western blot analysis indicate that the GFP fusion protein locates on the cell wall of T. Reesei. [ Conclusion ] According to these results, the GPI signal from A. Fumigatus can be recognized in T. Reesei and the expression system constructed in this study can be used to express heterogeneous protein in the cell wall of T. Reesei.%[目的]烟曲霉(Aspergillus fumigatus)的AfMp1p是一种通过糖基磷脂酰肌醇( glycosylphosphatidylinositol,GPI)修饰定位于细胞壁上的蛋白,其细胞壁定位信号位于蛋白质的C末端.里氏木霉(Trichoderma reesei)是一种重要的工业生产菌种.构建里氏木霉的细胞表面表达系统具有十分重要的意义.[方法]我们将AfMp1p的细胞壁定位GPI信号肽和烟曲霉几丁质酶AfChiB1的N端信号肽分别与绿色荧光蛋白(green fluorescent protein,GFP)的C末端和N末端融合并转化里氏木霉.本文首先对木霉遗传转化系统进行了优化；随后通过Real-time PCR和蛋白定量,对GFP融合蛋白在里氏木霉中不同时期的表达情况进行了研究；最后对里氏木霉表达的GFP融合蛋白进行细胞定位研究.[结果]荧光观察结合Western blot的结果表明,在平台期中期和后期,带有GPI信号的GFP融合蛋白定位于细胞壁.[结论]烟曲霉来源的GPI信号可被里氏木霉识别,本论文所构建的表达系统可用于外源蛋白在里氏木霉中的细胞壁定位表达.