目的针对植物生物反应器中外源基因表达水平普遍较低的问题,探索和评价丝状真菌瑞氏木霉(Trichode rma re e s e i)编码的小分子疏水蛋白(Hydrophobin,HFBI)作为融合标签在植物生物反应器中提高系统表达量的应用潜力;分析其提高外源基...目的针对植物生物反应器中外源基因表达水平普遍较低的问题,探索和评价丝状真菌瑞氏木霉(Trichode rma re e s e i)编码的小分子疏水蛋白(Hydrophobin,HFBI)作为融合标签在植物生物反应器中提高系统表达量的应用潜力;分析其提高外源基因表达水平的可能机制。方法以绿色荧光蛋白(GFP)为报道基因,采用基因体外合成技术和亚克隆技术构建GFP和GFPHFBI融合植物表达载体。利用农杆菌渗滤技术接种植物本明烟(Nicotiana be nthamiana)。通过GFP荧光观察,荧光显微镜镜检,Western blot,ELISA和RT-PCR等实验手段测定报告基因GFP在植物中的表达情况,探明HFBI融合标签在植物中表达外源基因的作用效果和特点并分析其可能的作用机制。结果 HFBI融合标签对植物细胞无明显的细胞毒性;GFP-HFBI融合蛋白在植物中的积累水平显著高于对照;GFP-HFBI融合蛋白在细胞内形成致密的蛋白质颗粒。结论 HFBI融合标签能够显著提高外源基因在植物中的积累水平。推测形成的蛋白质颗粒隔绝了细胞内源性蛋白酶对目的表达产物的降解,进而提高了外源基因产物在细胞中的积累。展开更多
Hydrophobins are a type be used to modify material surfaces of small amphipathic proteins with a and adsorb enzymes, antibodies and unique self-assembly property, which can even cells. In this study, a fusion protein ...Hydrophobins are a type be used to modify material surfaces of small amphipathic proteins with a and adsorb enzymes, antibodies and unique self-assembly property, which can even cells. In this study, a fusion protein consisting of hydrophobin HGFI and green fluorescent protein（GFP） was successfully obtained from Pichia patoris （P. pastoris）. Water contact angle（WCA） measurement proves that the wettability of the surfaces of different materials was changed. We further demonstrated the self-assembly ability of HGFI-GFP, which can be used to disperse the multi-walled carbon nanotubes（MWCNTs）. Finally, the adsorption of HGFI-GFP onto the surface of the tissue engineering material poly（ε-caprolactone）（PCL） was evaluated by detecting the fluorescence of the fusion protein itself. The resalt demonstrates that both the basic self-assembly activity of the HGFI domain and the functional activity of the GFP domain were still remained.展开更多
文摘目的针对植物生物反应器中外源基因表达水平普遍较低的问题,探索和评价丝状真菌瑞氏木霉(Trichode rma re e s e i)编码的小分子疏水蛋白(Hydrophobin,HFBI)作为融合标签在植物生物反应器中提高系统表达量的应用潜力;分析其提高外源基因表达水平的可能机制。方法以绿色荧光蛋白(GFP)为报道基因,采用基因体外合成技术和亚克隆技术构建GFP和GFPHFBI融合植物表达载体。利用农杆菌渗滤技术接种植物本明烟(Nicotiana be nthamiana)。通过GFP荧光观察,荧光显微镜镜检,Western blot,ELISA和RT-PCR等实验手段测定报告基因GFP在植物中的表达情况,探明HFBI融合标签在植物中表达外源基因的作用效果和特点并分析其可能的作用机制。结果 HFBI融合标签对植物细胞无明显的细胞毒性;GFP-HFBI融合蛋白在植物中的积累水平显著高于对照;GFP-HFBI融合蛋白在细胞内形成致密的蛋白质颗粒。结论 HFBI融合标签能够显著提高外源基因在植物中的积累水平。推测形成的蛋白质颗粒隔绝了细胞内源性蛋白酶对目的表达产物的降解,进而提高了外源基因产物在细胞中的积累。
基金Supported by the National Natural Science Foundation of China（No.31170066） and the Tianjin Key Research Program of Application Foundation and Advanced Technology, China（No. 12JCZDJC22600）.
文摘Hydrophobins are a type be used to modify material surfaces of small amphipathic proteins with a and adsorb enzymes, antibodies and unique self-assembly property, which can even cells. In this study, a fusion protein consisting of hydrophobin HGFI and green fluorescent protein（GFP） was successfully obtained from Pichia patoris （P. pastoris）. Water contact angle（WCA） measurement proves that the wettability of the surfaces of different materials was changed. We further demonstrated the self-assembly ability of HGFI-GFP, which can be used to disperse the multi-walled carbon nanotubes（MWCNTs）. Finally, the adsorption of HGFI-GFP onto the surface of the tissue engineering material poly（ε-caprolactone）（PCL） was evaluated by detecting the fluorescence of the fusion protein itself. The resalt demonstrates that both the basic self-assembly activity of the HGFI domain and the functional activity of the GFP domain were still remained.
