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制备重组蛋白pET28a-LHRH-PTD-GFP, 利用绿色荧光蛋白GFP, 研究LHRH的介导和PTD蛋白的转膜作用.以重组质粒pET28a-PTD-GFP为模板, 设计含有LHRH基因序列的PCR引物, 经过PCR扩增后双酶切, 克隆到原核表达载体p ET28 (a) 中, 构建重组表达质粒pET28a-LHRH-PTD-GFP, 并在大肠杆菌中诱导表达, 纯化目的蛋白.通过GFP的荧光特性, 检测LHRH介导的PTD蛋白的转膜作用.成功构建LHRH-PTD-GFP原核表达载体, 融合蛋白相对分子质量约为30 k, 纯化得到蛋白浓度为1.62 mg/m L的LHRH-PTD-GFP重组蛋白.荧光显微镜检测结果显示, He La细胞内显现明显的绿色荧光, 证明LHRH介导的PTD蛋白具有很好的跨膜特性.该研究结果为下一步构建以LHRH为靶向, 以PTD为转膜作用的肿瘤治疗药物提供了理论基础和技术支撑.“,”The aim is to study preparation recombinant protein of pET28a-LHRH-PTD-GFP, the use of green fluorescent protein GFP and LHRH-mediated and PTD protein transfection.The recombinant plasmid pET28a-PTD-GFP was used as a template to design a PCR primer containing the LHRH gene sequence.After double-digestion with PCR, the recombinant plasmid pET28aLHRH-PTD-GFP was constructed and expressed in Escherichia coli.The target protein was purified.Through the fluorescence characteristics of green fluorescent protein, LHRH-mediated transmembrane function of PTD protein was detected.The prokaryotic expression vector LHRH-PTD-GFP was successfully constructed.The relative molecular weight of the fusion protein was about 30 k.After purification, the LHRH-PTD-GFP recombinant protein with the protein concentration of 1.62 mg/m L was obtained.Fluorescence microscopy showed significant green fluorescence in He La cells, demonstrating that LHRH-mediated PTD protein had good transmembrane properties.The results of this study provide the theoretical basis and technical support for the next step to construct a tumor therapeutic drug targeting LHRH and PTD transmembrane action.