LHRH介导的TAT蛋白核心肽PTD的制备及转膜研究

Study on the Preparation and Transmembrane of TAT Protein Core Peptide PTD under LHRH Interference

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 pET28a- LHRH-PTD-GFP was constructed and expressed in Escherichia coli. The target protein was purified. Through the fluorescence charac- teristics of green fluorescent protein, LHRH-mediated transmembrane function of PTD protein was detected. The prokaryotic expres- sion 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/mL was obtained. Fluorescence microscopy showed significant green fluorescence in HeLa cells, demonstrating that LHRH-mediated PTD protein had good transmem- brane 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.