| 摘要: |
| 【目的】利用马铃薯X病毒(PVX)表达载体(pgR107),在本氏烟草中表达人成纤维细胞生长因子21（Fibroblast growth factor 21，FGF21），旨在寻求一种低成本、方便、安全，且保持hFGF21蛋白生物活性的方法。【方法】将包含组氨酸(His)标签序列的hFGF21-smGFP融合基因和hFGF21基因克隆进PVX表达载体，构建重组表达质粒pgR107-His-hFGF21-smGFP和pgR107-His-hFGF21。采用冻融法将质粒pgR107-His-hFGF21-smGFP、pgR107-smGFP和pgR107-His-hFGF21转入根瘤农杆菌GV3101中，通过农杆菌介导注射侵染接种本氏烟的叶片，采用SDS-PAGE、Western blotting和ELISA检测hFGF21-smGFP和hFGF21蛋白的表达情况。烟草叶片表达的融合蛋白经Ni-NTA柱纯化后，再用重组牛肠激酶（rbEK）裂解，获得纯度较高的重组hFGF21活性蛋白。hFGF21调节葡萄糖吸收活性用微量化的GOD-POD法检测。【结果】成功构建了植物病毒双元表达载体pgR107-His-hFGF21-smGFP和pgR107-His-hFGF21。表型观察发现，侵染第7天，烟草外源蛋白积累最高，smGFP和hFGF21-smGFP蛋白的积累主要在细胞质中。SDS-PAGE、Western blotting和ELISA分析表明，hFGF21重组蛋白可在烟草中有效表达，表达量高达500 μg/g (鲜叶质量)。生物活性检测结果表明，纯化的hFGF21蛋白可以促进3T3-L1脂肪细胞对葡萄糖的吸收。【结论】利用PVX病毒载体，在本氏烟草中表达的重组hFGF21蛋白具备生物活性。 |
| 关键词: 成纤维细胞生长因子21(FGF21) 融合蛋白 烟草 马铃薯X病毒 |
| DOI： |
| 分类号: |
| 基金项目:教育部科学技术研究重点项目（210156）；渭南职业技术学院院级重点项目（WZYZ201203）；东莞市科技计划项目（201010815209）；广东省湛江市科技攻关项目(2010C3104001) |
|
| Expression of human fibroblast growth factor 21 in transgenic tobacco (Nicotiana benthamiana) using potato virus X vector |
|
|
| Abstract: |
| 【Objective】In this study,we produced tobacco (Nicotiana benthamiana) plants with human recombinant FGF21 (hFGF21) via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107),aiming to seek a low-cost,convenient,and safe method that keeps the biological activity of hFGF21 protein.【Method】The plant expression vectors pgR107-His-hFGF21-smGFP and pgR107-His-hFGF21 were obtained by cloning hFGF21-smGFP fusion gene and hFGF21 gene into the pgR107 vector.After pgR107-His-hFGF21-smGFP and pgR107-His-hFGF21 were transferred into Agrobacterium tumefaciens strain GV3101,the recombinant plasmid was introduced into leaf cells of N.benthamiana via Agrobacterium-mediated agroinfiltration.The proteins were determined by SDS-PAGE,Western blotting and ELISA analysis.The fusion proteins were purified from plant tissues by Ni-NTA affinity chromatography and cleaved with recombinant bovine enterokinase (rbEK) to obtain pure and active hFGF21.Glucose uptake activity of hFGF21 was examined by glucose oxidase and peroxidase (GOD-POD) assay.【Result】The PVX-based binary vectors,pgR107-His-hFGF21-smGFP and pgR107-His-hFGF21,were successfully constructed.Phenotype observation found that the highest accumulation of smGFP and hFGF21-smGFP protein in N.benthamiana approximately appeared at the 7th day after agroinfection.The highest accumulation of smGFP and hFGF21-smGFP appeared in the cytoplasm.As determined by SDS-PAGE,Western blotting and ELISA of leaf extracts,the hFGF21 gene was correctly translated in tobacco plants.Moreover,the recombinant hFGF21 accumulated to as high as 500 μg/g fresh mass in leaves of agro-infected plants.The purified hFGF21 stimulated glucose uptake in 3T3-L1 cells.【Conclusion】This study indicated that the recombinant hFGF21 expressed via the PVX viral vector in N.benthamiana was biologically active. |
| Key words: fibroblast growth factor 21 (FGF21) fusion protein Nicotiana benthamiana potato virus X |
