马铃薯X病毒载体介导的人成纤维细胞生长因子21(hFGF21)在烟草中的表达

Expression of human fibroblast growth factor 21 in transgenic tobacco (Nicotiana benthamiana) using potato virus X vector

【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500μg/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。

[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 （pgRi07）,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 ofN. 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. bentharniana was biologically active.