【摘要】 目的:设计构建和表达新型融合蛋白谷胱甘肽-碱性成纤维细胞生长因子(Glutathione-basic fibroblast growth factor,GSH-b FGF),并检测其体外抗氧化、抗衰老能力.方法:采用RT-PCR,酶切,连接,转化等分子克隆技术构建新型融合蛋白GSH-b FGF,并通过镍柱亲和层析、阳离子交换层析纯化该目的蛋白.通过CCK-8试剂盒检测GSH-b FGF对鼠源成纤维细胞NIH-3T3的促增殖活性.同时,经总抗氧化检测试剂盒检测GSH-b FGF的抗氧化能力.结果:克隆表达并纯化获得融合蛋白GSH-b FGF.CCK-8促增殖结果显示质量浓度为20 ng/m L的GSH-b FGF可显著促进鼠源成纤维细胞NIH-3T3增殖.体外抗氧化结果显示GSH-b FGF有显著的抗氧化活性,纯化得到的GSHb FGF蛋白的GSH浓度约为(0.06±0.02)μmol/L,抗氧化能力为(0.56±0.23)mmol/g.结论:新型融合蛋白GSHb FGF有显著的体外促增殖能力及抗氧化活性.更多还原

【Abstract】 Aim: A novel fusion protein,Glutathione-basic fibroblast growth factor( GSH-b FGF) was constructed and expressed in BL21. The antioxidant activity of the purified fusion protein and its influence on cell proliferation was determined in vitro. Methods: The ORF of GSH and b FGF was obtained by RT-PCR and cloned into p ET-21 b. The fusion protein GSH-b FGF was expressed in BL21 and purified by Ni-NTA、cation exchange chromatography sequentially. Increased cell( NIH-3T3) proliferation was observed using CCK-8 assays and the antioxidant capacity of GSH-b FGF was determined using total antioxidant capacity assays. Results: The protein GSH-b FGF showed positive influence on the proliferation of cultured NIH-3T3 cells at 20 ng / m L using CCK-8 assays. A significant antioxidant activity of GSH-b FGF was also showed using total antioxidant capacity assays. GSH-b FGFs contained( 0. 06 ± 0. 02) μmol / L of GSH and the antioxidant activity was found at( 0. 56 ± 0. 23) mmol / g. Conclusion: Antioxidant activity and the activity to promote cell proliferation were well preserved in the novel fusion protein GSH-b FGFs.更多还原