快速筛选高水平稳定表达成纤维细胞生长因子-21真核细胞新株方法的建立

成纤维细胞生长因子-21（FGF-21）是成纤维细胞生长因子家族的一名新成员,其研究已成为世界糖尿病研究的新热点，并有望成为治疗2型糖尿病的新型药物.然而，应用真核表达系统，更加快速、高效生产更接近天然状态的FGF-21蛋白并对其生物学功能进行的研究，至今尚未报道，因此建立高效稳定的FGF-21真核表达系统至关重要.本研究以FGF-21为目的基因，利用谷氨酰胺合成酶基因（GS）和绿色荧光蛋白基因（EGFP）作为筛选标记，分别构建了单基因表达载体Pee124-FGF-21-Pee64-EGFP（Peedual）、Pee124-FGF-21-IRES-EGFP（PeeIRES）和双基因的真核表达载体Pee124-FGF-21-IRES-EGFP-Pee64-FGF-21(Peedual-IRES)，分别转染CHO-K1SV细胞后，通过GS加压和流式细胞术（在488 nm波长的激发光下能检测到EGFP绿色荧光）双筛选系统快速有效获得了稳定高效表达目的蛋白的细胞系.应用SDS-PAGE、Western印迹和ELISA检测细胞系目的蛋白的表达及差异，并通过HepG-2细胞糖吸收模型进行蛋白活性检测.研究结果表明：两个筛选系统结合使用，更好更快地筛选到了稳定转染并高效表达目的蛋白的细胞系，而且与单基因的载体相比，双基因载体Peedual-IRES在操作条件一致的情况下，目的蛋白表达量显著提高且均具有生物活性.本研究建立了省时、高效的真核表达平台，为FGF-21在真核水平上的高表达提供了一个新的方法.立了省时、高效的真核表达平台，为FGF-21在真核水平上的高表达提供了一个新的方法.

Establishment of a Novel Method for Rapid Screening of Eukaryotic Cell Lines Stably Expressing High Level FGF-21

Fibroblast growth factor-21(FGF-21) is a new member of the fibroblast growth factor family,which has the potential to become a potential therapeutic agent for type 2 diabetes.To establish an efficient and stable recombinant eukaryotic expression system,together with a high-throughput screening method for the study of the biological function of FGF-21 are very important and practical.We cloned FGF-21 into Peedual IRES eukaryotic expression vectors alone and with GS or EGFP as the selectable markers.The CHO-K1SV cells transfected with these vectors were able to express FGF-21 at high levels,and could be isolated by GS selection and flow cytometry efficiently.Western blotting and ELISA were used to analyze the expression of the target protein from the transfected cells.The activity of FGF-21 on glucose metabolism regulation was tested with HepG2 cells.The glucose uptake levels were measured using the mini-glucose oxidase-preoxydase(GOD-POD) method.The results showed that FGF-21 stable transfected cell lines were obtained with the GS plus EGFP dual system,and the expression with the double-gene-vector(PeedualIRES)was better than those with Peedual and PeeIRES backbones.In this study,we have established an eukaryotic expression system that can be used for the rapid screening of eukaryotic cell lines expressing a FGF-21 gene as the target protein.