本文選題：人B淋巴細胞刺激因子　切入點：綠色熒光蛋白　出處：《南京師范大學》2006年碩士論文

【摘要】：人B淋巴細胞刺激因子(hBAFF)是一種B淋巴細胞的共刺激因子，，屬TNF超家族成員。本文一方面利用巴斯德畢赤酵母(pichia pastoris)這一真核表達系統(tǒng)成功構(gòu)建、表達了hBAFF蛋白；同時對畢赤酵母分泌表達得到的hBAFF進行SDS-PAGE、Western blotting、ELISA等方面的檢測。另一方面將hBAFF基因通過柔性臂(Gly_4Ser)_2與增強型綠色熒光蛋白(EGFP)相連，通過大腸桿菌(E．coli)表達系統(tǒng)進行融合蛋白的表達；并對表達蛋白做Western blotting、熒光及B淋巴細胞增殖的活性測定。實驗設(shè)計及結(jié)果如下： 1 EGFP-hBAFF融合蛋白的表達純化及活性測定 將人B淋巴細胞刺激因子和增強型綠色熒光蛋白通過(Gly_4Ser)_2linker相連接，在大腸桿菌BL21(DE3)中誘導表達融合蛋白EGFP-hBAFF；并對該蛋白進行Ni~(2+)-IDA親和層析柱純化，純化的蛋白經(jīng)檢測具有EGFP的熒光活性和hBAFF的B淋巴細胞增殖活性。 2 hBAFF在畢赤酵母中的表達及產(chǎn)物鑒定 克隆得到hBAFF基因與穿梭載體pPIC9連接得到pPIC9-hBAFF重組質(zhì)粒，線性化后電轉(zhuǎn)酵母菌GS115中，挑選出陽性重組子進行誘導表達；并對表達條件優(yōu)化，表達產(chǎn)物進行Western blotting、ELISA方面的檢測。結(jié)果表明，當甲醇濃度為1％、誘導時間為72h、溫度為20℃時hBAFF蛋白表達量最高。
[Abstract]:Human B lymphocyte stimulating factor hBAFF is a costimulatory factor of B lymphocytes and belongs to the TNF superfamily. On the one hand, the eukaryotic expression system of Pichia pastoris, Pichia pastoris, was successfully constructed and expressed hBAFF protein. At the same time, the hBAFF secreted by Pichia pastoris was detected by SDS-PAGEG Western blotting Elisa. On the other hand, the hBAFF gene was linked to the enhanced green fluorescent protein (EGFP) via Gly4Sert2, and the fusion protein was expressed by E. coli expression system. The Western blotting, fluorescence and B lymphocyte proliferation activity of the expressed protein were determined. The experimental design and results were as follows:. Expression, purification and activity determination of 1 EGFP-hBAFF fusion protein. The fusion protein EGFP-hBAFFwas induced by human B lymphocyte stimulating factor and enhanced green fluorescent protein (EGFP-hBAFF) in E. coli BL21DE3, and purified by Ni~(2 affinity chromatography. The purified protein was tested for fluorescence activity of EGFP and B lymphocyte proliferation activity of hBAFF. Expression of 2 hBAFF in Pichia pastoris and identification of its product. HBAFF gene was ligated with shuttle vector pPIC9 to obtain pPIC9-hBAFF recombinant plasmid. After linearization, the positive recombinant plasmid was selected from yeast GS115 for induction and expression, and the expression conditions were optimized. The results of Western blotting Elisa showed that when methanol concentration was 1, induction time was 72 h, and temperature was 20 鈩

本文編號：1673267