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

【摘要】：人B淋巴細(xì)胞刺激因子(hBAFF)是一種B淋巴細(xì)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(dá)系統(tǒng)成功構(gòu)建、表達(dá)了hBAFF蛋白；同時(shí)對(duì)畢赤酵母分泌表達(dá)得到的hBAFF進(jìn)行SDS-PAGE、Western blotting、ELISA等方面的檢測(cè)。另一方面將hBAFF基因通過(guò)柔性臂(Gly_4Ser)_2與增強(qiáng)型綠色熒光蛋白(EGFP)相連，通過(guò)大腸桿菌(E．coli)表達(dá)系統(tǒng)進(jìn)行融合蛋白的表達(dá)；并對(duì)表達(dá)蛋白做Western blotting、熒光及B淋巴細(xì)胞增殖的活性測(cè)定。實(shí)驗(yàn)設(shè)計(jì)及結(jié)果如下： 1 EGFP-hBAFF融合蛋白的表達(dá)純化及活性測(cè)定 將人B淋巴細(xì)胞刺激因子和增強(qiáng)型綠色熒光蛋白通過(guò)(Gly_4Ser)_2linker相連接，在大腸桿菌BL21(DE3)中誘導(dǎo)表達(dá)融合蛋白EGFP-hBAFF；并對(duì)該蛋白進(jìn)行Ni~(2+)-IDA親和層析柱純化，純化的蛋白經(jīng)檢測(cè)具有EGFP的熒光活性和hBAFF的B淋巴細(xì)胞增殖活性。 2 hBAFF在畢赤酵母中的表達(dá)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(yáng)性重組子進(jìn)行誘導(dǎo)表達(dá)；并對(duì)表達(dá)條件優(yōu)化，表達(dá)產(chǎn)物進(jìn)行Western blotting、ELISA方面的檢測(cè)。結(jié)果表明，當(dāng)甲醇濃度為1％、誘導(dǎo)時(shí)間為72h、溫度為20℃時(shí)hBAFF蛋白表達(dá)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(hào)：1673267