人B淋巴细胞刺激因子的克

发布时间：2018-03-27 21:13

  本文选题：人B淋巴细胞刺激因子　切入点：绿色荧光蛋白　出处：《南京师范大学》2006年硕士论文

【摘要】：人B淋巴细胞刺激因子(hBAFF)是一种B淋巴细胞的共刺激因子，，属TNF超家族成员。本文一方面利用巴斯德毕赤酵母(pichia pastoris)这一真核表达系统成功构建、表达了hBAFF蛋白；同时对毕赤酵母分泌表达得到的hBAFF进行SDS-PAGE、Western blotting、ELISA等方面的检测。另一方面将hBAFF基因通过柔性臂(Gly_4Ser)_2与增强型绿色荧光蛋白(EGFP)相连，通过大肠杆菌(E．coli)表达系统进行融合蛋白的表达；并对表达蛋白做Western blotting、荧光及B淋巴细胞增殖的活性测定。实验设计及结果如下： 1 EGFP-hBAFF融合蛋白的表达纯化及活性测定 将人B淋巴细胞刺激因子和增强型绿色荧光蛋白通过(Gly_4Ser)_2linker相连接，在大肠杆菌BL21(DE3)中诱导表达融合蛋白EGFP-hBAFF；并对该蛋白进行Ni~(2+)-IDA亲和层析柱纯化，纯化的蛋白经检测具有EGFP的荧光活性和hBAFF的B淋巴细胞增殖活性。 2 hBAFF在毕赤酵母中的表达及产物鉴定 克隆得到hBAFF基因与穿梭载体pPIC9连接得到pPIC9-hBAFF重组质粒，线性化后电转酵母菌GS115中，挑选出阳性重组子进行诱导表达；并对表达条件优化，表达产物进行Western blotting、ELISA方面的检测。结果表明，当甲醇浓度为1％、诱导时间为72h、温度为20℃时hBAFF蛋白表达量最高。
[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

本文编号：1673267