GST融合蛋白原核表达条件的优化及纯化分析

文档编号:SW229  文档字数:20576,页数:28

摘 要：本文档主要研究人CD154蛋白的原核表达。在GST原核表达最优条件下进行大量表达，并经过GST柱层析纯化，以获得纯度较高的目的蛋白用于免疫学实验。本研究是根据目的蛋白CD154基因序列设计合成特异性引物，PCR扩增基因，并插入到融合蛋白原核表达载体pGEX－4T－1中，得到重组表达质粒pGEX－4T－1/CD154，用此重组质粒转化大肠杆菌DH5-α细胞，转化菌落经BamHⅠ、EcoRⅠ双酶切鉴定。并通过采用不同IPTG诱导浓度及不同的培养温度和时间，使GST融合蛋白在大肠杆菌DH5-α中得到最大表达，并采集样品进行SDS-PAGE电泳鉴定表达产物。通过对转化后的大肠杆菌进行SDS-PAGE电泳分析，发现明显的出现了54kD蛋白带。且最佳表达条件：37℃，IPTG诱导浓度0.2mM，诱导时间4h。本研究成功构建了GST原核表达质粒，并在大肠杆菌中表达出GST融合蛋白，经过提纯的GST融合蛋白可以用于更深层次的免疫实验。
关键词：GST融合蛋白；原核表达；大肠杆菌；SDS-PAGE

Abstract：The present paper mainly studies the human CD154 protein the nucleus expression. Carries on the massive expressions under the GST nucleus expression most superior condition, and after the GST column chromatographic analysis purification, obtains the purity high goal protein to use in the immunology experiment. This research is directs the thing according to the goal protein CD154 gene sequence design synthesis specificity, PCR to expand increases the gene, and inserts to the fusion protein nucleus expresses in carrier pGEX-4T-1, obtains the reorganization to express material particle pGEX-4T-1/CD154,Reorganizes the material particle transformation backwoods E.coli DH5-α; cell with this, the transformed colony after BamHⅠ、EcoRⅠ;The double enzyme cuts the appraisal. And through uses the different IPTG induction density and the different raise temperature and the time, causes GST to fuse the protein DH5-α; to obtain the biggest expression at the backwoods E.coli, and gathers the sample to carry on the SDS-PAGE electrophoresis appraisal to express the product. Through to transforms after the backwoods coli to carry on the SDS-PAGE electrophoretic analysis, discovered the obvious appearance 54kD protein leucorrhea. Also best expression condition: 37℃, IPTG induction density 0.2mM, induction time 4h.This studied constructs the GST nucleus to express the material particle successfully, and expressed the GST fusion protein in the backwoods coli, might use in a deeper level after the depuration GST fusion protein the immunity experiment.
keywords： GST fusion protein, expression, E.Coli , SDS-PAGE

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