GST融合蛋白原核表達(dá)條件的優(yōu)化及純化分析

論文編號:SW229  論文字?jǐn)?shù):20576,頁數(shù):28

摘 要：本論文主要研究人CD154蛋白的原核表達(dá)。在GST原核表達(dá)最優(yōu)條件下進(jìn)行大量表達(dá)，并經(jīng)過GST柱層析純化，以獲得純度較高的目的蛋白用于免疫學(xué)實(shí)驗(yàn)。本研究是根據(jù)目的蛋白CD154基因序列設(shè)計(jì)合成特異性引物，PCR擴(kuò)增基因，并插入到融合蛋白原核表達(dá)載體pGEX－4T－1中，得到重組表達(dá)質(zhì)粒pGEX－4T－1/CD154，用此重組質(zhì)粒轉(zhuǎn)化大腸桿菌DH5-α細(xì)胞，轉(zhuǎn)化菌落經(jīng)BamHⅠ、EcoRⅠ雙酶切鑒定。并通過采用不同IPTG誘導(dǎo)濃度及不同的培養(yǎng)溫度和時(shí)間，使GST融合蛋白在大腸桿菌DH5-α中得到最大表達(dá)，并采集樣品進(jìn)行SDS-PAGE電泳鑒定表達(dá)產(chǎn)物。通過對轉(zhuǎn)化后的大腸桿菌進(jìn)行SDS-PAGE電泳分析，發(fā)現(xiàn)明顯的出現(xiàn)了54kD蛋白帶。且最佳表達(dá)條件：37℃，IPTG誘導(dǎo)濃度0.2mM，誘導(dǎo)時(shí)間4h。本研究成功構(gòu)建了GST原核表達(dá)質(zhì)粒，并在大腸桿菌中表達(dá)出GST融合蛋白，經(jīng)過提純的GST融合蛋白可以用于更深層次的免疫實(shí)驗(yàn)。
關(guān)鍵詞：GST融合蛋白；原核表達(dá)；大腸桿菌；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