發(fā)布時間：2018-04-02 06:32

  本文選題：Survivin　切入點：原核表達　出處：《細胞與分子免疫學(xué)雜志》2013年08期

【摘要】：目的構(gòu)建人腫瘤抗原survivin的原核表達載體,優(yōu)化在大腸桿菌中的表達條件,并對survivin/His融合蛋白進行純化和抗原活性鑒定。方法設(shè)計針對survivin基因序列的特異引物,通過聚合酶鏈式反應(yīng)(PCR)擴增人survivin全長基因序列(538 bp)克隆至原核表達載體pET28a(+),構(gòu)建重組表達載體pET28a-survivin,并將該載體轉(zhuǎn)化大腸桿菌BL21(DE3),經(jīng)IPTG誘導(dǎo)表達survivin/His融合蛋白,并采用Ni親和層析凝膠純化重組蛋白。純化后的重組蛋白經(jīng)Western blot法、ELISA鑒定其抗原活性。結(jié)果重組表達載體經(jīng)BamHⅠ和HindⅢ鑒定正確;IPTG誘導(dǎo)后經(jīng)SDS-PAGE分析表明獲得了相對分子質(zhì)量(M r)24 000大小的重組蛋白;純化后的蛋白純度達到90%。Western blot法和ELISA檢測證實純化的survivin蛋白能夠與特異性抗體發(fā)生反應(yīng),表明其具有良好的抗原活性。結(jié)論成功構(gòu)建了原核表達載體pET28a-survivin,利用大腸桿菌表達系統(tǒng)實現(xiàn)了融合蛋白的可溶性表達并進行純化,純化后survivin蛋白經(jīng)鑒定具備較高的抗原活性。
[Abstract]:Objective to construct the prokaryotic expression vector of human tumor antigen survivin, optimize the expression conditions in Escherichia coli, and purify and identify the antigen activity of survivin/His fusion protein.Methods specific primers for survivin gene sequence were designed.The full-length human survivin gene was amplified by polymerase chain reaction (PCR) and cloned into prokaryotic expression vector pET28a (pET28a). The recombinant expression vector pET28a-survivin was transformed into Escherichia coli BL21DE3, and the survivin/His fusion protein was induced by IPTG.The recombinant protein was purified by Ni affinity chromatography gel.The purified recombinant protein was identified by Western blot Elisa.Results the recombinant expression vector was identified by BamH 鈪，

本文編號：1699165