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腺病毒5型EIA(Ad5EM)作为一个抑癌基因,由于具有较强抑癌作用及显著的化放疗增敏作用而受到人们的关注.但由于它能整合到宿主细胞基因组中长期表达,并能使Rb基因失活,使动物细胞永生化等而引起人们的担心.为了避免EIA在基因治疗中存在的问题,设想以EIA蛋白进行探索.首先构建了EIA真核表达载体(pPIC9/EIA),转化毕赤酵母(GS115),筛选高表达重组菌株.经摇瓶试验,甲醇诱导表达3 d后,分泌型的EIA蛋白经HiTrap Q和HiTrap SP两步柱层析,获得了较高纯度的EIA蛋白,并经SDs-PAGE和Western blot鉴定.EIA蛋白经Chariot介导能顺利进入细胞,并大部分定位在细胞核,使LN686细胞发生明显的G2/M期阻滞,体外能显著抑制LN686肿瘤细胞生长.为利用EIA蛋白在肿瘤治疗中应用的可能性提供了实验依据.
As a tumor suppressor gene, adenovirus type 5 EIA (Ad5EM) has drawn much attention due to its strong anti-tumor effect and significant chemoradiotherapy, but since it can be integrated into the host cell genome for long-term expression, So that the Rb gene inactivation, the immortalization of animal cells caused by people’s concerns.In order to avoid the EIA in the gene therapy exists, to explore the EIA protein to explore.Established the EIA eukaryotic expression vector (pPIC9 / EIA) The recombinant E. coli was transformed into Pichia pastoris (GS115) and screened for high expression of recombinant strains.After induced by methanol for 3 days in shake flasks, the secreted EIA protein was purified by HiTrap Q and HiTrap SP two-step chromatography to obtain higher purity EIA Protein, and identified by SDs-PAGE and Western blot.EIA protein by Chariot mediated can successfully enter the cells, and most of the localization in the nucleus, LN686 cells significantly G2 / M phase arrest, in vitro can significantly inhibit LN686 tumor cells The experimental data provided the experimental basis for the possibility of using EIA protein in tumor therapy.