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OALib Journal期刊
ISSN: 2333-9721
费用：99美元
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融合蛋白rgd-trail在毕赤酵母中的表达及活性
, PP. 552-559
Keywords: rgd,肿瘤坏死因子相关凋亡诱导配体,细胞凋亡,融合蛋白
Abstract:
本文分别利用大肠杆菌和毕赤酵母表达系统构建表达rgd-trail蛋白,比较两种表达系统对该蛋白活性的影响,并对毕赤酵母表达的rgd-trail进行活性分析。利用基因工程方法分别构建重组质粒pet30-rgd-trail和phbm-rgd-trail,于大肠杆菌和毕赤酵母中诱导表达。采用mtt法、elisa法、划痕及transwell小室和hoechst33342染色检测rgd-trail对肿瘤细胞增殖抑制、亲和活性、迁移和凋亡的影响。westernblotting检测相关凋亡蛋白的表达。重组蛋白rgd-trail于大肠杆菌中以包涵体形式表达,在毕赤酵母中分泌表达。mtt结果显示,毕赤酵母表达的rgd-trail抑制肿瘤细胞增殖活性明显强于大肠杆菌表达的rgd-trail。elisa实验表明,酵母表达的rgd-trail可与表达αv和dr4、dr5的肿瘤细胞发生特异性结合。划痕和transwell实验证明,rgd-trail能抑制肿瘤细胞a549和ht1080迁移。经过rgd-trail作用后,荧光显微镜下可观察到肿瘤细胞明显发生凋亡,westernblotting检测parp及caspase-3均出现剪接体。研究结果提示,毕赤酵母表达系统更适合于rgd-trail蛋白的表达,且能够很好地发挥靶向作用及杀伤活性。
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