基础医学与临床 ›› 2010, Vol. 30 ›› Issue (9): 902-906.

• 研究论文 • 上一篇    下一篇

穿膜肽-EGFP融合蛋白的表达、纯化与鉴定

周洁 郭晓云 冷静 万彦彬   

  1. 广西医科大学第一附属医院 广西医科大学第一附属医院 广西医科大学 广西医科大学
  • 收稿日期:2009-06-12 修回日期:2009-12-14 出版日期:2010-09-05 发布日期:2010-09-05
  • 通讯作者: 周洁

Expression, Purification and Identification of CPPs-EGFP Fusion Protein

Jie ZHOU, Xiao-yun GUO, Jing LENG, Yan-bin WAN   

  1. the First Affiliated Hospital, Guangxi Medical University
  • Received:2009-06-12 Revised:2009-12-14 Online:2010-09-05 Published:2010-09-05
  • Contact: Jie ZHOU,

摘要: 目的 构建EGFP-CPPs融合基因载体,表达、纯化并鉴定EGFP-CPPs融合蛋白。方法 通过碱基合成的方法合成含有CPPs基因序列的EGFP上游引物,PCR扩增后收集CPPs-EGFP融合基因片段并电泳鉴定,将融合基因表达载体转染大肠杆菌并扩增表达,收集表达蛋白后过层析柱纯化,蛋白电泳检测表达蛋白分子量,蛋白飞行质谱分析检测蛋白序列,体外细胞实验验证其穿膜功能。结果 电泳检测可见与CPPs-EGFP分子量对应的电泳条带,构建质粒测序含有目的基因序列。蛋白电泳检测到纯度较高的融合蛋白,其蛋白序列与目的序列一致。体外细胞实验证实了融合蛋白的穿膜功能。结论 成功制备了具有穿膜功能的CPPs-EGFP融合蛋白,为其促进抗体-蛋白微球偶联物进入肿瘤细胞发挥治疗作用的研究奠定了基础。

关键词: 穿膜肽, 增强型绿色荧光蛋白, 膀胱肿瘤

Abstract: Objective To construct EGFP-CPPs fusion genetic carrier,express,purify and identify EGFP-CPPs fusion protein.Methods Upstream primer containing CPPs gene sequence of green fluorescent was synthesized by basic radical synthesise methods.CPPs-EGFP fusion gene fragment was amplificatied by PCR methods and was indentified by electrophoresis. Bacillus coli were transfected with the fusion gene express vector and the fusion gene was amplificated and expressed.Fusion protein was collected and purified by column chromatography.Protein molecular mass was detected by protein electrophoresis.Protein sequence was detected by TOF analysis.Cell-penetrating function was tested by vitro cell experiment. Results Electrophoresis site corresponding to EGFP-CPPs was displayed in electrophoresis;The gene sequence of constructed plasmid contained interest gene sequence.Purified fusion protein was displayed in protein electrophoresis.Protein sequence was in accord with interested protein sequence. CPPs-EGFP fusion protein displayed Cell-penetrating function in vitro. Conclusions CPPs-EGFP fusion protein with cell-penetrating function was prepared successfully in the experiment.It will establish basis for study of promoting antibody-albumin nanosphere conjugate to enter tumor cell and bring into full play therapeutical effect.

Key words: CPPs, EGFP, Bladder tumor