穿膜肽-EGFP融合蛋白的表达、纯化与鉴定

Expression,purification and identification of CPPs-EGFP fusion protein

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质量,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分子质量对应的电泳条带,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。

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 then 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 cell experiment in vitro.Results Electrophoresis site corresponding to EGFP-CPPs was displayed in electrophoresis;The gene sequence of constructed plasmid contained the target 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.Conclusion CPPs-EGFP fusion protein with cell-penetrating function was successfully prepared in the experiment.It is a solid plateform to support the study of bringing antibody-albumin nanosphere conjugate into tumor cell with potertial therapeutical effect.