【摘要】 目的:构建重组原核表达载体pGEX-5x-1-p65,诱导GST-p65融合蛋白的表达并观察其包涵体的显微结构。方法:应用PCR技术扩增得到p65全长序列,并亚克隆至带有GST标签的pGEX-5x-1载体中。经酶切、测序鉴定后,在原核细胞中诱导表达GST-p65融合蛋白并将诱导后的菌体制作透射电镜标本,观察菌体内部显微结构。结果:成功构建表达载体pGEX-5x-1-p65,原核细胞中诱导表达、凝胶电泳后未见可溶性融合蛋白的高效表达。透射电镜观察到在承载有重组载体的菌体内部出现大量高电子密度的包涵体。结论:成功构建了原核表达载体pGEX-5x-1-p65,电子显微镜观察并证实在原核细胞内p65蛋白诱导表达形成包涵体。更多还原

【Abstract】 Objective: To construct the expression plasmid of pGEX-5X-1-p65 and observe the microstructure of induced fusion protein.Methods: The p65 coding sequence was amplified by polymerase chain reaction and subcloned into pGEX-5X-1 vector.After identification by ensymes digesting and sequencing,the vector was induced to express the GST-p65 protein.Prokaryotic cells was made to TEM sample,and the internal microstructure was observed.Results: The pGEX-5X-1-p65 vector has been constructed successfully.The segments digested by enzymes and the results by sequence both consistent with expectations.Induced by IPTG,the fusion protein was expressed as the form of inclusion body and observed generous electron-dense structure through transmission electron microscope.Conclusion: The human p65 gene was subcloned into pGEX-5X-1 successfully and the TEM observation indicated that the fusion protein expressed in inclusionbody form in prokaryotic cell.更多还原