人p65真核表达载体的构建及鉴定  

Construction and Identification of Human p65 Eukaryotic Expression Vector

作　　者：杨莞琦 王瑞峰 程龙 牛畅[1] 

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]军事医学研究院生物工程研究所,北京100850

出　　处：《生物技术通讯》2018年第1期55-58,90,共5页Letters in Biotechnology

摘　　要：目的:构建带Myc标签的人p65真核表达载体,对其在人宫颈癌He La细胞中的定位进行检测,并研究p65对核因子κB(NF-κB)转录活性的影响。方法:构建带Myc标签的p65真核表达载体pc DNA3.1-Myc-p65,质粒测序验证正确后脂质体法瞬时转染人胚肾HEK293T细胞,Western印迹鉴定p65的表达;细胞免疫荧光实验检测p65的亚细胞定位;重组质粒与NF-κB-Luc报告基因质粒共转染HEK293T细胞,加TNFα刺激后检测萤光素酶报告基因的活性。结果:测序结果证实pc DNA3.1-Myc-p65真核表达载体构建成功;脂质体法转染HEK293T细胞后检测到Myc-p65蛋白的表达;细胞免疫荧光实验显示Myc-p65蛋白定位于He La细胞的细胞质中,当加入TNFα刺激后大部分Myc-p65蛋白进入细胞核;萤光素酶活性检测结果显示,提高细胞内p65水平或加入TNFα刺激均可明显激活NF-κB信号通路的活性。结论:构建了带Myc标签的p65真核表达载体,并使其在HEK293T细胞中表达;正常情况下,Myc-p65蛋白定位于宫颈癌He La细胞的细胞质中,TNFα促进Myc-p65入核;Myc-p65能够以TNFα不依赖的方式激活NF-κB信号通路。pc DNA3.1-Myc-p65真核表达载体的构建,为进一步筛选NF-κB的相互作用蛋白及功能研究奠定了基础。Objective: To construct human p65 eukaryotic expression vector with Myc tag and detect localization and transcriptional activity of Myc-p65 in He La cells and 293 T cells. Methods: p65 was constructed into eukaryotic expression vector pc DNA3.1-Myc, and subjected to gene sequencing,transfection into HEK293 T cells and detection by Western blotting. Subcellular localization of Myc-p65 was measured by cell immunofluorescence, and transcriptional activity of Myc-p65 was determined by cotransfection with NF-κB-Luc in HEK293 T cells with or without TNFα. Results: Myc-p65 protein localized in cytoplasmic in He La cells and translocated to nucleus after TNFα stimulation. Luciferase reporter assay showed that Myc-p65 and TNFα enhanced the activity of NF-κB signaling pathway. Conclusion: Construction of pc DNA3.1-Myc-p65 laid a foundation for further screen and study of interaction proteins with p65.

关 键 词：P65 真核表达载体 亚细胞定位 NF-ΚB信号通路 

分 类 号：Q78[生物学—分子生物学] Q24