GST-pulldown体外研究LRP16与NF-κB/p65相互作用的功能表位

Investigate the Interaction Between LRP16 and Function Epi-Position of NF-κB / p65 by GST-Pulldown in vitro

目的:构建核转录因子NF-κB/p65亚基的功能表位载体,采用GST-pulldown探究p65与LRP16体外相互作用的功能表位。方法:以真核表达质粒pcDNA3.1-p65为模板,PCR扩增一组p65功能表位的基因片段,经测序后,克隆至pcDNA3-flag载体上,用BamHⅠ/XhoⅠ双酶切重组质粒鉴定目的片段大小,然后采用GST-pulldown实验验证LRP16与p65之间的相互作用。结果:构建了p65的5个功能表位的表达载体且可用于体外转录翻译,GST-pulldown体外研究表明,p65蛋白N端RHD结构域是与LRP16相互作用所必需的,p65的RHD结构域的缺失完全消除了p65与LRP16的体外相互作用。结论:GST-pulldown实验验证了LRP16与p65存在体外直接相互作用,并具体分析了LRP16与p65相互作用的表位。

Objective： To construct recombinant vectors of function epi-position of p65 subunit of NF-κB so as to investigate the interaction between LRP16 and function epi-position of p65 by GST-pulldown in vitro.Methods： The function epi-positions of p65 were respectively amplified from plasmid pcDNA3.1-p65 by using PCR tech-nique,and those DNA fragments were finally connected with pcDNA3-flag vector after sequencing.The recombinant plasmids were extracted and confirmed by digestion with BamHⅠ / XhoⅠ enzymes.Then,the interaction between LRP16 and p65 was investigated by GST-pulldown.Results： The series of p65 deletion mutants were successfully constructed and used in vitro transcription.The N-terminal RHD region of p65 was necessary for p65 to interact with LRP16.Meanwhile,the deletion mutants of RHD region in p65 completely abolished the interaction with LRP16.Conclusion： We validated the direct interaction of LRP16 and p65 via GST-pulldown,and also analyzed the function epi-position of p65.