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BamH Ⅰ/Xho Ⅰ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-kB so as to investigate the interaction between LRP16 and function epi-position of p6S by GST-pulldown in vitro. Methods: The function epi-positions of p65 were respectively amplified from plasmid pcDNA3.1-p65 by using PCR technique, and those DNA fragments were finally connected with pcDNA3-flag vector after sequencing. The recombinant plasmids were extracted and confirmed by digestion with BanM I /Xho I 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.
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