目的克隆NF-κB(p65)基因,构建真核表达载体pcDNA3.1/NF-κB(p65),并进行体外表达研究。方法提取B16总RNA,RT-PCR扩增出NF-κB(p65)基因片段,并将其克隆到pcDNA3.1载体中进行序列分析,转染到B16细胞中,Real-time PCR和Western-blot检测NF-κB(p65)的抗原性和表达量。结果获得高质量的B16总RNA,RT-PCR扩增出1.65kb的cDNA片段,成功构建了pcDNA3.1/NF-κB(p65)载体,Blast序列分析与GenBank中NM_009045.4完全一致。NF-κB(p65)重组蛋白具有抗原性,其基因和蛋白表达高于B16细胞组和空质粒pcDNA3.1组。结论成功构建pcDNA3.1/NF-κB(p65)表达载体和表达出具有NF-κB(p65)抗原性的重组蛋白。 Objective To clone the gene of NF-κB (p65) and construct the eukaryotic expression vector pcDNA3.1 / NF-κB (p65). Methods The total RNA of B16 was extracted and the gene fragment of NF-κB (p65) was amplified by RT-PCR. The fragment was cloned into pcDNA3.1 vector and sequenced. The recombinant plasmid was transfected into B16 cells. Real-time PCR and Western-blot The antigenicity and the expression level of NF-κB (p65) were detected. Results High quality B16 total RNA was obtained. A 1.65 kb cDNA fragment was amplified by RT-PCR. The pcDNA3.1 / NF-κB (p65) vector was successfully constructed. Blast sequence analysis was identical with that of NM_009045.4 in GenBank. The NF-κB (p65) recombinant protein has antigenicity and its gene and protein expression is higher than that of the B16 cell group and the empty plasmid pcDNA3.1 group. Conclusion The pcDNA3.1 / NF-κB (p65) expression vector and the recombinant protein with NF-κB (p65) antigen were successfully constructed.