pcDNA3.1/NF-κB(p65)表达载体的构建及蛋白表达

Construction and expression of recombinant pcDNA3.1/NF-κB(p65) eukaryotic expression vector

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 NF-κB（p65） cDNA and to construct a pcDNA3.1/NF-κB（p65） eukaryotic expression vector and assay NF-κB（p65） expression in vitro. Methods Total RNA was extracted from B16 cell by TRIZOL reagents, NF-κ B（p65） cDNA fragment was amplified by RT-PCR and cloned into pcDNA3.1 vector and then sequenced. Recombinant vector pcDNA3.1/ NF-κB（p65） was transfected into B16 cells, and the antigenicity and expression of NF- K B（p65） were analyzed by Real-time PCR and Western-blot. Results Total RNA was extracted from B16 cell by TRIZOL reagents, NF-κB（p65） eDNA fragment was amplified by RT-PCR and cloned into peDNA3.1 vector and then sequenced. Recombinant vector pcDNA3.1/NF-κ B（p65） was transfected into B16 cells, and the antigenicity and expression of NF-κB（p65） were analyzed by Real-timHigh-quality total RNA was extracted from B16 cells, a 1.65kb fragment of human NF- κ B（p65） gene was amplified by RT-PCR. pcDNA3.1/NF-κ B vector was constructed successfully, and the result of Blast sequence analysis is in accordance with that of NM_009045.4 in GenBank. The expression levels of NF-κ B（p65） recombinant protein and gene were much higher compared to B16 cells group and empty plasmid peDNA3.1 group, e PCR and Western-blot. Conclusion pcDNA3.1/NF-κB（p65） vector was constructed successfully and NF-κB（p65） recombinant protein and gene were expressed successfully.