摘要
目的:构建NF-κB的抑制蛋白IκBα突变体(IκBαM)的真核表达载体,检测其在内皮细胞中的表达。方法:从克隆质粒pSP64TL-IκBαM中用限制性内切酶双酶切IκBαM cDNA片段,克隆到真核表达质粒pcDNA 3.1/myc-His A内构建pcDNA 3.1/myc-His A-IκBαM质粒,并酶切鉴定及测序分析。以脂质体法转染内皮细胞,观察其表达及活性。结果:pcDNA 3.1/myc-His A-IκBαM质粒含有大小正确的IκBαM cDNA碱基片段,其碱基序列为编码目的基因的正确序列。pcDNA 3.1/myc-His A-IκBαM质粒能在内皮细胞中表达,并抑制IL-6分泌。结论:真核表达质粒pcDNA 3.1/myc-His A-IκBαM构建成功,IκBαM蛋白在内皮细胞中表达,并具有良好的生物学活性。
Objective： To construct eukaryotic expression plasmid of IκBα mutant （IκBαM）gene and to investigate its expression in endothelial cells. Methods：IκBαM cDNA was obtained from plasmids pSP64TL- IκBα M by digestion with Hind Ⅲ and Xba Ⅰ, and cloned into pcDNA 3. 1/myc-His to construct pcDNA 3.1/myc-His - IκBαM eukaryotic expression vector. The sequence of the plasmid was confirmed by restricting enzyme digestion and DNA sequencing. Then the plasmid was transfected into endothelial cells by liposome-mediated gene transfer method,and IκBαM protein and its activity were determined. Results. pcDNA 3. 1/myc-His -IκBαM contained the correct nucleotide sequence of the full length of IκBαM eDNA fragment. The IκBαM eDNA fragment had been inserted into the eukaryotic expression plasmid pcDNA 3.1/myc-His A, and was formed into the pcDNA 3. 1/myc-His A-IκBαM. The IκBαM gene was expressed successfully in the transfected endothelial cells. The level of IL-6 secreted by the transfected cells was decreased. Conclusion： The pcDNA 3.1/myc-His A- IκBαM,an eukaryotic expression plasmid for IκBαM was constructed successfully. The IκBαM gene was expressed successfully in the transfected endothelial cells with good biological activity.
出处
《武汉大学学报（医学版）》
CAS
2006年第1期9-12,共4页
Medical Journal of Wuhan University
基金
武汉大学人才专项基金资助(编号:502273106)
