应用巢式PCR法克隆N端部分缺失的IκBα基因

Cloning of N-terminal deleted IκBα gene by nested PCR

目的 克隆中国人N端部分缺失的IκBα基因 ,为进一步应用其进行抗肿瘤基因治疗研究打下基础。方法 从中国人外周血中分离单核细胞 ,采用多聚赖氨酸贴壁刺激 0 .5h后提取总RNA。应用逆转录PCR法 ,以自行设计的引物扩增全序IκBα基因 ,应用巢式PCR法扩增N端部分缺失的IκBα基因 ,用TA克隆技术将目的基因连接到pGEM T质粒载体中 ,进行测序并与已知IκBα序列进行同源性分析。结果 测序结果表明我们所克隆的目的基因 ,其基因序列与GenBank中人IκBα基因序列仅有 5个碱基不同但所编码氨基酸仅 1个发生改变。结论 已成功克隆了中国人N端部分缺失的IκBα基因 ,为进一步应用N端部分缺失的IκBα基因进行抗肿瘤基因治疗研究打下了基础。

Objective To clone the N terminal deleted IκBα gene for further study.Methods The peripheral blood mononuclear cells (PBMCs) were isolated by gradient fractionation and stimulated by adherence to polylysine coated culture dishes. Total RNA was isolated from the stimulated PBMCs with RNeasy Total RNA kit and transcribed to cDNA with Ready To Go T Primed first strand kit. The IκBα gene was amplified by nested PCR using the primers based on the published sequence of IκBα.The nested PCR product was cloned into pGEM T vector by TA cloning strategy and the recombinant plasmid was screened by restriction analysis and direct sequencing. Results Sequencing analysis revealed that the amino acid sequence of N terminal deleted IκBα gene had only one amino acid residue displace from isoleucine to threonine (compared with that of published sequence) although there were five variations in nucleotide sequence.Conclusion The N terminal deleted IκBα gene is successfully cloned from a Chinese donor, which provide a solid basis for the study of IκBα mediated antitumor genetherapy.