摘要
目的克隆中国人IκBα基因，了解其与欧美人种有无不同, 为进一步的研究工作打下基础。方法从中国人外周血中分离单个核细胞，刺激细胞贴壁30 min后 提取总RNA。应用RT-PCR法扩增目的基因，胶回收法纯化。将其连接到pGEM-T质粒载体，进行测序并与已知IκBα 序列进行同源性分析。结果中国人IκBα基因与genebank中发表的人IκB α基因序列仅2个碱基不同且所编码氨基酸无改变。结论我们已成功克隆了中国人IκBα基因 。同源性分析表明，其与 genebank中所报道的基因序列基本一致。
Objective To clone the Chinese IκBα gene and analysis whether or not the sequence of Chinese IκBα gene is different with other races. Methods The peripheral blood mononuclear cells(PBMCs) was isolated with Ficoll-Hypaque 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 RT-PCR using the primers based on the published sequence o f IκBα(genebank accession number M69043). The 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 sequence of Chinese IκBα gene is identical to that of published sequence although there are two variations in nuc leotide sequence. Conclusions We have successful cloned the Chinese IκBα gene. The cloning of IκBα gene will provide a solid basis for the study of IκBα-mediated anti-tumor gene therapy.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2002年第2期81-84,共4页
Immunological Journal
基金
国家自然科学基金面上项目资助(30070297)
