IκBα缺失突变体真核表达载体的构建、表达及其生物活性检测  

Construction and Expression of Deletion Mutant of IκBα and Its Bioactivity

作　　者：黄思超[1] 杜军[2] 邱胜红[1] 徐俊[1] 傅刘鹏[1] 马锦珊[1] 蔡绍晖[1] 

机构地区：[1]暨南大学药学院,广州510632 [2]中山大学药学院,广州510006

出　　处：《生物技术通报》2009年第11期83-88,共6页Biotechnology Bulletin

基　　金：国家自然科学基金(30672510);广东省科技计划重点资助项目(7112208000694)

摘　　要：旨在构建缺失N端前36个氨基酸的IκBα突变体真核表达载体,并对其表达及生物学活性进行检测。从人源子宫颈癌细胞HeLa中提取总RNA,利用RT-PCR的方法获得IκBα缺失突变体的cDNA,将其克隆至真核表达载体pcDNA3.1/myc-HisA中,构建重组载体pcDNA3.1-IκBαΔN。通过PCR方法、NcoⅠ酶切以及核酸测序分析对其进行鉴定;采用WesternBlot检测IκBα缺失突变体蛋白在HeLa细胞中的表达。将pcDNA3.1-IκBαΔN和pNF-κB-Luc共转染HeLa细胞,经TNF-α诱导后,利用萤光素酶报告系统来检测重组载体对NF-αB的抑制活性。结果表明,经PCR方法、NcoⅠ酶切鉴定及核酸测序分析后,证实成功构建了重组载体pcDNA3.1-IκBαΔN;IκBα缺失突变体蛋白在HeLa细胞中高效表达,并对NF-κB有显著的抑制活性(P<0.01)。因此,真核表达载体pcDNA3.1-IκBαΔN构建成功,为一步研究NF-κB信号传导通路及其相关疾病提供有效的分子工具。It was to construct a eukaryotic expression vector of IκBα mutant deleted N-terminal aminos from 1 to 36,and to identify its expression and bioactivity. Total RNA was extracted from human cervical cancer HeLa cells, and cDNA of IκBα deletion mutant was obtained by RT-PCR method. The mutant gene was cloned into the eukaryotic expression plasmid pcDNA3. 1/ myc-His （A） to construct the recombinant vector pcDNA3. 1-IκBα△Nq. PCR method,Nco I digestion and DNA sequencing analysis were - employed to identify the recombinant vector. The expression of IκBα deletion mutant was detected by Western Blot. HeLa cells were co-transfected with pcDNA3.1-IκBα△N and pNF-κB-Luc. Then, after the induction of TNF-α,its inhibitory effect on NF-κB was tested by Luciferase Assay System. Result indicated that the recombinant vector pcDNA3. 1-IκBαAN was confirmed by PCR method, Nco I digestion and DNA sequencing analysis. The IκBα△N gene was expressed in HeLa cells and the deletion mutant protein had displayed obvious inhibitory effect on NF-KB （ P 〈 0.01 ）. Therefore, the pcDNA3.1 -IκBα△N, a eukaryotic expression vector of IκBα deletion mutant,was constructed successfully. It provides a potent molecular tool for further study of NF-κB signal transduction pathway and related diseases.

关 键 词：IΚBΑ 缺失突变体 载体构建 NF-ΚB 

分 类 号：Q78[生物学—分子生物学]