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

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-His A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Western Blot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.

Construction and Expression of Deletion Mutant of IκBα and Its Bioactivity

It was to construct a eukaryotic expression vector of IκBα mutant deleted N-terminal aminos from 1 to 36, and to i-dentify 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 plasm id pcDNA3. 1/ myc-His (A) to construct the recombinant vector pcDNA3. 1-IκBα△N. 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 -IκBα△N 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-κB (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-kB signal transduction pathway and related diseases.