编码N端截短的IκBα突变体重组腺病毒的构建与鉴定

Construction and identification of recombinant adenovirus vector carrying a N-terminal phosphorylation sites-deleted human IκBα mutant gene

目的:通过去除N端丝氨酸32/36磷酸化位点,获得人胎盘组织IκBα突变体(IκBαM)基因,构建其复制缺陷型重组腺病毒(AdIκBαM),并进行体外表达和活性检测。方法:PCR定点克隆IκBαM基因(203-1003bp),亚克隆至pShuttle和pGEM-T,进行PCR、双酶切、DNA测序和同源性分析。将重组质粒pShuttle-IκBαM中含CMV启动子、IκBαMcDNA和PolyA信号的表达单元定向插入Ad5腺病毒载体,构建成重组腺病毒AdIκBαM,再经脂质体介导共转染293细胞进行包装。Western blotting检测AdIκBαM在293细胞中蛋白表达情况,电泳迁移率实验观察AdIκBαM抑制佛波酯诱导的ECV304细胞核因子κB(NF-κB)激活的作用。结果:成功克隆长801bp的新型IκBαM基因,与Gen Bank中登陆的IκBα基因(接受号M69043)相应核苷酸序列一致。所制备的AdIκBαM滴度为4.0×1012pfu/L。AdIκBαM介导IκBαM基因在293细胞中表达,并以剂量依赖性方式显著抑制佛波酯诱导的ECV304细胞NF-κB活化。结论:AdIκBαM有效介导IκBαM基因表达并特异性抑制NF-κB活性,有望应用于哮喘的基因治疗。

AIM： To optimize the IκBα mutant （IκBαM） gene derived from human placenta tissue by deleting N - terminal phosphorylation sites of serine 32/36, and to construct and identify its replication - deficient recombinant adenovirus （AdIκBαM）. METHODS： The IκBαM gene （203 -1 003 bp） was acquired by positional cloning, followed by subcloning it into pShuttle and pGEM -T vectors for further PCR, double digestion, DNA sequencing and homology analysis. Subsequently, the expression unit of pShuttle - IκBαM containing CMV promoter, IκBαM cDNA and poly A signals was inserted into AdS vector, after which the resultant recombinant adenovirus AdIκBαM was packaged in 293 cells by cotransfection with lipofectamine. Western blotting analysis and electrophoretic mobility shift assay were utilized to detect the AdIκBαM -mediated expression of IκBαM gene in 293 cells and its suppressive effect on phorbol myristate acetate （PMA） -induced nuclear factor KB （NF-κB） activation in ECV304 cells, respectively. RESULTS： The relevant nucleotide and amino acid sequence of IκBαM gene was consistent with that of GenBank （accession number M69043 ）. The titer of the prepared AdIκBαM was 4.0 × 10^12 pfu/L. Moreover, the IκBαM gene was expressed in 293 cells, and potently inhibited the PMA - induced NF - κB activation in ECV304 cells in a dose - dependent manner. CONCLUSION ： AdIκBαM is a nonvel vector for both efficient transfer and expression of IκBαM gene as well as specific inhibition of NF - κB activity, providing a promising future for gene therapy of asthma.