带组氨酸标签的斑马鱼p53真核表达载体的构建及表达鉴定

Construction and Expressional Analysis of Eukaryotic Expression Vector for His-tagged Zebrafish p53 Protein

构建斑马鱼p53基因的真核表达系统,为下一步p53体内、外功能研究,以及为斑马鱼作为抗肿瘤药物筛选模型的构建和应用奠定基础。采用RT-PCR法从斑马鱼胚胎中扩增获得p53基因编码区,定向克隆到真核表达载pcDNA3.1上,构建真核表达质粒pcDNA3.1/his-p53,在起始密码前加入增强翻译的Kozak序列,并在终止密码前引入组氨酸标签便于检测和纯化,脂质体介导质粒转染HeLa细胞,RT-PCR和Western blotting检测基因表达情况。结果表明,成功构建了斑马鱼p53真核表达载体,RT-PCR扩增出1 100 bp的转录产物,表达产物能被抗his单克隆抗体特异性识别,Western blotting呈现53 kD左右单一条带。斑马鱼p53蛋白在HeLa细胞中成功表达。

It was to construct eukaryotic expression vector of zebrafish p53 gene for further study the biological function of zebrafish p53 in vitro or in vivo.p53 encoding region was obtained from zebrafish embryos by RT-PCR,and then the p53 fragment was subcloned into pcDNA3.1 vector to construct the recombinant plasmid pcDNA3.1/his-p53.A Kozak sequence was inserted before the start codon to enhance translation of p53 protein in HeLa cells.A his-tag was added before the stop codon.The recombinant plasmid pcDNA3.1/his-p53 was transfected into HeLa cells.RT-PCR and Western blotting were used to detect the expression of p53 in HeLa cells.The results showed that recombinant plasmid pcDNA3.1/his-p53 was successfully constructed.A transcriptional product corresponding to the encoding region of of p53 gene was detected by RT-PCR amplification.Expressed his-tagged p53 protein was specifically recognized by anti-his monoclonal antibody and displayed one single band about 53 kD by Western blotting.