γ-干扰素基因pIRES改进型黄色荧光蛋白载体的构建

Construction of the Enhanced Yellow Fluorescent Protein Expression Vector Carrying IFN-γ Gene

【目的】构建携带γ 干扰素基因 (ifn γ)的pIRES改进型黄色荧光蛋白载体 (pIRES EYFPIFN γ) ,为γ 干扰素基因的体内外表达及蛋白定位提供理想的标记。【方法】根据已知γ 干扰素基因序列 ,设计在目的片段两端分别带EcoRⅤ和NotⅠ酶切位点序列的 2条引物 ,用PCR技术从质粒 pcDNA3IFN γ中扩增出ifn γ(499bp) ,PCR产物用 EcoRⅤ和 NotⅠ双酶切后回收纯化 ,定向克隆至商品质粒 pIRES EYFP ,重组质粒 pIRES EYFPIFN γ用限制性内切酶酶切和DNA序列分析鉴定。同时以脂质体为介导 ,将 pIRES EYFPIFN γ转染正常人眼Tenon囊成纤维细胞 ,4 8h后于荧光倒置显微镜下观察基因的表达。【结果】DNA序列分析证明PCR产物为ifn γ ,将重组质粒转染正常人眼Tenon囊成纤维细胞 ,荧光倒置显微镜下可见黄色荧光蛋白的表达 ,转染率约为 4 3%。【结论】成功构建 pIRES EYFPIFN γ载体为利用黄色荧光蛋白标记在体内外进行γ

To construct the enhanced yellow fluorescent protein (EYFP) vector carrying interferon γ gene (ifn γ) in order to provide an ideal reporter in the expression of ifn γ and identify the location of protein in vitro and in vivo According to the nucleotide sequence of ifn γ gene, a pair of oligonucleotides was designed as primer which contained nucleotide sequence of EcoRⅤ and NotⅠ restriction endonuclease at the two end respectively The gene encoding for inf γ (499 bp) was amplified using PCR technique The PCR product was digested with EcoRⅤ and NotⅠ, and then cloned into the plasmid pIRES EYFP The recombinant plasmid pIRES EYFPIFN γ was identified by restriction endonuclease enzyme analysis and partial DNA sequence analysis And the recombinant plasmid pIRES EYFPIFN γ was transfected into human Tenon's capsule fibroblasts with Lipofectamine, the expression of yellow fluorescent protein was observed after 48 h The ifn γ was successfully amplified and verified by partial DNA sequence analysis The yellow fluorescent protein was expressed after transfection The transfecting rate was about 43% [Conclusion] The EYFP expression vector carrying ifn γ gene is successfully established This research work has formed a base for monitoring the ifn γ gene expression and identifying protein position in living cells