论文部分内容阅读
目的构建人细胞外基质金属蛋白酶诱导因子(matrix metalloproteinase inducer,EMMPRIN)真核表达质粒,并在COS-7细胞中表达。方法采用巢式PCR法从人结肠癌SW-480细胞中扩增EMMPRIN基因,插入pEGFP-N1质粒,构建重组真核表达质粒pEGFP-N1-EMMPRIN,转染COS-7细胞,48 h后,于倒置荧光显微镜下观察绿色荧光蛋白的表达,RT-PCR法和Western blot法分别检测转染细胞中EMMPRIN基因mRNA的转录和蛋白的表达。结果重组真核表达质粒pEGFP-N1-EMMPRIN经菌落PCR、酶切及测序证实构建正确,测序结果经比对分析,仅发现1个碱基突变,即534位:GCC→GCT,为无义突变;pEGFP-N1-EMMPRIN转染的COS-7细胞能表达绿色荧光蛋白,且能检测到EMMPRIN基因mRNA的转录和蛋白的表达。结论成功构建了人EMMPRIN真核表达质粒,并在COS-7细胞中表达了EMMPRIN,为进一步研究EMMPRIN在恶性肿瘤发生发展中的作用及其基因治疗奠定了基础。
Objective To construct eukaryotic expression plasmid of human extracellular matrix metalloproteinase inducer (EMMPRIN) and express in COS-7 cells. Methods EMMPRIN gene was amplified from human colon cancer SW-480 cells by nested PCR and inserted into pEGFP-N1 plasmid. Recombinant eukaryotic expression plasmid pEGFP-N1-EMMPRIN was constructed and transfected into COS-7 cells. After 48 h, The expression of green fluorescent protein (EGFP) was observed under an inverted fluorescence microscope. The transcription and protein expression of EMMPRIN mRNA were detected by RT-PCR and Western blot respectively. Results The recombinant eukaryotic expression plasmid pEGFP-N1-EMMPRIN was confirmed by colony PCR, restriction enzyme digestion and sequencing. The result of sequencing showed that there was only one base mutation, ie, 534 GCC → GCT, which was a nonsense mutation ; COS-7 cells transfected with pEGFP-N1-EMMPRIN could express green fluorescent protein and could detect the mRNA transcription and protein expression of EMMPRIN gene. Conclusion The eukaryotic expression plasmid of human EMMPRIN was successfully constructed and EMMPRIN was expressed in COS-7 cells, which laid a foundation of further study on the role of EMMPRIN in the genesis and development of malignant tumor.