人细胞外基质金属蛋白酶诱导因子真核表达质粒的构建及表达  

Construction and expression of eukaryotic expression vector for human extracellular matrix metalloproteinase inducer

作　　者：亢渝俊[1] 王川[1] 姜政[2] 王丕龙[2] 

机构地区：[1]重庆市第三人民医院消化科,重庆400014 [2]重庆医科大学附属第一医院消化内科,重庆400016

出　　处：《中国生物制品学杂志》2013年第9期1218-1221,1227,共5页Chinese Journal of Biologicals

摘　　要：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 the eukaryotic expression vector for human extracellular matrix metalloproteinase in- ducer （EMMPRIN） and express in COS-7 cells. Methods EMMPRIN gene was amplified by nested PCR from human colon cancer SW-480 cells, and inserted into vector pEGFP-N1, and the constructed recombinant plasmid pEGFP-N1- EMMPRIN was transfected to COS-7 cells. Forty-eight hours later, the expression of green fluorescent protein （GFP） was observed by fluorescent microscopy, while the mRNA transcription and the protein expression of EMMPRIN by RT-PCR and Western blot respectively. Results Recombinant plasmid pEGFP-N1-EMMPRIN was constructed correctly as proved by colony PCR, restriction analysis and sequencing. Sequencing result showed only one base mutation, i. e. GCC-~GCT at site 534, which was nonsense mutation. GFP was expressed in COS-7 cells transfected with pEGFP-N1-EMMPRIN, while the mRNA transcription and protein expression of EMMPRIN were observed. Conclusion The eukaryotic expres- sion vector for human extracellular EMMPRIN was constructed successfully and expressed in COS-7 cells, which laid a foundation of further study on role of EMMPRIN in onset, progress and gene therapy of malignant tumors.

关 键 词：细胞外基质金属蛋白酶诱导因子 真核细胞 基因表达 恶性肿瘤 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] Q786[医药卫生—基础医学]