TIMP1基因重组真核表达质粒的构建及其在MCF-7细胞中的表达  

Construction of Eukaryotic Expression Vectors for Tissue Inhibitor of Metalloproteinase 1 Gene and Their Expressions in MCF-7 Cells

作　　者：李鹏[1,2] 马艳娇[1,2] 赵娜[2] 袁婷[2] 宫鹏涛[2] 李建华[2] 欧阳红生[2] 张西臣[2] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]吉林大学畜牧兽医学院,长春130062

出　　处：《中国生物制品学杂志》2011年第6期649-652,共4页Chinese Journal of Biologicals

基　　金：国家重大基础研究计划资助项目(2006CB910505)

摘　　要：目的构建3种TIMP1基因重组真核表达质粒,并在人乳腺癌MCF-7细胞中表达。方法将TIMP1基因分别插入3种真核表达载体pcDNA3.1(+)、pVAXⅠ和pEGFP-C1,构建3种重组表达质粒pcDNA3.1(+)-TIMP1、pVAXⅠ-TIMP1和pEGFP-C1-TIMP1,转染MCF-7细胞,采用Real-time PCR和Western blot法检测TIMP1基因在MCF-7细胞中的表达。结果 3种重组表达质粒经双酶切及测序证实构建正确;重组表达质粒pEGFP-C1-TIMP1转染MCF-7细胞48 h后,在荧光显微镜下可观察到绿色荧光,而pcDNA3.1(+)-TIMP1和pVAXⅠ-TIMP1转染的细胞则观察不到绿色荧光;在转染的MCF-7细胞中,TIMP1基因mRNA转录水平和蛋白表达水平从高到低依次为pcDNA3.1(+)-TIMP1、pVAXⅠ-TIMP1和pEGFP-C1-TIMP1。结论已成功构建了3种TIMP1基因重组真核表达质粒,其在MCF-7细胞中的表达量有差异。Objective To construct three eukaryotic expression vectors for tissue inhibitor of metalloproteinase 1（TIMP1） and express in MCF-7 cells.Methods TIMP1 gene was inserted into plasmids pcDNA3.1（＋）,pVAXⅠand pEGFP-C1,and the constructed recombinant plasmids pcDNA3.1（＋）-TIMP1,pVAXⅠ-TIMP1 and pEGFP-C1-TIMP1 were transfected to MCF-7 cells respectively.The expressed TIMP1 was identified by real-time PCR and Western blot.Results Restriction analysis and sequencing proved that the three recombinant plasmids were constructed correctly.Green fluorescence was observed in the MCF-7 cells 48 h after transfection with recombinant plasmid pEGFP-C1-TIMP1,while was not observed in those with pcDNA3.1（＋）-TIMP1 or pVAXⅠ-TIMP1.In the turn of TIMP1 mRNA transcription and protein expression levels,the MCF-7 cells were those transfected with pcDNA3.1（＋）-TIMP1,pVAXⅠ-TIMP1 and pEGFP-C1-TIMP1.Conclusion Three eukaryotic expression vectors for TIMP1 were successfully constructed,of which the expression levels in MCF-7 cells were different.

关 键 词：基质金属蛋白酶抑制剂1 真核细胞 基因表达 乳腺肿瘤 

分 类 号：Q782[生物学—分子生物学] Q786