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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 recombinant eukaryotic expression plasmids of TIMP1 and express them in human breast cancer MCF-7 cells. Methods Three kinds of recombinant plasmids pcDNA3.1 (+) - TIMP1, pVAXⅠ-TIMP1 and pEGFP-C1-TIMP1 were constructed by inserting TIMP1 gene into three eukaryotic expression vectors pcDNA3.1 (+), pVAXⅠ and pEGFP- Transfected MCF-7 cells, the expression of TIMP1 gene in MCF-7 cells was detected by Real-time PCR and Western blot. Results The recombinant plasmids pEGFP-C1-TIMP1 were transfected into MCF-7 cells 48 h after transfection and confirmed by fluorescence microscope. However, pcDNA3.1 (+ ) -TIMP1 and pVAXⅠ-TIMP1 transfected cells showed no green fluorescence; in transfected MCF-7 cells, TIMP1 mRNA transcription level and protein expression levels from high to low were pcDNA3.1 (+) - TIMP1, pVAXI-TIMP1 and pEGFP-C1-TIMP1. Conclusion Three TIMP1 gene eukaryotic expression plasmids have been successfully constructed and their expression levels are different in MCF-7 cells.