MMP-13基因真核表达载体的构建及其功能研究

Construction of MMP-13 eukaryotic expression vector and research on its function

目的为了研究MMP-13蛋白在瘢痕形成的作用,我们构建MMP-13基因真核重组表达质粒,检测并鉴定了MMP-13基因在转染了重组质粒的皮肤角质形成细胞HaCat中的表达。方法通过RT-PCR方法将MMP-13基因克隆到真核表达载体pcDNA3.1(+)中,构建其真核表达重组质粒pcDNA3.1(+)/MMP-13,基因测序鉴定。重组质粒以阳离子脂质体2000介导转染HaCat细胞,RT-PCR检测外源基因的表达,间接免疫荧光和Western blot检测外源蛋白的表达。以明胶酶谱法和MTT法分别检测MMP-13的活性以及其对HaCat细胞增殖的影响。结果通过RT-PCR检测到MMP-13基因在重组质粒转染后的HaCat细胞中大量表达,以间接免疫荧光反应和Western blot可检测到MMP-13蛋白在在重组质粒转染后的HaCat细胞中呈阳性,明胶酶谱法和MTT法检测显示真核表达质粒pcDNA3.1(+)/MMP-13表达的MMP-13蛋白具有活性,并且可以抑制HaCat细胞的增殖。结论构建的重组质粒pcDNA3.1(+)/MMP-13能在Hacat细胞中表达活性蛋白MMP-13,并可以抑制HaCat细胞的增殖,这为研究MMP-13在瘢痕形成中的作用奠定了基础,从而为瘢痕治疗提供有效靶标。

To investigate the roles of MMP-13 protein in scar formation,we constructed MMP-13 eukaryotic recombinant vector and detected its expression in HaCat cells.MMP-13 gene was cloned into eukaryotic expression vector pcDNA3.1（＋） by RT-PCR to construct eukaryotic expression plasmid pcDNA3.1（＋） / MMP-13,then confirmed by gene sequencing.The recombinant vector was transfected into HaCat cell with Lipofectamine 2000.RT-PCR demonstrated that MMP-13 mRNA expressed abundantly in the HaCat cells,while indirect immunofluorescence reaction and Western blot confirmed the MMP-13 protein expression in the transformed HaCat cells.Zymography and MTT assay showed the expressed MMP-13 protein is competent and can inhibit cell proliferation HaCat.All the results indicate that active MMP-13 protein can be expressed in HaCat cells and can inhibit the cell proliferation of HaCat cells,which laid a basis for studying the protein＇s role in scar formation,so as to provide an effective scar treatment target.