人MT1-MMP真核表达载体的构建及其在HepG2细胞中的表达及意义

目的：克隆人膜型基质金属蛋白酶1（MT1-MMP）基因，构建真核表达载体，并检测其在人肝癌细胞HepG2中的表达。方法：采用逆转录聚合酶链式反应（RT-PCR）从人正常肝组织中扩增MT1-MMP全长cDNA，将之与pMD18-T载体连接，测序后将该片段亚克隆至真核表达载体pcDNA3.1中。将构建好的pcDNA3.1/MT1-MMP真核表达载体经酶切鉴定后，采用脂质体法转染入HepG2细胞，经G418筛选，得到阳性克隆细胞株。应用RT-PCR检测转染前后该细胞株MT1-MMP mRNA表达水平。结果：①RT-PCR获得长度约为704 bp的目的片段，与预期片段相符;②将pcDNA3.1/MT1-MMP真核表达载体经EcoRⅠ和BamHⅠ双酶切鉴定，得到约5 400 bp和704 bp两个片段，测序结果证实所插入目的片段与GenBank中MT1-MMP cDNA序列匹配；③重组质粒转染株MT1-MMP mRNA表达水平（1.66±0.43）明显高于空质粒组（1.21±0.25）和对照组（1.19±0.18)(P<0.01）。结论：成功构建pcDNA3.1/MT1-MMP真核表达载体，并在人肝癌细胞株HepG2中稳定表达。

Construction of eukaryotic expression vector of human MT1-MMP and its expression in HepG2 cells and significance

Objective To clone human membrane type-1 matrix metalloproteinase(MT1-MMP) gene and construct its eukaryotic expression vector,then detect its expression in human hepatoma cell line HepG2.Methods Full-length human MT1-MMP cDNA was amplified from normal liver by RT-PCR and cloned into pMD18-T simple vector.After sequencing,the fragment was subcloned into the pcDNA3.1 vector and the recombinant eukaryotic expression vector was constructed.MT1-MMP mRNA level of HepG2 cells was evaluated by RT-PCR.Results ① A 704 bp fragment was obtained by PCR,which was the same as the expected fragment.②Two fragments of PCR products(5400 bp and 704 bp) of eukaryotic expression vector pcDNA3.1/MT1-MMP were obtained by EcoRⅠ and BamHⅠ restriction enzyme digestion.The sequencing result of MT1-MMP cDNA was identical with that reported in GenBank.③ MT1-MMP mRNA level of HepG2 cells transfected with the recombinant vector was obviously higher(1.66±0.43) than those of empty vector group(1.21±0.25) and control group(1.19±0.18)(P<0.01).Conclusion The recombinant vector pcDNA3.1/MT1-MMP is successfully constructed and expressed stably in HepG2 cells.