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  

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

作　　者：孟刚[1] 张扬[2] 蔺勇[3,4] 王广义[1] 

机构地区：[1]吉林大学第一医院普通外科,吉林长春130021 [2]吉林大学药学院微生物与生化药学教研室,吉林长春130021 [3]吉林大学第一医院神经内科,吉林长春130021 [4]中国医科大学附属第一医院神经内科,辽宁沈阳110001

出　　处：《吉林大学学报（医学版）》2009年第1期64-67,195,共5页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科研基金资助课题(20070729-07)

摘　　要：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5400 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。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. ② Two fragments of PCR products （5400 bp and 704 bp） of eukaryotic expression vector pcDNA3.1/MT1-MMP were obtained by EcoR Ⅰ and BamH Ⅰ 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.

关 键 词：膜型基质金属蛋白酶1 基因转染 真核表达 

分 类 号：R394.2[医药卫生—医学遗传学] Q78[医药卫生—基础医学]