人Ⅰ型基质金属蛋白酶基因真核表达重组质粒的构建

Construction of Eukaryotic expression Recombinant Plasmid with Human Matrix Metalloproteinase Ⅰ Gene

【目的】构建人Ⅰ型基质金属蛋白酶基因 (MMP1)真核表达重组质粒 ,并进行序列分析。【方法】用逆转录聚合酶链反应扩增人Ⅰ型基质金属胶原酶cDNA ,获得目的基因片段 (140 7bp)连接至 pcDNA3载体 ,并转化大肠杆菌DH5α,筛选阳性克隆并鉴定 ,并对片断全长进行DNA序列测定。【结果】所克隆人Ⅰ型基质金属胶原酶cDNA ,含全长MMP1编码区 ,与GeneBank公布序列比较 ,仅 1318位的胞苷酸C突变为腺苷酸A。并成功构建了含有MMP1的真核表达质粒 pcDNA3 MMP1。【结论】以逆转录聚合酶链反应方法成功构建了人Ⅰ型基质金属胶原酶cDNA克隆 ,并获得该基因真核表达质粒pcDNA3 MMP1,从而为下一步抗肝纤维化基因治疗研究提供物质基础。

To construct expression recombinant plasmid and to analyze the sequence of human matrix metalloproteinase Ⅰ gene (MMP1). The full human MMP1 cDNA was amplified by reverse transcription polymerase chain reaction (RT PCR). The obtained fragment of 1 407 bp was inserted into pcDNA3 vector and transformed into E.Coli DH5α and positive clone was selected. Also the automatic DNA sequencer sequenced the full fragment. A full human MMP1 fragment was obtained, and a base substitution(C to A) of coding region at the position 1 318 was compared with the GeneBank information. Eukaryotic expression recombinant plasmid with MMP1 fragment was successfully constructed. [Conclusion] The human MMP1 cDNA clone is constructed by RT PCR, and recombinant plasmid pcDNA3 MMP1 is successfully constructed;It may provide a basis for further anti liver fibrosis gene therapy.