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【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。
【Objective】 To construct the eukaryotic expression plasmid of human type Ⅰ matrix metalloproteinase (MMP1) and analyze the sequence of the recombinant plasmid. 【Method】 The cDNA of human type Ⅰ matrix metalloproteinase was amplified by reverse transcription polymerase chain reaction (PCR). The target gene fragment (140 7 bp) was ligated into pcDNA3 vector and transformed into E. coli DH5α. The positive clones were screened and identified. Long DNA sequencing. 【Results】 Compared with the published sequence of GeneBank, the cloned human type Ⅰ matrix metalloproteinase cDNA contained the full-length MMP1 coding region, only the 1318-mer C was mutated to adenylate A. The eukaryotic expression plasmid pcDNA3 MMP1 containing MMP1 was successfully constructed. 【Conclusion】 The cDNA clone of human type Ⅰ matrix metalloproteinase was successfully constructed by reverse transcription polymerase chain reaction and the eukaryotic expression plasmid pcDNA3 MMP1 was obtained, which provided the material basis for the further study of anti-hepatic fibrosis gene therapy.