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目的 构建一个包含人巨噬细胞金属弹性蛋白酶 (HME)基因全部功能区的重组真核表达载体pcDNA3 .1(-) /HME ,为进一步研究人巨噬细胞金属弹性蛋白酶对体内肿瘤血管生成的影响奠定基础。方法 用Trizol试剂法从经体外纯化培养的人巨噬细胞中提取总RNA ,经逆转录 聚合酶链式反应 (RT PCR)扩增目的片段 ,PCR产物及真核表达载体分别双酶切后进行连接 ,并转化入大肠杆菌DH5α扩增以获得重组载体。结果 RT PCR获得预期大小的特异性DNA片段 ,经双酶切鉴定及测序证实已将人巨噬细胞金属弹性蛋白酶基因cDNA片段正确插入真核表达载体中。结论 成功获得pcDNA3 .1(-) /HME重组真核表达载体。
Objective To construct a recombinant eukaryotic expression vector pcDNA3.1 (-) / HME containing all functional regions of human macrophage metalloelastase (HME) gene in order to further study the effect of human macrophage metalloelastin on tumor angiogenesis in vivo Lay the foundation. Methods Trizol reagent was used to extract total RNA from human macrophages purified in vitro. The target fragment was amplified by reverse transcription-polymerase chain reaction (RT PCR). The PCR products and the eukaryotic expression vector were double digested respectively Ligated and transformed into E. coli DH5α for amplification to obtain a recombinant vector. Results The specific DNA fragments of the expected size were obtained by RT-PCR. The cDNA fragments of human macrophage metalloelastase gene were correctly inserted into the eukaryotic expression vector by double enzyme digestion and sequencing. Conclusion The pcDNA3. 1 (-) / HME recombinant eukaryotic expression vector was successfully obtained.