人巨噬细胞金属弹性蛋白酶基因真核表达载体的构建及其鉴定

Construction and identification of human macrophage metalloelastase gene recombinant eukaryotic expressing vector

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 expressing vector-pcDNA3.1(-)/HME including functional region of human macrophage metalloelastase gene (HMME) to provide a basis for further study on the role of HMME in neovascularization.Methods Total RNA was extracted by using TRIZOL one-step method from human macrophage which was purified in vitro.CDNA was amplified by using reverse transcriptional polymerase chain reaction (RT-PCR).Product of PCR,amplified by transformed into Escherichia coli DH5α,was inserted into the eukaryotic expression vector after digestion and ligation by using restriction endonucleases and ligase.Results The correct HME cDNA clone was obtained and it was confirmed that HME cDNA wasinserted into the eukaryotic expression vector correctly by using digestion identification and sequencing.Conclusion The recombinant eukaryotic expression vector pcDNA3.1(-)/HME is successfully constructed.