5-LO真核表达载体的构建及其在RAW264.7细胞中的表达的初步研究

目的构建5-LO真核表达载体并初步研究其在RAW264．7细胞中的表达，为建立5-LO高表达转基因小鼠模型提供表达载体。方法从人全血标本中提取细胞总RNA，以逆转录聚合酶链反应（RT-PCR）法扩增5-LO基因，分别构建5-LO目的基因的克隆载体与表达载体，提取质粒，进行酶切、PCR和DNA测序鉴定，将重组融合表达载体pEGFP-5-LO稳定转染RAW264．7细胞，RT-PCR、western blot方法检测pEGFP-5-LO的表达情况。结果构建了重组克隆载体pUCm-5-LO和真核表达载体pEGFP-5-LO，经酶切、PCR及测序鉴定正确；转染细胞后，在荧光显微镜下观察到pEGFP-5-LO融合蛋白的表达，RT—PCR和Westemblot结果表明转染pEGFP．5-LO的RAW264．7细胞中有5-LO的表达。结论成功克隆了5-LO基因，构建的表达载体pEGFP-5-LO在RAW264．7细胞中表达5-LO蛋白，为进一步5-LO高表达转基因鼠模型的建立和临床应用奠定了基础。

Construction of Eukaryotic Expression Plasmid for 5-Lipoxygenase Gene and its Expression in RAW264.7 Cell

Objective To construct 5-Lipoxygenase gene eukaryotic expression vector and study its expression in R AW264.7cell. Methods The gene of 5-LO was amplified from human peripheral blood by reverse trameriptage-pelymerase chain reaction（RT-PCR）, and then inserted it into cloning vector pUCm-T, and eukaryotic expressing vector pEGFP-C2 respectively. Recombinant plasmids were trans- formed and screened, and identified by PCR, restriction enzyme digesdon and DNA sequencing. The recombinant epressing plasmid 5-LO was transfected into RAW264.7 cells. Then detected the expres- sion of the interesting gene 5-LO. Result The recombinant plasmid pUCm-5-LO and pEGFP-5-LO were successfully constructed, and the correct sequence of 5-LO identified by PCR, restriction enzyme, digestion and DNA sequencing. The recombinant expressing plasmid pEGFP-5-LO was successfully transfected into RAW264.7 cells and it could be effectively expressed which was also testified by RT-PCR and Western blot. Conclusion The recombinant eukaryotic expressing plasmid ofpEGFP-5-LO is successfully constructed and effectively expressed in RAW264.7 cells, and it may be useful for further research of 5-LO transgenic mice.