关键词: 5-脂氧化酶, 基因克隆, 真核表达, RAW264.7细胞

Abstract: 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.

Key words: 5-Lipoxygenase； Gene cloning, Eukaryoiic expressing, RAW264.7 cell