目的尝试构建人血凝素样氧化型低密度脂蛋白受体1(LOX-1)基因双顺反子表达载体并检测其在293T细胞中的表达情况和生物学活性,为深入探讨LOX-1在动脉粥样硬化中的作用和以LOX-1为靶点建立干预机制治疗动脉粥样硬化奠定基础。方法首先根据Primer5.0软件设计引物,采用聚合酶链反应法以LOX-1 c DNA片段为模板扩增LOX-1基因完整编码区,克隆至T载体,经测序成功后亚克隆到双顺反子真核表达载体p IRES2-Ac GFP1-Nuc。利用脂质体转染法将双顺反子重组表达质粒转染至293T细胞,倒置荧光显微镜检测质粒转染情况。采用逆转录聚合酶链反应和免疫印迹法鉴定LOX-1基因在核酸和蛋白水平的表达,采用激光共聚焦检测LOX-1基因在293T细胞膜上的表达,激光共聚焦和流式细胞术检测表达在293T细胞膜上的LOX-1结合氧化型低密度脂蛋白(ox-LDL)的生物学活性。结果成功构建p IRES2-LOX-1双顺反子重组表达载体,将其转染293T细胞后,观察到绿色荧光蛋白基因的表达,初步表明LOX-1基因转染至293T细胞。进一步分子水平鉴定结果表明LOX-1基因在293T细胞核酸和蛋白水平均得到表达,激光共聚焦结果证明LOX-1基因在293T细胞膜上表达。最后激光共聚焦和流式细胞术结果证实表达在293T细胞膜上的人LOX-1基因可以结合氧化型低密度脂蛋白。结论成功构建人LOX-1基因双顺反子表达载体,在此基础上证明其在293T细胞膜上得到表达,并且具有结合ox-LDL的生物学活性,为后续体外研究其在动脉粥样硬化中的作用以及以此为靶点建立干预机制奠定了基础。 OBJECTIVE: To construct a bicistronic expression vector of human hemagglutinin-like oxidized low density lipoprotein receptor 1 (LOX-1) gene and to detect its expression and biological activity in 293T cells. To investigate the expression of LOX-1 In the role of atherosclerosis and LOX-1 as the target to establish an intervention mechanism for the treatment of atherosclerosis and lay the foundation. Methods Primer5.0 software was used to design the primers. Polymerase chain reaction (PCR) was used to amplify the complete coding region of LOX-1 gene with LOX-1 c DNA fragment and cloned into T vector. After successful sequencing, Eukaryotic expression vector pIRES2-Ac GFP1-Nuc. The bicistronic recombinant plasmids were transfected into 293T cells by lipofection method. The transfection conditions of plasmids were detected by inverted fluorescence microscope. The expression of LOX-1 gene at the level of nucleic acid and protein was identified by reverse transcription polymerase chain reaction and Western blotting. The expression of LOX-1 gene on 293T cell membrane was detected by laser confocal laser scanning confocal microscope and flow cytometry LOX-1 binds to the biological activity of oxidized low-density lipoprotein (ox-LDL) on 293T cell membranes. Results The recombinant eukaryotic expression vector pIRES2-LOX-1 was successfully constructed and transfected into 293T cells. The expression of green fluorescent protein (GFP) gene was observed and the LOX-1 gene was transfected into 293T cells. Further molecular profiling results showed that LOX-1 gene was expressed in 293T cells at both nucleic acid and protein levels. Confocal laser scanning results showed that LOX-1 gene was expressed on 293T cell membrane. The results of laser confocal and flow cytometry confirmed that the human LOX-1 gene expressed on 293T cell membrane could bind to oxidized low density lipoprotein. CONCLUSION: The bicistronic expression vector of human LOX-1 gene was successfully constructed and proved to be expressed on the membrane of 293T cells. The biological activity of ox-LDL was also demonstrated in this study. In order to further study its expression in atherosclerosis As well as the establishment of a mechanism of intervention as the basis for this.