携带Tum5基因的慢病毒表达载体的构建及其在人脐静脉血管内皮细胞中的表达

Construction of Tum5 gene lentiviral vector and its expression in human umbilical vein endothelial cells

目的:构建及鉴定Tum5基因慢病毒表达载体,并观察在体外人血管内皮细胞中的表达。方法:采用PCR技术从含有tumstatin基因的质粒克隆模板pSPORT1-Sfi钓取Tum5基因,并将基因克隆到慢病毒载体表达质粒pGC-FU(含EGFP基因)中,构建慢病毒载体表达质粒pGC-FU-Tum5,通过酶切、测序验证Tum5基因后,将pGC-FU-Tum5质粒和包装质粒pHelper1.0,pHelp-er2.0共同转染人胚胎肾上皮细胞系293T细胞,获得携带Tum5基因和EGFP基因的重组慢病毒GC-FU-Tum5,并转染靶细胞人脐静脉血管内皮细胞。通过检测标志蛋白-增强型绿色荧光蛋白(EGFP)和目的蛋白Tum5进一步验证pGC-FU-Tum5在靶细胞中Tum5的表达。结果:(1)pGC-FU-Tum5中携有正确的Tum5基因,并能在人类细胞中表达;pGC-FU-Tum5共转染包装细胞293T能产生重组病毒GC-FU-Tum5;(2)目的基因Tum5能被重组慢病毒高效地转导入靶细胞人脐静脉血管内皮细胞,并达到稳定的表达,荧光显微镜下能直接观察到GFP,Western blotting能检测到Tum5蛋白在靶细胞中的表达。结论:成功构建了携带Tum5基因的重组慢病毒载体,转染人脐静脉血管内皮细胞后能够稳定表达Tum5基因,为进一步研究Tum5的功能和眼部新生血管疾病的治疗奠定基础。

AIM： To construct a lentiviral vector carrying Tum5 gene and investigate its expression in human umbilical vein endothelial cells（HUVEC）. METHODS： Tum5 gene was amplified from plasmid pSPORT1-Sfi by PCR technique and subcloned into the expression plasmid of lentiviral vector, pGC-FU, to generate the lentiviral expression vector, pGC-FU-Tum5. The correct Tum5 gene was confirmed by endoenzyme digestion and sequencing. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pGC-FU-Tum5 and packaging plasmids--pHelper 1.0 and pHelper2.0. The resulting recombinant lentiviruses （GC-FU-Tum5） which carried Tum5 and EGFP gene were then used to infect human umbilical vein endothelial cells. EGFP and Tum5 protein expressions in 293T and human umbilical vein endothelial cells were dectected by fluorescent microscope and Western blotting analysis. RESULTS ： 1） plasmid pGC-FU-Tum5 carried the correct Tum5 gene and could be expressed in human cells. The recombinant lentiviruses GC-FU-Tum5 which carried Tum5 could be produced by co-transfection of pGC-FU-Tum5 and packaging plasmid to 293T; 2） The EGFP and Tum5 protein were dectected by fluorescent microscope and Western blotting analysis in 293T and human umbilical vein endothelial cells. The recombinant lentiviruses which carried Tum5 could transfect and deliver Tum5 genes to HUVEC, and Tum5 can be expressed efficiently in HUVEC. CONCLUSION ： The recombinant lentiviruse GC-FU-Tum5 was produced successfully and it can deliver target gene Tum5 to HUVEC.