B7-1／GFP基因真核表达载体的构建及其在骨肉瘤细胞中的表达(英文)

Construction of Eukaryotic Expression Vector Containing B7-1/GFP Gene and Its Expression in Osteosarcoma Cell Line

目的构建含B7-1基因与绿色荧光蛋白(GFP)基因的融合表达载体,并转染LM8骨肉瘤细胞,研究获得其在LM8细胞中高效稳定表达的方法。方法用RT-PCR法扩增B7-1基因片断,应用含GFP基因的真核表达质粒构建融合表达载体,酶切、测序鉴定构建质粒;采用脂质体介导方法将其转染到LM8细胞,并用荧光显微镜及Western blot检测其在肿瘤疫苗细胞中的表达。结果经PCR及酶切鉴定,证实成功构建了含B7-1基因的真核表达重组体pEGFP-C1／B7,重组子测序结果与Genebank中mB7-1 序列相符。用荧光显微镜观察到转染骨肉瘤细胞中有GFP表达,RT-PCR检测到转染骨肉瘤细胞中B7-1基因的表达．Western blot 发现转染骨肉瘤细胞中有B7蛋白的表达。结论 B7-1重组真核绿色荧光表达载体pEGFP-C1／B7已成功构建,并成功转染LM8细胞。

Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.