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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 a fusion expression vector containing B7-1 gene and green fluorescent protein (GFP) gene and transfect it into LM8 osteosarcoma cells to obtain a highly efficient and stable expression vector in LM8 cells. Methods The B7-1 gene fragment was amplified by RT-PCR, and the fusion expression vector was constructed by using the eukaryotic expression plasmid containing GFP gene. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The plasmid was transfected into LM8 cells by lipofectamine , And its expression in tumor vaccine cells was detected by fluorescence microscopy and Western blot. Results The recombinant plasmid pEGFP-C1 / B7 containing the B7-1 gene was successfully constructed by PCR and restriction enzyme digestion. The sequencing result of the recombinant plasmid was consistent with the sequence of mB7-1 in Genebank. The expression of GFP was detected by fluorescence microscopy in transfected osteosarcoma cells, and the expression of B7-1 gene was detected by RT-PCR in transfected osteosarcoma cells. The expression of B7 protein in transfected osteosarcoma cells was detected by Western blot. Conclusion B7-1 recombinant eukaryotic expression vector pEGFP-C1 / B7 has been successfully constructed and successfully transfected into LM8 cells.