B7-1转基因骨肉瘤疫苗的制备及鉴定

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，转染LM8骨肉瘤细胞制备骨肉瘤疫苗。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LCM结合蛋白免疫印记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，重组子测序结果与国际基因文库中mB7—1序列相符。用荧光显微镜观察到疫苗细胞中有GFP表达，RT—PCR检测到疫苗细胞中B7—1基因的表达．westernblot发现疫苗细胞中有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，转染，LM8细胞成功制备了骨肉瘤细胞疫苗。

Preparation and identification of B7-1 gene modified osteosarcoma vaccine

Objective:To prepare and indentify the osteosarcoma vaccine of transfered gene of the B7-1.Methods:By using gene cloning technique,eukaryote expression vector pEGFP-C1 were used to prepare the murine B7-1 recombinant plasmid(pEGFPC1/B7).Recombinant plasmid was transferred into LM8 cells with liposome and confirmed by restriction endonuclease digestion and DNA sequencing.The expression of the fusion protein was detected using fluorescence microscope and Western blot.Results:The analysis of sequence were corresponded to the data from Genebank.The pEGFP-C1/B7 was confirmed to be constructed successfully by RT-PCR and restriction enzymes analysis.Green fluorescence could seen in the vaccine cells under fluorescence microscope.The expected B7-1 and green fluorescent protein(GFP)fusion protein was detected by Western blot.Conclusion:B7-1 gene modified murine LM8 cells vaccine can be successfully prepared.