稳定表达人G250基因小鼠renca细胞株的建立

目的建立能够稳定表达人G250基因的renca细胞株。方法采用PCR扩增出人G250基因编码区长度的互补序列,根据DNA重组技术定向插入到pIRES-neo真核表达载体中,得到重组表达质粒pIRES-neo-G250。用阳离子脂质体介导法将该质粒稳定转染到renca小鼠细胞中,通过调整G418的浓度筛选出阳性的克隆,蛋白免疫印迹和免疫荧光测试验证人G250基因转染小鼠renca细胞株后的表达情况。结果经过限制性内切酶鉴定和序列分析发现pIRES-neo-G250重组质粒构建正确,蛋白免疫印迹结果表明,可以从稳定转染pIRES-neo-G250的小鼠renca细胞中检测到G250的蛋白条带,流式细胞仪和激光共聚焦显微镜检测结果表明,稳定转染pIRES-neo-G250的renca细胞用人的G250特异性抗体检测到有高效的荧光表达。结论成功建立了稳定、高效表达pIRES-neo-G250分子的人G250基因renca细胞株,为后续的肾癌疫苗研究奠定了基础。

Establishment of renca cell strain stably expressing human G250 gene

Objective To establish the renca cell strain stably expressing human G250 gene.Methods The complementary sequence of human G250 gene length was amplified by PCR and inserted into the eukaryotic expression vector pIRES-neo using recombinant DNA technology to obtain the recombinant plasmid pIRES-neo-G250.The recombinant plasmid was stably transfected into the mouse renca cells with Lipofectamine 2000.Expression of renca cell strain in mice transfected with the human G250 gene was detected by Western blot and immunofluorescence assay,respectively,after the positive clone was screened by adjusting the G418 concentration.Results Restriction enzyme digestion and sequence analysis revealed that the recombinant plasmid pIRES-neo-G250 was successfully constructed.Western blot showed that the G250 protein was expressed in mouse renca cells stably transfected with pIRES-neo-G250.Flow cytometry and laser confocal microscopy demonstrated that the fluorescence was highly expressed in mouse renca cells stably transfected with pIRES-neo-G250.Conclusion The renca cell strain we established can stably and effectively express the human G250 gene transfected with pIRES-neo-G250 molecules,thus laying a foundation for further study of renal tumor vaccine.