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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。
Objective To establish a renca cell line capable of stably expressing human G250 gene. Methods The complementary sequence of human G250 gene coding region was amplified by PCR and inserted into pIRES-neo eukaryotic expression vector according to DNA recombination technology to obtain the recombinant plasmid pIRES-neo-G250. The plasmid was transfected into renca mouse cells by cationic liposome-mediated method. Positive clones were screened by adjusting the concentration of G418. The western blot and immunofluorescence assay were used to confirm the human G250 gene transfected mouse renca cell line After the expression of the situation. Results The recombinant plasmid pIRES-neo-G250 was constructed correctly by restriction enzyme analysis and sequence analysis. Western blotting results showed that G250 protein bands could be detected from mouse renca cells stably transfected with pIRES-neo-G250 Band, flow cytometry and laser scanning confocal microscopy showed that the renca cells stably transfected with pIRES-neo-G250 detected highly efficient fluorescent expression with human G250-specific antibodies. Conclusion The human G250 gene renca cell line stably and efficiently expressing pIRES-neo-G250 molecule was successfully established, which laid the foundation for the subsequent study of renal cell carcinoma vaccine.