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目的构建人survivin真核表达载体,稳定转染入小鼠黑素瘤B16细胞,建立稳定表达人survivin的小鼠黑素瘤细胞系。方法采用PCR方法扩增出人survivin全长基因的cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo中,并加入酶切位点和6×his标签,得到重组表达质粒pIRES-neo-SUR-(his)6。利用阳离子脂质体介导法将其稳定转染入小鼠黑素瘤B16细胞,经G418加压筛选出阳性克隆。利用RT-PCR、蛋白免疫印迹及免疫荧光等检测方法验证人survivin基因在稳定转染B16细胞株中的表达。结果经限制性内切酶鉴定及序列分析,pIRES-neo-SUR-(his)6重组体构建正确。RT-PCR结果表明:从稳定转染pIRES-neo-SUR-(his)6的B16细胞所抽提的总RNA中能够扩增出survivin基因(约530bp);蛋白免疫印迹结果表明:利用特异抗体能够从稳定转染pIRES-neo-SUR-(his)6的B16细胞中检测到survivin蛋白条带,而对照空质粒转染后的细胞则没有相应的条带;流式细胞仪和激光共聚焦显微镜检测结果显示,稳定转染pIRES-neo-SUR-(his)6的B16细胞用特异性抗体检测到高效的荧光表达,其中5号单克隆的荧光表达率为91.38%,而对照空质粒转染后的细胞则没有相应的荧光表达。结论成功构建了真核表达载体pIRES-neo-SUR-(his)6,建立了稳定转染高效表达人survivin的小鼠黑素瘤细胞系。
Objective To construct a human survivin eukaryotic expression vector and stably transfected it into mouse melanoma B16 cells to establish a mouse melanoma cell line stably expressing human survivin. Methods The full-length cDNA of human survivin gene was amplified by PCR and inserted into the eukaryotic expression vector pIRES-neo by DNA recombination. The restriction enzyme sites and 6 × his tag were added to obtain recombinant plasmid Expression plasmid pIRES-neo-SUR- (his) 6. The positive cells were stably transfected into mouse melanoma B16 cells by cationic liposome-mediated method and positive clones were screened by G418. The expression of survivin gene in stable transfected B16 cells was verified by RT-PCR, Western blot and immunofluorescence. Results The recombinant pIRES-neo-SUR- (his) 6 was confirmed by restriction enzyme analysis and sequence analysis. The results of RT-PCR showed that the survivin gene (about 530 bp) was amplified from the total RNA extracted from B16 cells stably transfected with pIRES-neo-SUR- (his) 6. Western blot results showed that using specific antibody The survivin protein band was detected from B16 cells stably transfected with pIRES-neo-SUR- (his) 6, while the control transfected cells had no corresponding bands. Flow cytometry and confocal laser scanning microscopy The results of microscopy showed that B16 cells stably transfected with pIRES-neo-SUR- (his) 6 detected highly efficient fluorescent expression with specific antibodies, in which the fluorescent expression rate of No. 5 monoclonal antibody was 91.38%, whereas the control null plasmid Dyed cells do not have the corresponding fluorescent expression. Conclusion The eukaryotic expression vector pIRES-neo-SUR- (his) 6 was successfully constructed and a mouse melanoma cell line stably transfected with survivin was established.