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目的:建立稳定表达萤火虫荧光素酶的人肺癌细胞系并进行体外验证。方法:亚克隆萤火虫荧光素酶基因到真核表达载体pRc/CMV2上,构建重组质粒pRc/CMV2-luc+,转染到肺癌细胞株spc-a-1,经过G418筛选获得单细胞抗性克隆。抗性克隆连续传代,并通过荧光素酶活性检测筛选出高水平表达荧光素酶的稳定细胞克隆,在体外检测细胞的生物发光能力与细胞数量的相关性。结果:构建的pRc/CMV2-luc+真核表达质粒转染spc-a-1细胞后,经G418筛选出多个抗性克隆;传代至40代时确定有3株细胞的荧光素酶活性最高;活体影像系统体外检测证实阳性细胞株发光强度与数量呈正相关。结论:结果表明成功构建了稳定表达萤火虫荧光素酶的spc-a-1细胞系。
Objective: To establish a human lung cancer cell line stably expressing firefly luciferase and to verify it in vitro. Methods: The firefly luciferase gene was subcloned into the eukaryotic expression vector pRc / CMV2 to construct the recombinant plasmid pRc / CMV2-luc +, which was then transfected into the lung cancer cell line spc-a-1. Single cell resistant clones were obtained through G418 screening. Resistant clones were passaged in succession and stable luciferase-expressing cell clones were screened by luciferase activity assay, and the correlation between the bioluminescence ability and the number of cells was examined in vitro. Results: The constructed pRc / CMV2-luc + eukaryotic expression plasmid transfected spc-a-1 cells, a number of resistant clones were screened by G418; 3 cells were determined to have the highest luciferase activity from passage 40 to passage 40; In vivo imaging system in vitro tests confirmed that the positive cell line luminescence intensity and the number was positively correlated. Conclusion: The results show that spc-a-1 cell line stably expressing firefly luciferase was successfully constructed.