细菌荧光酶的融合luxAB基因在肝癌细胞中的表达  

Expression of fused luxAB gene of bacterial luciferase in liver carcinoma cells

作　　者：茆灿泉[1] 杨树德[1] 赵玫[2] 范云霞[2] 徐枫[2] 杜菲[2] 杨华卫[1] 袁兴华[2] 黄常志[2] 

机构地区：[1]卫生部北京医院 [2]中国医学科学院中国协和医科大学肿瘤研究所肿瘤医院

出　　处：《中华肿瘤杂志》2001年第5期359-362,共4页Chinese Journal of Oncology

基　　金：国家自然科学基金资助项目 ( 396 70 6 96 )

摘　　要：目的　探索细菌荧光酶的融合luxAB基因作为新的报告基因在肝癌细胞中表达的可行性。方法　基于PCR的位点突变技术 ,构建了含有细菌荧光酶融合AB基因的哺乳动物表达载体pcDNA3 luxAB ,通过脂质体转染 ,使其在肝癌细胞BEL740 2中稳定表达。以序列测定和PCR分别确定融合luxAB基因构建的正确性和质粒转染的成功。以MTT和生物发光法分别测定细胞群体生长曲线和细菌荧光酶发光强度。结果　构建的融合luxAB基因是单顺反子 ,与设计完全一致 ;pcDNA3 luxAB的转染对BEL740 2肝癌细胞的群体生长无显著影响 ;表达载体在BEL740 2细胞中可稳定表达 ,在细胞水平可检测到 (8.71± 1.2 1)mV/ 40 μg蛋白的细菌荧光酶发光强度。Objective This work was done to look into the expression of fused luxAB gene of bacterial luciferase as a reporter gene in liver carcinoma cells. Methods The mammalian expression vector pcDNA 3 luxAB gene, constructed by the fusion of luxA and luxB genes, were amplified in the polymerase chain reaction (PCR) directed site mutagenesis from the vibrio harveyi 1600 strain and inserted into the plasmid of pcDNA 3. This analysis was to confirm the fused luxAB gene and the positive clones obtained by the G418 resistant stable selection and transfected by lipofectin, when they were confirmed by the PCR. The growth curve of cell population and luminescence of bacterial luciferase was obtained through MTT and bioluminescence, respectively. Results The fused luxAB gene, being a monocistron, completely agreed with the design. No significant difference in the growth curves of cell population was observed between the transfected cells and untransfected ones. The recombinant plasmid was likely to be expressed in a stable fashion in the BEL7402 cell. Meanwhile, the maximum cellular level in terms of vitro bioluminescent strength reached the point of (8.71±1.21) mV/40 μg protein.Conclusion Bacterial luciferase luxAB gene may become the first choice as a new, sensitive and non invasive reporter gene in the research on liver cancer cells. [

关 键 词：报告基因 细菌荧光酶 luxAB基因 基因转染 肝癌 luxAB基因 

分 类 号：R735.7[医药卫生—肿瘤]