带新霉素抗性的萤光素酶报告载体的构建

Construction of a luciferase reporter vector with resistance of neomycin

目的:构建带新霉素抗性的萤光素酶报告载体。方法:设计扩增新霉素抗性基因neo,克隆入萤光素酶报告载体pGL3,得到pGL3-neo。分别比较pGL3、pGL3-CMV promoter瞬时转染组和经G418筛选的pGL3-neo、pGL3-neo-CMV promoter稳定转染组细胞萤光素酶活性。结果:PCR扩增出neo表达单元;酶切和DNA测序分析鉴定得到带新霉素抗性的萤光素酶报告质粒pGL3-neo。G418筛选可获得稳定转染pGL3-neo细胞株和稳定转染pGL3-neo-CMV promoter的细胞株。荧光检测显示:瞬时转染pGL3组、pGL3-CMVpromoter组及稳定转染pGL3-neo组、pGL3-neo-CMVpromoter组萤光素酶活性值分别为1047±86、1504±107和34819±1479、456109±15791,稳定转染组萤光素酶活性均高于瞬时转染组,P<0.05。结论:成功构建了带新霉素抗性的萤光素酶报告基因载体,使报告基因检测数据的敏感性和可靠性显著提高。

Aim： To construct a luciferase reporter vector with neomycin resistance. Methods：To design and amplify neomycin resistance gene, then clone the gene into lnciferase reporter vector pGL3, and obtain the luciferase reporter vector pGL3-neo. To compare the luciferase activity of cell lines transiently transfected by pGL3 and pGL3-CMV promoter, and cell line lasting transfected by pGL3-neo and pGL3-nco-CMV promoter after screening. Results： The neo gene was obtained by PCR ; screening by restriction enzyme and sequencing analysis showed that the luciferase reporter vector with neomycin resistance had been constructed successfully. The cell lines lasting transfected by pGL3-neo and pGL3-neo-CMV promoter were separately obtained by G418 screening. The luciferase activity of the groups transiently transfected by pGL3 and pGL3- CHY promoter was I 047 ± 86 and 1 504 ± 107 ,lower than that of groups transfected by pGL.3-neo and pGL3-neo-CMV promotet （34 819± 1 479 and 456 109±15 791 ） （ P 〈 0.05 ）. Conclusion ：The luciferase reporter vector with resistance of neomycin has been successfully constructed, which remarkably elevates the sensibility and dependability of lucisferase repotter gene detection.