摘要
目的 探寻人类 β -珠蛋白基因 5′ -帽位上游新转录调控元件 ;方法 利用重组DNA技术 ,构建及克隆 3个含有人类 β -基因不同 5’帽位上游片段的荧光素酶报告基因重组载体 (pGL2 -promoter/SB -βRHB734、pGL2 -promoter/SB - βRAB573、pGL2 -promoter/SB - βRHfB2 63) ,分别将其导入Hela细胞中 ,采用 β -半乳糖苷酶报告基因为转移效率内对照 ,空白的荧光素酶基因载体为基础对照 ,进行荧光素酶活性比较。结果 3个基因片段载体构建符合设计 ,其相对抑制活性分别为 59.74 %、80 .97%、79.4 2 % ;结论 初步提示人类 β -基因 5’帽位上游 - 2 132～ - 182 2bp片段及 - 182 2～ - 15559bp片段内可能存在有起负调控作用的顺式作用元件 (抑制子 ) ,而 - 2 2 77bp～ - 2 132bp片段间未检出抑制子或增强子活性存在。此初步结果尚需进一步研究证实和阐明其结构与功能的关系。
The luciferase reporter gene(Luc) recombinant vectors (pGL 2-promoter/SB-β RHB734、pGL 2-promoter/SB-β RAB573 and pGL 2-promoter/SB-β RHfB263) were constructed ,which contain different length of the 5'flankling sequences of β-globin gene by using deletion and recombinant DNA technique.Then these recombinant vectors were transfected into the Hela cells separately. The expressed luciferase activities of the recombinant vectors were measured by counting the single photon intensity (cpm)in the Liquid scintillometer, and the gene transfer efficiency was calibrated by transfection of a recombinant vector of the β-galactosidase gene and measurement of its expression activity at the same time with the luciferase repoter gene. The results show that a silencer activity onto the luc gene expression may be possible present in both of the -2 132～-1 822 and -1 822～-1 559 bp regions 5'-upstream to the cap site of human β-globin gene , but there is neither silencer nor enhancer activation detectable in-2277～-2132bp fragment.The inhibitilities of three vector are 59.74%,80.97%,79.42%.
出处
《南华大学学报（医学版）》
2001年第2期109-112,119,共5页
Journal of Nanhua University(Medical Edition)
基金
国家自然基金!课题 (编号 :3940 0 0 72 )
关键词
珠蛋白基因
表达调控
报告基因
抑制子
globin gene
expression regulation
luciferase reporter gene
silencer
